The Interactive Fly

Genes involved in tissue and organ development

Oogenesis and the Oocyte


The process of oogenesis
A combinatorial code for pattern formation in Drosophila oogenesis
Long-term live imaging provides new insight into stem cell regulation and germline-soma coordination in the Drosophila ovary
Mei-p26 cooperates with Bam, Bgcn and Sxl to promote early germline development in the Drosophila ovary
mRNA localization and translational control during oogenesis
Independent and coordinate trafficking of single Drosophila germ plasm mRNAs
Drosophila protein kinase N (Pkn) is a negative regulator of actin-myosin activity during oogenesis
Evidence for the mechanosensor function of filamin in tissue development
The Drosophila putative histone acetyltransferase Enok maintains female germline stem cells through regulating Bruno and the niche
H3K36 trimethylation-mediated epigenetic regulation is activated by Bam and promotes germ cell differentiation during early oogenesis in Drosophila
gone early, a novel germline factor, ensures the proper size of the stem cell precursor pool in the Drosophila ovary
Regulation of ribosome biogenesis and protein synthesis controls germline stem cell differentiation
DNA damage-induced CHK2 activation compromises germline stem cell self-renewal and lineage differentiation
Identification of germline transcriptional regulatory elements in Aedes aegypti
Aging and insulin signaling differentially control normal and tumorous germline stem cells
Insulin signaling acts in adult adipocytes via GSK-3beta and independently of FOXO to control Drosophila female germline stem cell numbers
Protein competition switches the function of COP9 from self-renewal to differentiation
Evidence for chromatin-remodeling complex PBAP-controlled maintenance of the Drosophila ovarian germline stem cells
Pleiotropic functions of the chromodomain-containing protein Hat-trick during oogenesis in Drosophila melanogaster
The Drosophila LIN54 homolog Mip120 controls two aspects of oogenesis
Specification and spatial arrangement of cells in the germline stem cell niche of the Drosophila ovary depend on the Maf transcription factor Traffic jam
The NuRD nucleosome remodelling complex and NHK-1 kinase are required for chromosome condensation in oocytes
A genetic mosaic screen reveals ecdysone-responsive genes regulating Drosophila oogenesis
A visual screen for diet-regulated proteins in the Drosophila ovary using GFP protein trap lines
The genetic architecture of ovariole number in Drosophila melanogaster: Genes with major, quantitative, and pleiotropic effects
Long Oskar controls mitochondrial inheritance in Drosophila melanogaster
DIP1 modulates stem cell homeostasis in Drosophila through regulation of sisR-1
Cyclin B3 deficiency impairs germline stem cell maintenance and its overexpression delays cystoblast differentiation in Drosophila ovary
Quantitative microscopy of the Drosophila ovary shows multiple niche signals specify progenitor cell fate
A targeted RNAi screen reveals Drosophila female-sterile genes that control the size of germline stem cell niche during development
Stonewall and Brickwall: Two partially redundant determinants required for the maintenance of female germline in Drosophila

Meiosis
Meiosis in females
TORC1 regulators Iml1/GATOR1 and GATOR2 control meiotic entry and oocyte development in Drosophila
A pathway for synapsis initiation during zygotene in Drosophila oocytes
A Balbiani body and the fusome mediate mitochondrial inheritance during oogenesis
A maternal screen for genes regulating Drosophila oocyte polarity uncovers new steps in meiotic progression
Quantitative proteomics reveals the dynamics of protein changes during Drosophila oocyte maturation and the oocyte-to-embryo transition
Developments between gametogenesis and fertilization: Ovulation and female sperm storage
Sperm-storage defects and live birth in Drosophila females lacking spermathecal secretory cells
Severe fertility effects of sheepish sperm caused by failure to enter female sperm storage organs in Drosophila melanogaster
Cleavage of the Drosophila seminal protein Acp36DE in mated females enhances its sperm storage activity
Dynamic Notch signaling specifies each cell fate in Drosophila spermathecal lineage
Mechanical stimulation by osmotic and hydrostatic pressure activates Drosophila oocytes in vitro in a calcium-dependent manner
Calcium waves occur as Drosophila oocytes activate
The coevolutionary period of Wolbachia pipientis infecting Drosophila ananassae and its impact on the evolution of the host germline stem cell regulating genes
The Octopamine receptor Octβ2R regulates ovulation in Drosophila melanogaster
Adipocyte amino acid sensing controls adult germline stem cell number via the amino acid response pathway and independently of Target of Rapamycin signaling in Drosophila
Tight coordination of growth and differentiation between germline and soma provides robustness for Drosophila egg development
The temporally controlled expression of Drongo, the fruit fly homolog of AGFG1, is achieved in female germline cells via P-bodies and its localization requires functional Rab11
Evidence for the mechanosensor function of filamin in tissue development

Nurse Cells
Drosophila protein kinase N (Pkn) is a negative regulator of actin-myosin activity during oogenesis
The ABC transporter Eato promotes cell clearance in the Drosophila melanogaster ovary

Follicle cells
Populations of follicle cells
Epithelial rotation promotes the global alignment of contractile actin bundles during Drosophila egg chamber
Rab10-mediated secretion synergizes with tissue movement to build a polarized basement membrane architecture for organ morphogenesis
Influence of ovarian muscle contraction and oocyte growth on egg chamber elongation in Drosophila
Coupling of Hedgehog and Hippo pathways promotes follicle stem cell maintenance by stimulating proliferation
Development of the dorsal appendages
Proteomics analysis identifies orthologs of human chitinase-like proteins as inducers of tube-morphogenesis defects in Drosophila
Regulation of epithelial stem cell replacement and follicle formation in the Drosophila ovary
Gene amplification in follicle cells: Isolation of developmental amplicons
DNA sequence templates adjacent nucleosome and ORC sites at gene amplification origins in Drosophila
Replication fork progression during re-replication requires the DNA damage checkpoint and double-strand break repair
Systematic analysis of the transcriptional switch inducing migration of border cells
Tousled-like kinase regulates cytokine-mediated communication between cooperating cell types during collective border cell migration
The Hippo pathway controls border cell migration through distinct mechanisms in outer border cells and polar cells of the Drosophila ovary
An atypical tropomyosin in Drosophila with intermediate filament-like properties
DRP1-dependent mitochondrial fission initiates follicle cell differentiation during Drosophila oogenesis
Lamellipodin and the Scar/WAVE complex cooperate to promote cell migration in vivo
Drosophila eggshell production: identification of new genes and coordination by Pxt
Simple expression domains are regulated by discrete CRMs during Drosophila oogenesis
Three-dimensional epithelial morphogenesis in the developing Drosophila egg
A dynamic population of stromal cells contributes to the follicle stem cell niche in the Drosophila ovary
A genome-scale in vivo RNAi analysis of epithelial development in Drosophila identifies new proliferation domains outside of the stem cell niche
Coordinated niche-associated signals promote germline homeostasis in the Drosophila ovary
Stage-specific plasticity in ovary size is regulated by Insulin/Insulin-Like growth factor and Ecdysone signalling in Drosophila
Adipocyte metabolic pathways regulated by diet control the female germline stem cell lineage in Drosophila
Impact of gut microbiota on the fly's germ line
Phantom, a cytochrome P450 enzyme essential for ecdysone biosynthesis, plays a critical role in the control of border cell migration in Drosophila
Ecdysone response gene E78 controls ovarian germline stem cell niche formation and follicle survival in Drosophila.
GTP exchange factor Vav regulates guided cell migration by coupling guidance receptor signalling to local Rac activation
Discs large 5, an essential gene in Drosophila, regulates egg chamber organization
Drosophila glob1 is required for the maintenance of cytoskeletal integrity during oogenesis
Targeted downregulation of s36 protein unearths its cardinal role in chorion biogenesis and architecture during Drosophila melanogaster oogenesis
Increased intracellular pH is necessary for adult epithelial and embryonic stem cell differentiation
Neutral competition for Drosophila follicle and cyst stem cell niches requires vesicle trafficking genes
The retromer subunit Vps26 mediates Notch signaling during Drosophila oogenesis
Collective growth in a small cell network
Outer nuclear membrane protein Kuduk modulates the LINC complex and nuclear envelope architecture
Variations in basement membrane mechanics are linked to epithelial morphogenesis
Meiosis in females

In the Drosophila oocyte, meiosis is arrested in the first division of metaphase, when a tapered spindle aligned parallel to the egg surface forms. The chromosomes are therefore located in the cortical region near the anterior pole, whereas fusion of parental complements occurs in the inner ooplasm. How does the female pronucleus reach the interior of the egg? The second meiotic spindles are arranged in tandem, end to end, and disposed perpendicular to the longitudinal axis of the egg with the innermost spindle carrying the female pronucleus. This pattern of spindle organization is probably involved in the migration of the female pronucleus deeper into the egg near the cytoplasmic domain of the male pronucleus. The precise time at which the mitotic spindle of Drosophila changes orientation is unknown. However, spindle rotation from a position parallel to the egg surface to a radial orientation presumably occurs during or shortly after the oocyte passes through the oviduct. How spindle orientation is achieved and maintained during meiosis is an intriguing question. Microtubules linking spindle poles to the oocyte surface have been implicted in the rotation and anchoring of the meiotic apparatus in Xenopus oocytes and in other organisms, but this does not seem to be the case in the Drosophila oocyte, since the meiotic spindles lack astral microtubels. However, the observation that a transient array of microtubules links the meiotic apparatus to discrete subcortical foci suggests that in Drosophila the orientation of the spindle also requires a functional interaction between the spindle and the oocyte cortex (Riparbelli, 1996 and references).

The microtubule array of mitosis II observed between the twin spindles at metaphase, anaphase and telophase might be an intermediate between the anastral poles of the meiotic I spindles and the astral poles of the mitotic spindles in early embryos. A complex pathway of spindle assembly takes place during resumption of meiosis at fertilization, consisting of a transient array of microtubules radiating from the equatorial region of the spindle toward discrete foci in the egg cortex. A monastral array of microtubules is observed between twin metaphase II spindles in fertilized eggs. These microtubules originating from disc-shaped material stain with Rb188 antibody specific for an antigen asssociated with the centrosome of Drosophila embryos (DMAP190 or CP190). Therefore, the Drosophila egg contains a maternal pool of centrosomal components undetectable in mature inactivated oocytes. These components nucleate microtubues in a monastral array after activation, but are unable to organize bipolar spindles (Riparbelli, 1996).

The meiosis II spindle of Drosophila oocytes is distinctive in structure, consisting of two tandem spindles with anastral distal poles and an aster-associated spindle pole body between the central poles. Assembly of the anastral:astral meiosis II spindle occurs by reorganization of the meiosis I spindle, without breakdown of the meiosis I spindle. The unusual disc- or ring-shaped central spindle pole body forms de novo in the center of the elongated meiosis I spindle, followed by formation of the central spindle poles. gamma-Tubulin transiently localizes to the central spindle pole body, implying that the body acts as a microtubule nucleating center for assembly of the central poles. The first step in formation of the central pole body is the appearance of puckers in the center of the the meiosis I spindle, followed by the pinching out from the spindle of a disc or ring of microtubules that becomes the central pole body. The manner in which the central spindle pole body forms suggests the involvement of a microtubule motor. If so, the motor involved is likely to be different from Ncd (Nonclaret disjunctional), since loss of Ncd function does not seem to prevent its formation. Following the formation of the central spindle pole body, the microtubules arrayed to either side of the central body narrow into poles, forming the mature meiosis II spindle. The central poles become more tapered during progression through meiosis II, and the central spindle pole body also changes in morphology: the disc or ring becomes asterlike, then enlarges into a ring that lies between the two central telophase II nuclei (Endow, 1998).

Localization of gamma-tubulin to the meiosis II spindle is dependent on the microtubule motor protein, Ncd. Absence of Ncd results in loss of gamma-tubulin localization to the spindle and destabilization of microtubules in the central region of the spindle. Likewise, during meiosis I, the minus-end motility of Ncd and its crosslinking activity are probably needed to focus microtubules into spindle poles for the correct functioning of meiosis I. Assembly of the anastral:astral meiosis II spindle probably involves rapid reassortment of microtubule plus and minus ends in the center of the meiosis I spindle. This can be accounted for by a model that also accounts for the loss of gamma-tubulin localization to the spindle and destabilization of microtubules in the absence of Ncd (Endow, 1998).

A model for assembly of the Drosophila oocyte meiosis II spindle is suggested: gamma-Tubulin is first recruited or relocalized, possibly as gamma-TuRC, to the midbody of the meiosis I spindle, where it functions to nucleate microtubules for formation of the meiosis II central spondle poles. The loss of gamma-tubulin localization to the spindle in the absence of Ncd suggests that the Ncd motor serves to recruit or anchor gamma-tubulin to the center of the spindle. The Ncd motor would then stabillize newly nucleated microtubule minus ends and focus the microtubules into poles. The unstabilized plus ends of the microtubules in the center of the spindle (remaining from meiosis I) would undergo rapid depolymerization as a consequence of dynamic instability. Stabilization of the newly nucleated microtubule minus ends and depolymerization of the plus ends would cause a rapid sorting out of the microtubules in the center of the meiosis I spindle, replacing microtubule plus ends with minus ends. The distal poles of the meiosis II spindle would be retained from the meiosis I spindle and maintained by the same forces that originally formed them: the crosslinking activity and minus-end movement of Ncd along spindle microtubules (Endow, 1998).

TORC1 regulators Iml1/GATOR1 and GATOR2 control meiotic entry and oocyte development in Drosophila

In single-cell eukaryotes the pathways that monitor nutrient availability are central to initiating the meiotic program and gametogenesis. In Saccharomyces cerevisiae an essential step in the transition to the meiotic cycle is the down-regulation of the nutrient-sensitive target of rapamycin complex 1 (TORC1; see Drosophila Tor pathway) by the increased minichromosome loss 1/ GTPase-activating proteins toward Rags 1 (Iml1/GATOR1) complex in response to amino acid starvation. How metabolic inputs influence early meiotic progression and gametogenesis remains poorly understood in metazoans. This study defined opposing functions for the TORC1 regulatory complexes Iml1/GATOR1 and GATOR2 during Drosophila oogenesis. As is observed in yeast, the Iml1/GATOR1 complex inhibits TORC1 activity to slow cellular metabolism and drive the mitotic/meiotic transition in developing ovarian cysts. In iml1 germline depletions, ovarian cysts undergo an extra mitotic division before meiotic entry. The TORC1 inhibitor rapamycin can suppress this extra mitotic division. Thus, high TORC1 activity delays the mitotic/meiotic transition. Conversely, mutations in Tor, which encodes the catalytic subunit of the TORC1 complex, result in premature meiotic entry. Later in oogenesis, the GATOR2 components Missing oocyte (Mio) and Seh1 are required to oppose Iml1/GATOR1 activity to prevent the constitutive inhibition of TORC1 and a block to oocyte growth and development. These studies represent the first examination of the regulatory relationship between the Iml1/GATOR1 and GATOR2 complexes within the context of a multicellular organism. The data imply that the central role of the Iml1/GATOR1 complex in the regulation of TORC1 activity in the early meiotic cycle has been conserved from single cell to multicellular organisms (Wei, 2014b).

In yeast, the inhibition of the nutrient-sensitive target of rapamycin complex 1 (TORC1) in response to amino acid limitation is essential for cells to transit from the mitotic cycle to the meiotic cycle. In response to amino acid starvation, the Iml1 complex, comprising the Iml1, Nitrogen permease regulator-like 2 (Npr2), and Nitrogen permease regulator-like 3 (Npr3) proteins in yeast and the respective orthologs DEPDC5, Nprl2, and Nprl3 in mammals, inhibits TORC1 activity. The Iml1 complex, which has been renamed the 'GTPase-activating proteins toward Rags 1' (GATOR1) complex in higher eukaryotes, functions as a GTPase-activating protein complex that inactivates RagsA/B or Gtr1 in mammals and yeast, respectively, thus preventing the activation of TORC1. In the yeast Saccharomyces cerevisiae, mutations in the Iml1 complex components Npr2 and Npr3 result in a failure to down-regulate TORC1 activity in response to amino acid starvation and block meiosis and sporulation. As is observed in yeast, in Drosophila, Nprl2 and Nprl3 mediate a critical response to amino acid starvation (Wei, 2014a). However, their roles in meiosis and gametogenesis remain unexplored (Wei, 2014b).

Recent reports indicate that the Iml1, Npr2, and Npr3 proteins are components of a large multiprotein complex originally named the 'Seh1-associated' (SEA) complex in budding yeast and the 'GATOR' complex in higher eukaryotes. The SEA/GATOR complex contains eight highly conserved proteins. The three proteins described above, Iml1/DEPDC5, Npr2/Nprl2, and Npr3/Nprl3, form the Iml1/GATOR1 complex and inhibit TORC1. The five remaining proteins in the complex, Seh1, Sec13, Sea4/Mio, Sea2/WDR24, and Sea3/WDR59, which have been designated the 'GATOR2' complex in multicellular organisms, oppose the activity of Iml1/GATOR1 and thus promote TORC1 activity (Wei, 2014b).

Little is known about the physiological and/or developmental requirements for the GATOR2 complex in multicellular organisms. However, in Drosophila the GATOR2 components Mio and Seh1 interact physically and genetically and exhibit strikingly similar ovarian phenotypes, with null mutations in both genes resulting in female sterility (Senger, 2011; Wei, 2014a). In Drosophila females, oocyte development takes place within the context of an interconnected germline syncytium, also referred to as an 'ovarian cyst'. Ovarian cyst formation begins at the tip of the germarium when a cystoblast, the daughter of a germline stem cell, undergoes four synchronous divisions with incomplete cytokinesis to produce 16 interconnected cells. Actin-stabilized cleavage furrows, called 'ring canals', connect cells within the cyst. Each 16-cell cyst develops with a single oocyte and 15 polyploid nurse cells which ultimately are encapsulated by a somatically derived layer of follicle cells to produce an egg chamber. Each ovary is comprised of ∼15 ovarioles that consist of a single germarium followed by a string of egg chambers in successively older stages of development. In mio- and seh1-mutant egg chambers, the oocyte enters the meiotic cycle, but as oogenesis proceeds, the oocyte fate and the meiotic cycle are not maintained stably (Senger, 2011; Wei, 2014a). Ultimately, a large fraction of mio and seh1 oocytes enter the endocycle and develop as polyploid nurse cells. A mechanistic understanding of how mio and seh1 influence meiotic progression and oocyte fate has remained elusive (Wei, 2014b).

This study demonstrates that the Iml1/GATOR1 complex down-regulates TORC1 activity to promote the mitotic/meiotic transition in Drosophila ovarian cysts. Depleting iml1 in the female germ line delays the mitotic/meiotic transition, so that ovarian cysts undergo an extra mitotic division. Conversely, mutations in Tor result in premature meiotic entry before the completion of the four mitotic divisions. Moreover, it was demonstrated that in the female germ line, the GATOR2 components Mio and Seh1 are required to oppose the TORC1 inhibitory activity of the Iml1/GATOR1 complex to prevent the constitutive down-regulation of TORC1 activity in later stages of oogenesis. These studies represent the first examination of the regulatory relationship between Iml1/GATOR1 and GATOR2 components within the context of a multicellular animal. Finally, these data reveal a surprising tissue-specific requirement for the GATOR2 complex in multicellular organisms and suggest a conserved role for the SEA/GATOR complex in the regulation of TORC1 activity during gametogenesis (Wei, 2014b).

Previous work demonstrated that in Drosophila the Iml1/GATOR1 complex mediates an adaptive response to amino acid starvation. This study tested the hypothesis that the Iml1/GATOR1 complex also has retained a role in the regulation of the early events of gametogenesis. Consistent with this model, this study found that in germline knockdowns of iml1, ovarian cysts delay meiotic entry and undergo a fifth mitotic division. This meiotic delay can be suppressed with the TORC1 inhibitor rapamycin. Thus, during Drosophila oogenesis the Iml1/GATOR1 complex promotes the transition from the mitotic cycle to the meiotic cycle through the down-regulation of the metabolic regulator TORC1. Increasing TORC1 activity by disabling its inhibitor delays meiotic progression, whereas germline clones of a Tor-null allele enter meiosis prematurely. Taken together, these data indicate that the level of TORC1 activity contributes to the timing of the mitotic/meiotic switch in Drosophila females and suggest that low TORC1 activity may be a conserved feature of early meiosis in many eukaryotes (Wei, 2014b).

However, in Drosophila, meiotic entry is not contingent on amino acid limitation at the organismal level. Indeed, the energy-intensive process of Drosophila oogenesis is curtailed dramatically when females do not have access to a protein source. Thus, to promote meiotic entry, Drosophila females must activate the Iml1/GATOR1 complex in a tissue-specific manner, using a mechanism that is independent of the overall nutrient status of the animal. At least two models can explain how Drosophila females might activate the Iml1/GATOR1 complex specifically in the germ line. In the first model, ovarian cysts locally experience low levels of amino acids during the mitotic cyst divisions and/or at the point of meiotic entry. These low levels of amino acids could reflect a non–cell-autonomous effect: The somatically derived escort cells that surround dividing ovarian cysts may function to create a low amino acid environment that triggers the activation of the Iml1/GATOR1 complex within developing ovarian cysts. Alternatively, the effect may be cell autonomous: The germ cells within dividing ovarian cysts may have a reduced ability to sense and/or import amino acids. In a second model, a developmental signaling pathway that is completely independent of local or whole-animal amino acid status directly activates the Iml1/GATOR1 complex. The identification of the upstream requirements for Iml1/GATOR1 activation in the female germ line will help distinguish between these two models (Wei, 2014b).

Although low TORC1 activity is required during early ovarian cyst development to promote the mitotic/meiotic switch, the dramatic growth of egg chambers later in oogenesis is a metabolically expensive process that is predicted to require high TORC1 activity. The current data indicate that the GATOR2 components Mio and Seh1 function to oppose the TORC1-inhibitory activity of the GATOR1 complex in the female germ line. In mio and seh1 mutants, TORC1 activity is constitutively repressed in the germ line of developing egg chambers, resulting in the activation of catabolic metabolism and the blocking of meiotic progression and oocyte development and growth (Wei, 2014b).

Previous data indicate that Mio and Seh1 act very early in oogenesis soon after the formation of the 16-cell cyst. The mio and seh1 ovarian phenotypes can be rescued by depleting the GATOR1 components nprl2, nprl3, or iml1 in the female germ line or by raising baseline levels of TORC1 activity by disabling an alternative TORC1 inhibitory complex, TSC1/2. These data are consistent with the model that the failure to maintain the meiotic cycle and the oocyte fate in mio and seh1 mutants is a direct result of inappropriately low TORC1 activity in the female germ line brought on by the deregulation of the Iml1/GATOR1 complex (Wei, 2014b).

Notably, null alleles of both mio and seh1 are viable, with many somatic tissues exhibiting no apparent developmental abnormalities and only limited reductions in cell growth. Thus, although Mio and Seh1 are critical for the activation of TORC1 and the development of the female gamete, these proteins play a relatively small role in the development and growth of many somatic tissues under nutrient-replete conditions. Whether this small role reflects the fact that components of the Iml1/GATOR1 complex are expressed at low levels in some somatic cell types or that the complex is present but needs to be activated by a signal, such as nutrient stress or a developmental signaling pathway, remains to be elucidated (Wei, 2014b).

In the future it will be important to gain a fuller understanding of the potential environmental and developmental inputs that regulate the activity of the Iml1/GATOR1 and GATOR2 complexes in multicellular organisms. These studies will provide much-needed insight into the basic mechanisms by which both environmental and developmental signaling pathways interface with the metabolic machinery to influence cell growth and differentiation (Wei, 2014b).

A pathway for synapsis initiation during zygotene in Drosophila oocytes

Formation of the synaptonemal complex (SC), or synapsis, between homologs in meiosis is essential for crossing over and chromosome segregation. How SC assembly initiates is poorly understood but may have a critical role in ensuring synapsis between homologs and regulating double-strand break (DSB) and crossover formation. This study investigated the genetic requirements for synapsis in Drosophila and found that there are three temporally and genetically distinct stages of synapsis initiation. In meiotic prophase 1 'early zygotene' oocytes, synapsis is only observed at the centromeres. It was also found that nonhomologous centromeres are clustered during this process. In 'mid-zygotene' oocytes, SC initiates at several euchromatic sites. The centromeric and first euchromatic SC initiation sites depend on the cohesion protein ORD. In 'late zygotene' oocytes, SC initiates at many more sites that depend on the Kleisin-like protein C(2)M. Surprisingly, late zygotene synapsis initiation events are independent of the earlier mid-zygotene events, whereas both mid and late synapsis initiation events depend on the cohesin subunits SMC1 and SMC3. It is proposed that the enrichment of cohesion proteins at specific sites promotes homolog interactions and the initiation of euchromatic SC assembly independent of DSBs. Furthermore, the early euchromatic SC initiation events at mid-zygotene may be required for DSBs to be repaired as crossovers (Tanneti, 2011).

Drosophila pro-oocytes develop within 16-cell cysts that are arranged in temporal order within the ovary. Each ovary contains several germaria, where pairs of pro-oocytes begin their development and enter prophase in region 2a and a single oocyte is selected by region 3. Oocytes are defined by the presence of the synaptonemal complex (SC), which is detected by antibodies to the transverse element C(3)G (Page, 2001), a coiled-coil protein similar to proteins in budding yeast (ZIP1), C. elegans (SYP-1, SYP-2), and mammals (SYCP1) (Page, 2004; Watts, 2011). Zygotene pro-oocytes were identified by their patchy C(3)G staining, as opposed to the thread-like staining typical of pachytene. Furthermore, by comparing the amount of synapsis to the relative positions of the pro-oocytes in the wild-type germarium, three stages of zygotene were defined (Tanneti, 2011).

First, early zygotene pro-oocytes have one or two patches of C(3)G that colocalize with CID, a centromere-specific histone H3. These pro-oocytes reside in the earliest (most anterior) part of region 2a, indicating that synapsis initiates at the centromeres before any other sites. These results were confirmed by comparing CID localization to histone modifications specific for the heterochromatin or euchromatin. Because there are four pairs of centromeres, the observation that most wild-type pro-oocytes have one or two CID foci indicates that nonhomologous centromeres cluster in meiotic prophase, confirming previous observations using electron microscopy (Tanneti, 2011).

Second, mid-zygotene pro-oocytes have the centromeric C(3)G staining plus approximately six additional sites in the euchromatin. Finally, late zygotene pro-oocytes contain many C(3)G foci but lack the continuous threadlike pattern of pachytene. Surprisingly, the mid-zygotene patches do not appear to get longer. Instead, there are more patches in late zygotene, suggesting that the progression from mid- to late zygotene involves the establishment of new SC initiation sites rather than polymerization from the small number of sites in mid-zygotene. It is suggested that the noncentromeric C(3)G sites in mid-zygotene represent the first euchromatic sites to initiate synapsis. This study provides evidence that the mid-zygotene sites have features in common with centromere synapsis sites but are mechanistically distinct and genetically separable from the additional synapsis initiation sites observed in late zygotene (Tanneti, 2011).

C(2)M is a lateral element component and is a member of the Kleisen family that includes Rec8 and Rad21 homologs (Schleiffer, 2003). In wild-type, C(2)M colocalizes with C(3)G in most locations except at the centromeres. In females lacking C(2)M, the first two stages of zygotene appear to occur normally. Early zygotene pro-oocytes exhibit one or two foci of CID that colocalize with C(3)G, showing that C(2)M is not required for centromere clustering or centromere synapsis. These results confirm previous observations (Khetani, 2007) that C(2)M is not required for centromere clustering in pachytene oocytes and are consistent with the observation that C(2)M does not localize to the centromeric regions. Early zygotene in c(2)M mutants is followed by cysts with several patches of euchromatic C(3)G staining that resemble wildtype cells in mid-zygotene. Synapsis in a c(2)M mutant does not, however, progress beyond this point. Examination of histone modifications in c(2)M mutants confirmed that synapsis is blocked in mid-zygotene with a small number of euchromatin initiation sites. Based on the similarities between wild-type mid-zygotene and c(2)M mutants, it is suggested that synapsis initiates in a c(2)M-independent manner at a small number of specialized sites on the chromosomes, which include approximately six euchromatic sites and the centromeres, and that C(2)M is required for additional initiation sites typical of late zygotene (Tanneti, 2011).

There is a striking similarity between the number of euchromatic synapsis initiation sites (~6) during mid-zygotene and the number of crossovers in Drosophila females. In order to determine the relationship between SC initiation sites and double-strand break (DSB) formation, c(2)M mutant oocytes were stained for C(3)G and γ-H2AV. DSBs in a c(2)M mutant are usually associated with a patch of C(3)G staining (55/56 γ-H2AV foci were touching or overlapped a patch of C(3)G). This experiment was also performed in an okr mutant background (okr encodes the Drosophila homolog of Rad54) where the DSBs are not repaired and γ-H2AV staining accumulates, allowing all DSBs to be counted. Most of the γ-H2AV foci in okr c(2)M mutant germaria colocalized with a patch of C(3)G, suggesting that the initiation of SC and recombination usually occur within the same region in c(2)M mutants. Indeed, MEI-P22, a protein required for DSB formation, also colocalizes with the SC in c(2)M mutant oocytes. It should be noted that previous observations showed that DSB formation is partially dependent on the SC. Indeed, the number of γ-H2AV foci in the okr c(2)M double mutant in region 3 oocytes was reduced compared to a okr single mutant. Overall, these results suggest that the SC, or a factor which stimulates SC formation, promotes recruitment of proteins required for DSB formation (Tanneti, 2011).

To investigate whether there is a connection between early SC initiation events and meiotic recombination, double mutants with c(2)M were constructed. Unlike wild-type, where γ-H2AV foci are not observed until pachytene, the block in synapsis observed in c(2)M mutants allowed examination of the relationship between SC initiation and DSB formation. By double staining with CID, it was found that eliminating meiotic DSBs with a mei-W68 mutation did not prevent formation of either the centromere and euchromatic SC in a c(2)M mutant. The small decrease in the number of euchromatic SC sites in the c(2)M mei-W68 double mutant may indicate that the number of initiation sites is sensitive to DSB formation. Furthermore, SC initiation is not grossly affected by a reduction in crossing over (mei-218), an increase in crossing over (TM6), or a defect in DSB repair (okr). DSBs do not occur in the heterochromatin; thus, it is not surprising that centromere SC is independent of DSB formation. However, these results show that the initiation of euchromatic synapsis at mid zygotene does not depend on DSBs or crossovers (Tanneti, 2011).

Because DSBs or recombination are not required for synapsis in wild-type or c(2)M mutants, tests were performed to see whether structural components of the meiotic chromosomes regulate SC initiation. ORD is a meiosis-specific protein required for cohesion and crossover formation that may be a component of the SC lateral elements. Although previous studies have shown that ord mutant oocytes generate threads of C(3)G staining that resemble pachytene, the effect of ord on zygotene progression has not been previously examined (Tanneti, 2011).

Consistent with previous results, this study found that centromere clustering is defective and the association of SC proteins with the centromeres is disrupted in ord mutant oocytes. Furthermore, zygotene appeared abnormal; rather than observing centromeric and euchromatic SC initiation sites typical of mid-zygotene in early region 2a, it was found that many ord mutant pro-oocytes with C(3)G staining only around the nuclear DNA. Of the 108 pro-oocytes examined in five germaria, 36 (33%) had no nuclear C(3)G. The remaining pro-oocytes [72, (67%)] either had a number of C(3)G patches that was more typical of late zygotene, usually in region 2a, or were in pachytene. It is concluded that the centromeric and euchromatic synapsis sites typical of early and mid zygotene are absent in ord mutants, suggesting that, in the absence of ORD, synapsis does not initiate normally (Tanneti, 2011).

Because ord mutants do eventually form threads of SC, it was difficult to be sure that SC initiation was defective. To test whether ord has a role in mid-zygotene synapsis, tests were performed to see whether the euchromatic patches of C(3)G in a c(2)M mutant depend on ord. Even though both single mutants exhibit at least some SC formation, most of the C(3)G staining in the c(2)M ord double mutant surrounded the DNA and within the nucleus. This nonchromosomal C(3)G localization in the c(2)M ord double mutant was much more pronounced than in the ord single mutant. In addition, C(3)G-staining ring-like structures were observed similar to what has been reported in some c(3)G missense mutants. All the nonchromosomal C(3)G staining may be due to polycomplex formation. c(2)M ord double mutant pro-oocytes were identified by the prominent C(3)G around the DNA, and the number of C(3)G patches on the chromosomes was found to be drastically reduced compared to wild-type zygotene or either single mutant (Tanneti, 2011).

These results demonstrate that ord is required for the centromeric and euchromatic synapsis sites observed in c(2)M mutants. Conversely, C(2)M is required for the threadlike synapsis observed in ord mutants. The synergistic phenotype of the double mutant suggests that there are two types of synapsis initiation - one depends on ORD (early and mid-zygotene) and the other depends on C(2)M (late zygotene) - and that these are independent events. In the absence of both types of synapsis initiation, C(3)G cannot load onto the chromosomes and accumulates in polycomplexes (Tanneti, 2011).

Like other Kleisin family members, C(2)M has been shown to physically interact with the cohesin subunit SMC3 (Heidmann, 2004). To determine whether C(2)M localization depends on an interaction with cohesin, oocytes lacking SMC1 and SMC3 were examined. To examine oocytes lacking SMC3 (encoded by cap), the recently developed short hairpin RNA (shRNA) resource, which allows RNA interference (RNAi) knockdown of gene expression in the Drosophila female germline, was used. Both the chromosomal localization of C(3)G and C(2)M were absent when cap shRNA was expressed in the germline. Furthermore, SMC1 staining was eliminated, suggesting that the RNAi was effective at knocking out SMC3 function. Like the c(2)M ord double mutant, most C(3)G staining accumulated around the periphery of the DNA, suggesting that the function of SMC3 in synapsis occurs through at least two independent interactions with C(2)M and ORD. Unlike the c(2)M ord double mutant, however, it was not possible to distinguish the pro-oocytes from the nurse cells because C(3)G staining was evenly distributed among the cells in each germarium cyst. Importantly, oocyte selection was not perturbed because one cell in each cyst accumulated ORB protein, a cytoplasmic marker for the oocyte. Thus, the loss of SMC3 may have a more severe phenotype than the c(2)M ord double mutant (Tanneti, 2011).

These results were confirmed with the analysis of SMC1 mutant germline clones. As with cap RNAi, there was an absence of nuclear C(2)M and C(3)G threads in oocytes lacking SMC1, indicating a complete block in synapsis. Also similar to cap RNAi, the accumulation of ORB in one cell indicated that an oocyte was established. The only difference compared to cap RNAi was that there was much less C(3)G staining around the periphery of the DNA. It is not known whether this minor difference is due to the different methods (RNAi versus germline clone) or distinct functions of the two SMC proteins. Nevertheless, the results of these two experiments demonstrate that SMC1 and SMC3 are required for synapsis (Tanneti, 2011).

It is concluded that synapsis initiation during zygotene in Drosophila females occurs in three stages. In early zygotene, the centromeres are the first sites to accumulate the transverse filament protein C(3)G. Indeed, cohesion proteins SMC1, SMC3, and ORD are detected at the centromeres before meiotic prophase (prior to or during premeiotic S phase), which could explain why synapsis is first observed at the centromeres. Interestingly, the SC also forms first at the centromeres in budding yeast and depends on cohesion proteins. In mid-zygotene, synapsis initiates at a small number of euchromatic sites. These first two steps depend on the ORD protein. Finally, in late zygotene, synapsis initiates at a larger number of euchromatic sites. This stage requires C(2)M and appears to occur through a new set of initiation events rather than extending synapsis, or 'zipping up,' from the mid-zygotene initiation sites. Indeed, the synapsis initiation events in mid and late zygotene are independent and genetically separable, supporting a model where synapsis occurs through two independent waves of initiation events. In the absence of ORD, early and mid-zygotene synapsis events are skipped and the late zygotene initiation events occur with normal kinetics. This is not without consequence, however, because at the electron microscopy level, this synapsis is abnormal and tripartite SC is not visible. Both waves of synapsis initiation depend on the cohesin proteins SMC1 and SMC3, which may interact independently with C(2)M and ORD (Tanneti, 2011).

In addition to its role in centromere synapsis, ORD and the SMC proteins are required for the pairing and clustering of centromeres, whereas the SC components C(2)M or C(3)G are not. Thus, cohesion proteins may be able to function in a pairing role independent of DSBs, as Rec8 does in budding yeast for centromere coupling. It is suggested that the first euchromatic sites to initiate SC assembly in Drosophila are in regions where cohesion proteins are most abundant. This model is attractive because it provides a mechanism for SC initiation in the absence of DSBs. Interestingly, the number of euchromatic initiation sites in mid-zygotene or in c(2)M mutants approximates the number of crossovers in the genome. Not only do these mid-zygotene sites depend on ORD, but in ord mutants, crossing over is reduced to less than 10% of wild-type, even though DSBs occur normally. It is suggested that the reduction in crossing over in ord mutants is due to the absence of the synapsis initiation sites at mid-zygotene. Whether the synapsis initiation sites actually correspond to crossover sites awaits further study (Tanneti, 2011).

ORD may have a function similar to yeast Rec8 because it is required for synapsis at the centromeres and a subset of euchromatic sites. Interestingly, the findings with C(2)M, which is not an ortholog of Rec8, are also probably relevant to other species. Several recent studies have revealed Non-Rec8 Kleisin homologs in mouse and C. elegans (COH-3 and COH-). These parallels between the synapsis pathway in flies and that of organisms that depend on DSBs for synapsis could reflect the existence of a conserved underlying mechanism of synapsis. If synapsis initiation sites can be marked prior to DSB formation in a process involving cohesion proteins, and if proteins like Zip3 can be recruited in the absence of DSBs, as is true in C. elegans and likely in Drosophila, the timing of the DSB then becomes less of a determining factor in the process of synapsis (Tanneti, 2011).

A Balbiani body and the fusome mediate mitochondrial inheritance during oogenesis

Maternally inherited mitochondria and other cytoplasmic organelles play essential roles supporting the development of early embryos and their germ cells. Using methods that resolve individual organelles, the origin of oocyte and germ plasm-associated mitochondria was studied during Drosophila oogenesis. Mitochondria partition equally on the spindle during germline stem cell and cystocyte divisions. Subsequently, a fraction of cyst mitochondria and Golgi vesicles associates with the fusome, moves through the ring canals, and enters the oocyte in a large mass that resembles the Balbiani bodies of Xenopus, humans and diverse other species. Some mRNAs, including oskar RNA, specifically associate with the oocyte fusome and a region of the Balbiani body prior to becoming localized. Balbiani body development requires an intact fusome and microtubule cytoskeleton since it is blocked by mutations in hu-li tai shao, while egalitarian mutant follicles accumulate a large mitochondrial aggregate in all 16 cyst cells. Initially, the Balbiani body supplies virtually all the mitochondria of the oocyte, including those used to form germ plasm, because the oocyte ring canals specifically block inward mitochondrial transport until the time of nurse cell dumping. These findings reveal new similarities between oogenesis in Drosophila and vertebrates, and support the hypothesis that developing oocytes contain specific mechanisms to ensure that germ plasm is endowed with highly functional organelles (Cox, 2003).

Drosophila oocytes contain a typical Balbiani body at the time follicles form in region 3 of the germarium. In a wide range of animal species, including Xenopus, chick, mouse and human, young oocytes at a similar developmental stage display these distinctive aggregates of mitochondria and other organelles near their germinal vesicles. In a typical Balbiani body, centrioles and associated cytoplasm are surrounded by a ring of Golgi bodies and encased in a large mass of mitochondria. As the oocyte grows, the mitochondria first spread around the nuclear periphery and later disperse throughout the oocyte cytoplasm (Cox, 2003).

Drosophila Balbiani bodies, like those described in other species, contain clustered mitochondria, Golgi vesicles and centrioles. Moreover, as young follicles develop from stage 1-6, the mitochondria move around the germinal vesicle and disperse after microtubules re-organize in stage 7 (Cox, 2003).

The studies reported here provide new insight into the origin of Balbiani bodies. Drosophila Balbiani bodies do not arise de novo within oocytes, but are built by the transport of organelles from neighboring cells within interconnected germline cysts. These experiments make clear that many components of oocyte cytoplasm arise in this manner (Cox, 2003).

Virtually all of the newly formed mitochondria in oocytes are derived from the Balbiani body. The great majority are transported from other cystocytes along the fusome but 1/16th or more might simply originate in the oocyte. Like oocyte determination itself, Balbiani body formation depends on the fundamental cyst polarity manifested in the fusome. Arising in embryonic germ cells, the fusome builds up a framework of cyst polarity during the cystocyte divisions. Fusome polarity probably acts directly to control centriole migration and the meiotic gradient, and acts indirectly to differentiate and maintain the oocyte by regulating the microtubule cytoskeleton. Deciphering the molecular mechanisms that define fusome polarity and allow the fusome to control microtubule organization remains a central issue for understanding Balbiani body formation and oocyte development (Cox, 2003).

Oocytes develop from germline cysts or syncytia in diverse species so Balbiani bodies may arise through intercellular transport in a wide range of organisms besides Drosophila. In both Xenopus and the mouse, mitochondrial clouds present within interconnected germ cells are thought to be precursors to the Balbiani bodies that arise shortly after the cysts break down and form primordial follicles. In Drosophila, the large chunk of fusome at the anterior of the early stage 1 oocyte contains clustered centrioles and is likely to act as a microtubule-organizing center. It may attract and retain mitochondria, Golgi and localized macromolecules as they enter the oocyte, thereby creating the Balbiani body. Xenopus Balbiani bodies may arise in a similar fashion as they have a similar organization consisting of a spectrin-rich zone, mitochondria, Golgi and the Metro region containing RNAs in transit. However, there has been insufficient study of the Xenopus larval ovary to identify a fusome or some other material with microtubule organizing properties that might play an analogous role. In most other systems whose Balbiani bodies share the same basic structure in young oocytes, very little is known about their origin during earlier stages of germ cell development (Cox, 2003).

The Balbiani bodies in many species contain structures resembling germinal granules. In Xenopus, these granules are found in a region containing specific RNAs that are also destined to be localized in the egg and incorporated in germ cells. Consequently, the Balbiani body has been proposed to function as a messenger transport organizer (METRO) that organizes and mediates the delivery of RNAs and germinal granules to the vegetal pole of the egg. Specific elements have been mapped in the 3' UTR of the Xcat2 mRNA that are sufficient for localization to the Balbiani body or to the germinal granules themselves (Cox, 2003).

The Drosophila Balbiani body may play a related role. oskar RNA, a key component that is capable of inducing germ plasm formation, is associated with the posterior segment of the Balbiani body in early stage 1 oocytes, much as Xcat2 is localized in the Xenopus Balbiani body. A few hours later, towards the end of stage 1, osk RNA moves to the oocyte posterior along with the other Balbiani-associated RNAs and proteins that have been studied, presumably in response to the shift in microtubule polarity that occurs at this time. Thus, at least some molecules that participate in germ plasm assembly associate with the Balbiani body in early Xenopus and Drosophila oocytes (Cox, 2003).

Drosophila RNAs that become associated with the Balbiani body, like organelles, first interact with the fusome during early stages of cyst development. However, there are significant differences in these fusome interactions with RNAs and organelles that probably reflect different molecular mechanisms of delivery to the Balbiani body. Organelles associate next to the fusome along much of its length and subsequently move toward the center, in concert with microtubule minus ends. By contrast, the RNAs associate with one or a few cells at the center of the fusome from the earliest stages they could be detected, and are located within it, as well as nearby. These observations suggest that localized RNAs may read the fusome polarity directly, and need not rely on changes in microtubule organizing activity to get to the oocyte or be stabilized within it (Cox, 2003).

Potentially significant differences exist in the role of RNA transport played by the Drosophila and Xenopus Balbiani bodies. The Drosophila Balbiani body associates with germ plasm RNAs for only 5-10 hours during early stage 1. By contrast, Xenopus Balbiani bodies associate throughout stage 1 of oogenesis, a process requiring many days, with at least 11 RNAs. When the RNAs leave the Drosophila Balbiani body, mitochondria mostly remain behind, only to follow much later in oogenesis. By contrast, in Xenopus, both mRNAs and mitochondria are reported to proceed together to the vegetal pole. These differences may simply reflect differences in the timing of cytoskeletal remodeling that control these events. Moreover, the observation that a small subset of mitochondria recognized by COXI antisera do translocate with the RNAs in stage 1 indicates that certain Drosophila mitochondria may follow a Xenopus-like pattern. However, it remains possible that RNAs in transit to the oocyte posterior may simply pass through the Balbiani body without being affected in any way (Cox, 2003).

Sponge-like structures have been described in the cytoplasm of stage 4-10 nurse cells that are associated with Exu protein, RNA, and (frequently) mitochondria and nuage. It has been proposed that these structures are analogous to classical Balbiani bodies and that they mediate transport of localized transcripts such as bicoid RNA. The current results suggest that the ooctye contains a true Balbiani body much earlier -- in stage 1 follicles. The sponge bodies more likely represent transport complexes organized at the surface of nurse cell nuclei that subsequently move through the follicle and into the ooctye. However, there may be structural and molecular similarities between nurse cell transport complexes and those mediating transport out of the Balbiani body (Cox, 2003).

These studies provide further evidence that the ring canals that join the cystocytes play an important role in regulating Balbiani body formation. Mitochondria appear to first enter the oocyte when fusome segments within the adjoining ring canals break apart, unplugging the channels. Subsequently, a novel mechanism blocks further mitochondrial passage through these canals, because large backups of mitochondria are observed outside each oocyte ring canal in young oocytes and a lack of mitochondrial movement into the oocyte has been documented in movies. Mitochondria do not accumulate in the same manner around the ring canals that join nurse cells, but are spread throughout the cell and in the nuclear periphery. This behavior has the effect of limiting the mitochondrial genotypes within the oocyte to those found in Balbiani body mitochondria until well after mitochondria have begun to associate with the germ plasm at the oocyte posterior pole. Despite the importance of these regulatory steps, little is known about how movement through ring canals is controlled (Cox, 2003).

These studies suggest that centrioles, mitochondria, Golgi, RNAs and other key components of oocyte cytoplasm are added to the Drosophila oocyte by a special mechanism that may have been widely conserved in evolution. It is remarkable that in the oocyte, the lone cell that will contribute cytoplasm for the next generation of organisms, many fundamental components of cytoplasm do not arise by random partitioning among daughter cells. Rather, an elaborate mechanism is used to transport materials from multiple cells and maintain them in a large aggregate for an extended period of time. It is possible that Balbiani bodies do not play a specific role in ooctye development, but represent a byproduct of the unusual centrosome behavior in these cells. However, an alternative hypothesis is favored. One of the potentially most interesting reasons that oocyte organelles might be delivered en mass via the fusome would be to increase organelle fitness. Mitochondrial DNAs are known to accumulate mutations that have frequently been postulated to affect the aging of cells and tissues. If only mitochondria with functional genomes are able to associate with the fusome and move into the oocyte, damaged genomes might be weeded out when they still represent a small fraction of the total. Such a system would be far more efficient than eliminating defective genomes by inducing the apoptosis of entire germ cells. A purifying mechanism based on organelle selection might be particularly important in organisms that need to produce eggs with a high average viability, or that must support long intergenerational life spans (Cox, 2003).

Several other observations may also be explained by the need to eliminate defective mitochondrial genomes. The exclusion of nurse cell mitochondria from passing through the oocyte ring canals prior to dumping would ensure that only the 'selected' mitochondria in the Balbiani body populate the germ plasm. Mitochondria may break up into small, nearly round, organelles during this period so that each will contain a single genome whose fitness can be tested. The cytoplasmic streaming of the ooctye may serve to mix the two populations of organelles so each somatic cell type inherits at least some of the selected mitochondrial population. Finally, a requirement for translation on mitochondrial ribosomes in the early embryonic germ plasm might serve as a concluding selective step to ensure that viable germ cells are well supplied with intact mitochondrial genomes. If female germ cells do possess mechanisms to remove defective mitochondria, they would probably have contributed to the evolutionary conservation of germ line cysts and Balbiani bodies (Cox, 2003).

A maternal screen for genes regulating Drosophila oocyte polarity uncovers new steps in meiotic progression

Meiotic checkpoints monitor chromosome status to ensure correct homologous recombination, genomic integrity, and chromosome segregation. In Drosophila, the persistent presence of double-strand DNA breaks (DSB) activates the ATR/Mei-41 checkpoint, delays progression through meiosis, and causes defects in DNA condensation of the oocyte nucleus, the karyosome. Checkpoint activation has also been linked to decreased levels of the TGFα-like molecule Gurken, which controls normal eggshell patterning. This easy-to-score eggshell phenotype was used in a germ-line mosaic screen in Drosophila to identify new genes affecting meiotic progression, DNA condensation, and Gurken signaling. One hundred eighteen new ventralizing mutants on the second chromosome fell into 17 complementation groups. This study describes the analysis of 8 complementation groups, including Kinesin heavy chain, the SR protein kinase cuaba (CG8174), the cohesin-related gene dPds5/cohiba, and the Tudor-domain gene montecristo. These findings challenge the hypothesis that checkpoint activation upon persistent DSBs is exclusively mediated by ATR/Mei-41 kinase and instead reveal a more complex network of interactions that link DSB formation, checkpoint activation, meiotic delay, DNA condensation, and Gurken protein synthesis (Barbosa, 2007),

In this study, a clonal screen was used to identify genes regulating meiotic progression in Drosophila. Instead of testing directly for defects in meiosis, an easy-to-score eggshell phenotype was used that is produced when the levels or activity of the morphogen Grk are affected. This allowed an efficient screen of a large number of mutant lines and identification of germ-line-specific genes as well as genes with essential functions. The number of new genes identified is likely less than the total number of 2R genes required for Grk synthesis and function since mutations were discarded that blocked oogenesis. Of the eight genes described in this study, five show meiotic phenotypes. dPds5, nds, and mtc delay meiotic restriction to the oocyte, although only dPds5 and nds genetically interact with mei-W68 and mei-41, respectively. trin and blv affect the morphology of the karyosome in spite of normal timing in meiotic restriction. This confirms the effectiveness of the screening method for meiotic genes. Genetic and developmental analysis of the newly identified genes provides evidence for new regulatory steps in a network that coordinates Drosophila meiosis and oocyte development (Barbosa, 2007),

One complementation group, cohiba, identifies the Drosophila homolog of Pds5p in Schizosaccharomyces pombe, Spo76 in Sordaria macrospore, and BimD in Aspergillus nidulans, which have been found associated with the cohesion complex of mitotic and meiotic chromosomes. Depletion of Pds5 affects not only cohesion but also condensation in meiotic prophase. The unique 'open chromatin' karyosome defect observed in dPds5cohiba mutants is consistent with a role of Pds5 in chromosome cohesion during Drosophila meiosis. Like Spo76, the dPds5cohiba phenotype is suppressed by Spo11 (mei-W68) mutations defective in DSB formation. This suggests that dPds5 is necessary to maintain the structure of the meiotic chromosomes after DSBs are induced. However, in contrast to known DSB repair genes, the meiotic delay and oocyte patterning defects of dPdscohiba mutants are not due to activation of ATR/Mei-41-dependent checkpoint. One possibility is that the ATR downstream effector kinase dChk2 is activated via an alternative pathway, such as the Drosophila ataxia-telangiectasia mutated (ATM) homolog, which indeed activates dChk2 in the early embryo independently of ATR. Alternatively, dPdscohiba mutants may activate a checkpoint that measures cohesion rather than DSB breaks. The only other cohesion protein characterized in Drosophila is the product of the orientation disruptor (ord). ORD plays a role in early prophase I by maintaining synaptic chromosomes and allowing interhomolog recombination. More importantly and perhaps similar to dPds5, ORD seems not to be required for DSB repair. However, in contrast to dPds5 mutants, karyosome morphology is normal in ord mutants, and an eggshell polarity phenotype has not been reported. Although required for chromatid cohesion, dPds5 and ORD might play complementary roles in SC dynamics: ORD may stabilize the SC in the oocyte, whereas dPds5 may be required for the disassembly of synapses as one of the pro-oocytes regresses from meiosis (Barbosa, 2007),

The screen identified mutations in montecristo (mtc) that affect the restriction of meiosis to the oocyte. It has been proposed that this delay reflects the activation of the ATR/Mei-41 checkpoint pathway. Similar to dPds5, Mtc may control the regression from pachytene in those cyst cells that will not adopt the oocyte fate. The delayed meiotic restriction observed in mtc mutants occurs, however, independently of DSB formation or Mei-41 checkpoint activation. Mtc contains a Tudor domain. In other Tudor-domain proteins, this domain has been shown to interact with methylated target proteins. Identification of specific Mtc targets may clarify its role in meiotic restriction and oocyte patterning (Barbosa, 2007),

A particularly intriguing and novel phenotype is uncovered by mutations in indios (nds). By delaying meiotic restriction and activating Mei-41 without affecting the karyosome morphology, nds mutants separate checkpoint activation leading to Grk decrease from checkpoint activation controlling karyosome compaction. The nds phenotype also occurs independently of DSBs, suggesting that the trigger that leads Nds to trigger checkpoint activation is not DNA breaks. The fact that nds mutants are extremely sensitive to Mei-41 dosage further suggests that Nds activity may specifically control a branch of the Mei-41 checkpoint regulating Grk activity. In contrast to nds, trin mutants do not delay meiotic restriction and show defects in the karyosome in spite of normal Grk levels. Like mutants in src64B and tec29, which show a similar phenotype, Trin may mediate chromatin remodeling in the oocyte by regulating the actin cytoskeleton. In this context, the DV phenotype of eggs from trin mutants may be an indirect effect due to defects in actin cytoskeleton function. The production of collapsed eggs by trin mutant germ-line clones is consistent with this idea (Barbosa, 2007),

Finally, blv mutants show striking similarity to vas mutants with respect to lack of sensitivity to DSB formation, no evident delays of meiotic restriction, or karyosome and Grk phenotypes. Blv may thus act downstream or independent of the Mei41/ATR checkpoint, and its further characterization may help to understand the effector side of the meiotic checkpoint pathway (Barbosa, 2007),

Previous knowledge pointed to Drosophila meiosis as a linear progression of events from homologous chromosome pairing and recombination to meiotic restriction, karyosome formation, and eggshell patterning, with DSB repair as the main checkpoint linking meiosis to Grk signaling. By uncoupling some of these events, this study suggests the existence of a more complex network that links the surveillance of meiotic progression to oocyte patterning (Barbosa, 2007),

Quantitative proteomics reveals the dynamics of protein changes during Drosophila oocyte maturation and the oocyte-to-embryo transition

The onset of development is marked by two major, posttranscriptionally controlled, events: oocytematuration (release of the prophase I primary arrest) and egg activation (release from the secondary meiotic arrest). Using quantitative mass spectrometry, proteome remodeling has been described during Drosophila egg activation. This study describes quantitative mass spectrometry-based analysis of the changes in protein levels during Drosophila oocyte maturation. This study presents the first quantitative survey of proteome changes accompanying oocyte maturation in any organism and provides a powerful resource for identifying both key regulators and biological processes driving this critical developmental window. Muskelin, found to be up-regulated during oocyte maturation, was shown to be required for timely nurse cell nuclei clearing from mature egg chambers. Other proteins up-regulated at maturation are factors needed not only for late oogenesis but also completion of meiosis and early embryogenesis. Interestingly, the down-regulated proteins are predominantly involved in RNA processing, translation, and RNAi. Integrating datasets on the proteome changes at oocyte maturation and egg activation uncovers dynamics in proteome remodeling during the change from oocyte to embryo. Notably, 66 proteins likely act uniquely during late oogenesis, because they are up-regulated at maturation and down-regulated at activation. This study found down-regulation of this class of proteins to be mediated partially by APC/CORT, a meiosis-specific form of the E3 ligase anaphase promoting complex/cyclosome (APC/C) (Kronja, 2014).

Developments between gametogenesis and fertilization: Ovulation and female sperm storage

Introduction

In animals with internal fertilization, ovulation and female sperm storage are essential steps in reproduction. While these events are often required for successful fertilization, they remain poorly understood at the developmental and molecular levels in many species. Ovulation involves the regulated release of oocytes from the ovary. Female sperm storage consists of the movement of sperm into, maintenance within, and release from specific regions of the female reproductive tract. Both ovulation and sperm storage elicit important changes in gametes: in oocytes, ovulation can trigger changes in the egg envelopes and the resumption of meiosis; for sperm, storage is a step in their transition from being 'movers' to 'fertilizers'. Ovulation and sperm storage both consist of timed and directed cell movements within a morphologically and chemically complex environment (the female reproductive tract), culminating with gamete fusion. Within the female D. melanogaster, both gamete maturation and sperm storage are triggered by male factors during and after mating, including sperm and seminal fluid proteins. Therefore, an interplay of male and female factors coordinates the gametes for fertilization (Qazi, 2003).

The Drosophila female reproductive tract

Mating initiates a series of events within the female reproductive tract, including ovulation and sperm storage. Ovulation and sperm storage occur in different regions of the female reproductive tract, but are coordinated for a connected fate: the fertilization of an egg. At the anterior of the female Drosophila reproductive tract are two ovaries, each composed of 10-20 ovarioles. The ovarioles are held together by a peritoneal sheath, containing a network of fine, branching muscle fibers. The base (proximal end) of each ovariole forms a small duct or pedicel. The pedicels of all the ovarioles in an ovary unite to form a calyx; each calyx opens into a lateral oviduct. The lateral oviducts fuse into a common oviduct that, more posteriorly, enlarges to form the uterus. The wall of the oviducts consists of epithelium surrounded by circular muscles that is intensively innervated. The uterus is a heavily muscularized and innervated structure that receives sperm during mating and also holds the egg in position for fertilization. At the anterior end of the uterus are the three sperm storage organs: a single seminal receptacle and the paired spermathecae, as well as the paired spermathecal glands (also called parovaria or female accessory glands). At its posterior end, the uterus narrows forming the vagina that exits the female reproductive tract. The distal end of the vagina, called the gonopore, serves for the discharge of eggs (Qazi, 2003 and references therein).

Ovulation

In many organisms, interaction between the gametes triggers a series of cellular responses in the egg ('egg activation') required to initiate embryonic development. Drosophila eggs also are triggered to initiate a suite of cellular responses analogous to those of activation in other organisms (relief of arrest in meiosis, ultimately leading to completion of meiosis, changes in egg coverings, and initiation of translation) in a process that has therefore also been called activation. This semantic convergence masks a distinct mechanistic difference in the trigger for activation in Drosophila and at least one other insect relative to that in echinoderms, nematodes, and vertebrates: egg activation in Drosophila and other insects initiates independent of sperm entry, perhaps not surprising in an Order that includes species in which haploid males can develop from unfertilized eggs. In Drosophila, changes in the egg envelope's permeability, one feature of activation, initiate during ovulation, even while most of the egg is still within the ovary (Heifetz, 2001b). The egg's covering becomes progressively more impermeable to small molecules as the egg proceeds down the oviduct and the process is complete by the time the egg arrives at the uterus. Cross-linking of vitelline membrane protein sV23 also increases progressively as the egg moves through the oviduct and the uterus. Ovulation also triggers meiosis to resume before the egg reaches the uterus (Heifetz, 2001b). Thus, ovulation causes changes in the egg that prepare it for subsequent embryogenesis, should fertilization occur in the uterus, including coordination between cell cycle status of the egg and the sperm nuclei. The third feature of activation, resumption of translation, also initiates without fertilization in Drosophila, though it is not yet known if it too is triggered by ovulation (Qazi, 2003 and references therein).

Drosophila females produce up to two final-stage (stage 14) oocytes in each ovariole daily (as many as 80 oocytes/day. Several control points and feedback mechanisms regulate the production of mature oocytes. If mature oocytes are not ovulated, each ovariole accumulates two or three late-stage oocytes. This blocks the maturation of additional oocytes. Mating, and specifically sperm and some male accessory gland proteins [e.g., Acp26Aa (ovulin)], induces ovulation of mature oocytes (Heifetz, 2000 and Heifetz, 2001b) that, in turn, contribute to stimulating oogenic progression via a feedback mechanism. Thus, ovulating females produce high numbers of mature oocytes and deposit high numbers of fertilized eggs. The feedback mechanism by which seminal fluid, sperm, and the act of mating itself increase oogenic progression rate is not known (Qazi, 2003 and references therein).

Drosophila virgin females retain mature oocytes in their ovaries. Ovulation initiates within 1.5 h after mating (Heifetz, 2000) during a time when sperm are still being stored. Thus, the initial eggs ovulated are released potentially prior to full completion of sperm storage. The number of progeny per number of eggs laid (hatchability) immediately after matings between wild-type males and females is lower than at later time points (Chapman, 2001). This lower hatchability appears to reflect a lower efficiency of fertilization of the first eggs released. By visualizing the fertilizing sperm tail within wild-type eggs, it was found that the first eggs laid had a lower fertilization efficiency (ratio of unfertilized/fertilized eggs = 1.5 at 3-4 h post-mating) than those laid subsequently (0.6 at 48-49 h, Chapman, 2001). Two possible explanations for the low hatchability are suggested; regulated ovulation plays a critical role in each of these. (1) Ovulation allows coordination of oocyte release with the rate of sperm release from storage. The first eggs ovulated may reach the fertilization site before sperm are prepared and in position to be released from storage. Thus, lower numbers of those eggs would be fertilized, leading to lower hatchability. (2) Since unmated females can accumulate eggs for several days, it is possible that the older eggs are 'stale' and cannot be fertilized. By ovulating those eggs quickly, even before sperm are fully stored, the female clears out any stale eggs without wasting sperm (Chapman, 2001). Understanding the mechanism that coordinates oocyte release from the ovary and sperm release from the sperm storage organs will provide insights into the first steps of sperm/egg interactions that lead to successful fertilization (Qazi, 2003 and references therein).

In insects, production and laying of eggs requires five steps within the female reproductive tract: (1) oogenesis (the generation of oocytes within the ovary); (2) ovulation (release of an oocyte from the ovary into the oviducts); (3) movement of the egg down the oviducts; (4) fertilization of the egg when it has come to rest in the uterus, and (5) deposition of eggs onto the substratum. These steps are sequential and thus somewhat coupled. Therefore, assays of oocyte/egg progression sometimes measure several of these steps at once (Qazi, 2003).

Ovulation itself results from a series of processes occurring both sequentially and simultaneously within several ovarian microenvironments. The oocyte is released from its follicle, squeezed out of the ovary, and pushed through a lateral oviduct into the common oviduct, coming to rest in the uterus. Within each Drosophila ovariole, oocytes develop in a sequential fashion. Each ovariole contains a series of four to six egg chambers at stages in developmental progression (stages 1-14), with oogonia at the apex of the ovariole and the most mature oocytes (stage 14) at the base of the ovariole. In each egg chamber, the oocyte, connected to its sister cells (the nurse cells), is surrounded by a monolayer of somatic follicle cells. Follicle cells synthesize some of the yolk protein that will be deposited into the oocyte, as well as the proteins of the vitelline envelope and chorion that will cover and protect the oocytes. Specialized follicle cells at the anterior end of the egg chamber synthesize the micropyle, the site on the egg through which the sperm will enter. When the adult female fly ecloses, the base of the ovariole is still plugged with cells that grew out of the pedicel; this plug must be breeched to allow ovulation (Qazi, 2003 and references therein).

The mechanism by which oocytes are released from the Drosophila ovary is not known, but results in other insect systems suggest possible mechanisms. Insect ovulation involves two distinct steps: the opening of the oocyte follicle and of the intermost cells in the pedicel region, which releases the oocyte from the ovariole, and contraction of ovarioles, pedicels, and oviduct musculature, which moves the oocyte into the lateral oviducts. These contractions do not appear to involve sphincter(s), which are absent between the pedicels and the lateral oviducts in Drosophila. Ultrastructural studies of ovulation in the large aquatic beetle Dysticus marginalis (Coleoptera) show that the appearance of the first mature oocyte at the pedicel region triggers autolysis of the pedicel's innermost cells, opening the plug and releasing the mature oocyte from the ovariole. In addition, there are cells in the pedicel region that produce vacuoles whose contents might help the mature oocyte to glide into the oviduct. In the mosquito Culiseta inornata (Diptera), histological sections of whole ovaries also show that the pedicel is destroyed during ovulation. Thus, though little is known about the process of ovulation in Drosophila, it is likely that ovulation occurs, as in other insects, by cell degeneration in the pedicel region and massive muscle contractions at the base of the ovary and oviducts. These move the oocyte from the ovariole into and down the oviducts, where it eventually becomes lodged in the uterus. The posterior end of the oocyte leaves the ovary first. Thus, when the egg comes to rest in the uterus, its anterior end is 'up' with the egg's micropyle adjacent to the opening of the sperm storage organs, allowing for efficient fertilization (Qazi, 2003 and references therein).

In some insects, ovulation initiates only after mating. In Drosophila, however, ovulation occurs at a low level in adult females even without mating. Although Drosophila mature virgin females spontaneously ovulate at a very low rate (~1 egg/day), mating dramatically increases female ovulation rate (Heifetz, 2000). This effect of mating is rapid; an increase in ovulation is evident by 90 min post-mating (Heifetz, 2000). Some insects, such as walking sticks, ovulate one oocyte at a time and their ovarioles function alternately or in sequence, although in other insects, such as Orthoptera, all ovarioles ovulate simultaneously. It is not known which mechanism operates in Drosophila (Qazi, 2003 and references therein).

In Drosophila females, mating stimulates an increase in ovulation rate. Since ovulation is one step in the multistep process of egg laying, an assay was needed to distinguish this from the larger egg laying process. By measuring the progression of egg movement through the female reproductive tract, Heifetz (2000) showed that, by 90 min after mating, 90% of the females had at least one egg in their reproductive tract, and most of those were in the lateral oviduct. Ovulation rate increases with time after mating, such that by 6 h after mating, 30% of females have more than one egg in their reproductive tract. Seminal fluid components, Acps (accessory gland proteins), and sperm that are transferred to the female during mating play an important role in increasing female ovulation rate (Qazi, 2003 and references therein).

One seminal fluid protein, the prohormone-like Acp26Aa (also called 'ovulin') that shows sequence similarity to the Aplysia egg laying hormone, is essential for stimulating ovulation. The ovulation rate of females mated to males that lack Acp26Aa is 44% lower than the ovulation rate of mates of wild-type males (Heifetz, 2000). Acp26Aa's effects are evident between 1.5 and 6 h post-mating, and the protein is detectable in mated females for only a few hours post-mating. Since Acp26Aa stimulates ovulation shortly after mating, it is thought to act in 'clearing' the mature oocytes from the ovary, allowing for coordination of fresh oocyte release with sperm release and for increased oogenic progression rate (Chapman, 2001 and Heifetz, 2000). It is not yet known how Acp26Aa mediates oocyte release. Some Acp26Aa localizes at the base of the ovary (Heifetz, 2000), where it is processed into bioactive peptides. Some Acp26Aa enters the circulatory system. Thus, it is possible that Acp26Aa acts locally within the reproductive tract and/or via the neuroendocrine system to mediate contractile activity of the ovary and the oviduct musculature (Qazi, 2003 and references therein).

Another Acp, the 'sex peptide' Acp70A, also causes an increase in the number of eggs laid and, in one assay, the number of eggs in the uterus of dissected females. Since this Acp is known to increase oogenic progression, it is presently unknown whether the increased number of eggs seen in the uterus following Acp70A induction is simply a secondary indirect consequence of the increased production of oocytes, or is due to a separate direct effect on ovulation (Qazi, 2003 and references therein).

Drosophila males produce a peptide related to Acp70A in their ejaculatory ducts (Fan, 2000 and Saudan, 2002). Injection of this peptide, Dup99B, into unmated females can stimulate egg production. As with Acp70A, it is not presently known how Dup99B stimulates egg production, and therefore whether its effect on ovulation is direct or indirect (Qazi, 2003 and references therein).

In the absence of sperm transfer, oocyte development (from rapid yolk accumulation to vitelline membrane development and chorion deposition) and egg laying rates are lower than wild-type levels (50% and 33%-70% lower, respectively. Females that receive no sperm also begin to deposit eggs later than females mated to wild-type males (6 h vs 3 h; (Heifetz, 2001a). Moreover, even when sperm are transferred, if they are not stored, egg laying is low (<50% wild-type levels). Finally, when a female uses up all sperm from her mating, her egg laying rate decreases to virgin levels. These results indicate that the transfer and storage of sperm is essential to elevate oogenesis and egg deposition rates and may also affect ovulation (Qazi, 2003 and references therein).

The presence of sperm in the reproductive tract may trigger changes in female fecundity by causing the release of female-derived activating substances or by releasing male-derived substances that have adhered to the sperm. Alternatively, the presence of many sperm could stimulate the female central nervous system via stretch receptors in the uterus or sperm storage organs (Qazi, 2003 and references therein).

Contraction of insect oviducts is under neural control. The Drosophila female reproductive tract is innervated by branches of the abdominal nerve center (AbTNv). Thus, it seems likely that neurotransmitters and/or neurohormones will regulate the contractile activity of the Drosophila ovary and oviduct musculature to cause the release of mature oocytes. Since the Drosophila female reproductive tract's epithelium has secretory characteristics, it is also likely that neurotransmitters or neurohormones released at the base of the ovary could affect the secretory activity of the epithelium, and thereby trigger the disintegration of the pedicel plug to release mature oocytes (Qazi, 2003 and references therein).

Although the identity of neurotransmitters, neuromodulators, or neurohormones that could mediate ovulation in Drosophila is unknown, findings in other insects point to likely candidates. In locusts (Locusta migratoria), for example, glutamate, octopamine, proctolin, and SchistoFLRFamide have been shown to mediate oviduct muscle contraction, aiding the movement of eggs. In other insects, such as the bed bug Rhodnius prolixus, the neurosecretory peptide myotropin mediates the ovarian contractile activity that releases mature oocytes. Myotropin is released from the corpus cardiacum in response to ecdysteroid levels in the female hemolymph (Qazi, 2003 and references therein).

In Drosophila, ovarian function and ovarian environment affect egg chamber development and therefore ovulation rate. Female age, available nutritional resources (e.g., yeast), and other environmental factors, such as circadian cues and humidity, affect egg production. For example, nutritional supplementation with yeast causes increased germ cell proliferation and decreased cell death of both early-stage and vitellogenic-stage oocytes. This results in increased egg production and egg deposition rates thereby (directly or indirectly) increasing ovulation rates. Although the mechanism of environmental interaction with ovulation physiology is still to be determined in Drosophila, again clues are available from other insects. For example, in the beetle Xyleborus ferrugineus, ecdysteroid titers are significantly higher in young fertile adult females than in aging females, suggesting that fewer eggs are produced, and hence ovulated in older females, due to declining ecdysteroid levels (Qazi, 2003 and references therein).

In Drosophila, several control points and feedback mechanisms regulate the production of mature oocytes, which affects female ovulation status. One such feedback prevents oocytes from entering the oviduct before completing oogenesis. Thus, a female will ovulate only if she has mature oocytes in her ovaries. Another control point in egg maturation is regulated by Acp70A. Acp70A stimulates oocytes to progress to later vitellogenic stages and thus override a control point in egg maturation between mid/late vitellogenic stages (stages 9/10). Thus, Acp70A can stimulate oogenic progression rate, allowing the female to ovulate at a high rate (Qazi, 2003 and references therein).

Female sperm storage

Sperm storage allows sperm from a given mating to be used long after the male and female have separated (~2 weeks), thus increasing female fertility. Of the ~4000 sperm transferred to a Drosophila female during mating between wild-type flies, ~80% are expelled from the uterus when the first egg is laid, and subsequent fertilizations rely on the 700-1000 sperm stored in the female. Females that receive normal quantities of sperm, but store few of them, produce few progeny (~10% wild-type levels). Compared with mammals and birds, whose efficiency of sperm use can be as low as 0.01% (reviewed in Neubaum and Wolfner, 1999b), Drosophila use sperm quite efficiently: of the 700-1000 sperm stored, ~400 are used to fertilize eggs (Qazi, 2003 and references therein).

Sperm storage potentially allows coordination of ovulation rate with sperm release from storage. This prevents gamete wastage, which would occur when gametes are released at different times and unable to unite successfully. The coordination of sperm release and ovulation could also decrease the incidence of polyspermy that also wastes eggs and sperm because it results in nonviable fertilizations. The controlled release of sperm from storage in Drosophila may be one reason why polyspermy is rare in this organism (less-than or equal to 1% (Qazi, 2003 and references therein).

Sperm storage allows females to retain sperm for more than one male within her reproductive tract. This provides 'fertility insurance,' should any of the males be infertile or genetically incompatible with the female. Storing sperm from multiple males has the potential additional advantage for females of generating progeny with a broader range of different genotypes. Finally, storing sperm from more than one male generates an opportunity for sperm competition and female sperm preference (Qazi, 2003 and references therein).

After mating, Drosophila females become unreceptive to courting males for several days (mating effect). Male seminal products such as Acp70A trigger the female's immediate reluctance to remate, but their action is short-term (<24 h). For the normal (approximately 1-1.5 week) depression of receptivity, females must also store sperm (sperm effect). The sperm effect on female receptivity may begin as early as 6-8 h postmating; it is clearly operating by 10-18 h after mating. Females that store few sperm are receptive to remating sooner than those storing larger number of sperm. The return of female receptivity correlates both with the decline in numbers of stored sperm as those sperm are released and used for fertilization and, in the laboratory, with longer periods of male-female pair confinement, continued availability of food, and population density (Qazi, 2003 and references therein).

Potential mechanisms for the sperm effect (depression of female receptivity) include: pressure from within the sperm storage organs exerted by sperm, movements of sperm within the storage organs, and/or distension of the uterine walls by sperm and semen. In the white cabbage butterfly Pieris rapae, receipt of sperm and seminal fluid products results in changes in female receptivity to mating. Distension of a female P. rapae uterus with saline causes a decrease in receptivity similar to that after mating. A similar mechanism in the Drosophila sperm storage organs could explain the Drosophila sperm effect. Since the sperm effect is established several hours after sperm storage is complete (10-18 h vs 1.5 h, respectively), the effect of sperm on female behaviors is proposed to involve an intermediary acting on the female central nervous system (CNS). This intermediary could be a molecule(s) secreted from the female's cells. For example, in the redbanded leafroller moth, Argyrotaenia velutinana, mating depresses female pheromone titer via a CNS-mediated release of the peptide PBAN (pheromone biosynthesis activating neuropeptide). When the female CNS is disrupted by cutting the ventral nerve cord, PBAN remains within female tissues, and is not released into the hemolymph, where it normally acts. Alternatively, the intermediary could be a molecule from the male's seminal fluid. Although most seminal proteins are not detectable in the female more than a few hours after mating, others may be stabilized by associating with sperm, allowing them to act within the female long after mating has ended (Qazi, 2003 and references therein).

In several organisms, matings are costly to females. In Drosophila, this cost arises from a combination of exposure to males, receipt of Acps, and allocation of resources to the increased egg production postmatin. Sperm themselves do not directly contribute to the cost of mating in Drosophila. However, by storing sperm, females reduce costs that accrue from repeated matings since they do not need to mate as many times to maintain fertility (Qazi, 2003 and references therein).

Despite the costs associated with multiple mating and the general reluctance of females to remate after mating, female promiscuity is well documented in Drosophila. When a female mates with more than one male, female sperm storage provides both a venue for sperm from different males to compete for access to fertilizations (sperm competition) as well as an arena for the female to select sperm from among the contributions of competing males (sperm preference or cryptic female choice). These processes may be important mechanisms preventing genetic incompatibility (the production of nonviable or infertile progeny) by selecting among male ejaculates. This is observed in Drosophila: when a D. simulans female mates with both a D. simulans and a D. mauritiana male, D. simulans sperm are preferentially stored and used for fertilization. The mechanisms of this conspecific male advantage include both the inactivation and physical displacement of D. mauritiana stored sperm (Qazi, 2003 and references therein).

Sperm competition and sperm preference have potentially important evolutionary consequences. When a female Drosophila mates with more than one male, the most recent partner usually sires more than 80% of the subsequent progeny ('last male precedence'). However, large variation exists in the fertilization success among last-mating males due to genetic differences among both males and females. Examining sperm storage sheds light on the mechanisms of sperm precedence (Qazi, 2003 and references therein).

Female sperm storage in Drosophila occurs in two types of specialized organs, located at the anterior end of the uterus. The single seminal receptacle is on the ventral side, and the paired spermathecae are on the dorsal side of the uterus. The two types of storage organs differ in morphology and in patterns of sperm storage (Qazi, 2003 and references therein).

Numerically, the seminal receptacle plays the biggest role in sperm storage, retaining 65%-80% of the stored sperm. Observations and counts of stored sperm over time indicate that sperm are released almost exclusively from the seminal receptacle for the first several days after mating. Then, as the seminal receptacle sperm become depleted, sperm are released from the spermathecae. The seminal receptacle's importance in sperm storage is supported by a phylogenetic analysis of sperm storage organ use among 113 species of Drosophila. Loss of the sperm storage function by the seminal receptacle is a rare evolutionary event that appears to have occurred only once; it characterizes only 3 (2.6%) of the species examined. In contrast, the spermathecae's sperm-storage function may have been lost as many as 13 different times affecting 38 (33.6%) of the examined species (Qazi, 2003 and references therein).

The seminal receptacle is a thin (5-20 microm) blind-ended tube that is coiled against the outer uterine wall. In Drosophila, the seminal receptacle is more than 2 mm long, slightly longer than a sperm (1.75-1.90 mm). A positive relationship also exists between sperm and seminal receptacle length among 44 other Drosophila species examined. This observation and other detailed evolutionary studies suggest that increases in seminal receptacle length drive the evolution of longer sperm (Pitnick, 1999) (Qazi, 2003 and references therein).

The lumen of the seminal receptacle is lined with a thin cuticle and is surrounded by a layer of nonsecretory cells, a basement membrane, and a helically coiled layer of muscle. There does not appear to be a sphincter separating the seminal receptacle from the uterus. At any time, only a few sperm are observed in the proximal half of the tube (that is, near its entry into the uterus). The majority of stored sperm appear partially extended and lying parallel to each other within the distal half of the seminal receptacle. This mass of sperm is curvilinear, and their tails can be seen to move, except when the seminal receptacle is very full of sperm (Qazi, 2003 and references therein).

No genes responsible for seminal receptacle length or morphology have been identified. However, results of quantitative genetic analysis of selection experiments for increased or decreased seminal receptacle length suggest that only a small number (2-5) of loci determine seminal receptacle length. Since their effect is largely additive, the loci appear to be independent (Qazi, 2003 and references therein).

Fewer sperm reside within the paired spermathecae than in the seminal receptacle (maximum of 135-449 sperm within both spermathecae). However, the paired spermathecae appear to have two important roles in sperm storage. (1) Spermathecae are the long-term storage organs. Sperm may accumulate more slowly in the spermathecae than in the seminal receptacle. As mentioned before, spermathecal sperm are apparently used for fertilization after the seminal receptacle's sperm have been depleted. (2) Spermathecae may secrete substances that maintain sperm viability within both the seminal receptacle and the spermathecae. Posterior to the spermathecae, but also opening into the dorsal side of the uterus, are two spermathecal glands whose secretions could also affect sperm storage (Qazi, 2003 and references therein).

Each spermatheca joins the uterus via a stalk composed of a thin lumen surrounded by a thick cuticular intima, epithelial cells, and a helically coiled layer of muscle cells. Like the seminal receptacle, the spermathecae do not appear to have a sphincter near their base. The spermatheca itself is an oval-shaped capsule formed from a cuticular intima. Thin ducts within the spermathecal intima open to secretory cells surrounding the exterior of the capsule. These cells produce a lamellar type of secretion that accumulates within the spermathecal lumen. Sperm move from the uterus into the spermathecal capsule through the stalk. Within the capsule, they wind around forming a toroidal mass (Qazi, 2003 and references therein).

Two genes have been identified that affect spemathecal development. Several alleles of lozenge (lz), which encodes a putative transcription factor, cause loss of the spermathecal glands and vary in the extent to which they affect spermathecal development. Some lz females have intact spermathecae, while others are missing capsules and have malformed or missing spermathecal stalks. These alleles also cause low fertility, apparently due to lower sperm storage in the seminal receptacle and the loss of motility of any stored sperm within a few days after mating. Analysis of lz mutants suggests that they impair dorsal cell migration in the early genital imaginal disc that is needed for subsequent spermathecal development. dachshund (dac), a nuclear protein, is important for appropriate formation of the spermathecal stalks. When dac expression is blocked during development, the spermathecal capsules appear normal, but they share a single stalk. In addition to these genes that affect spermathecal morphology, some genes control spermathecal number. At least two genes are responsible for the formation of extra (>2) spermathecae, but neither has yet been identified (Qazi, 2003 and references therein).

Why are there two types of sperm storage organs? Although the use of two types of storage organs is common (63.8% of 113 species examined), among Drosophila species, it is not clear why the spermathecae and seminal receptacle are both required for sperm storage. It has been suggested that the seminal receptacle might have evolved as a new organ, which is more efficient at storing sperm than are the spermathecae. The apparent coevolution of seminal receptacle length and sperm length among several Drosophila species suggests that the seminal receptacle may also provide more opportunity for female or male influence over sperm storage and sperm fate. Sperm displacement, as a result of multiple mating, appears to be primarily associated with the seminal receptacle. Residence in the spermathecae might protect sperm from displacement, and the secretions of the spermathecae might also be essential for viability (Qazi, 2003 and references therein).

When do sperm enter and exit storage? Sperm storage begins before the ~20-min copulation is complete. Sperm accumulate rapidly within the two types of storage organs, leveling off at ~700-1000 sperm less than 6 h after mating ends. The first egg laid after mating (~90 min) pushes out remaining unstored sperm. By 10 h after mating, seminal receptacle sperm numbers have noticeably declined due to the female's use of stored sperm to fertilize her eggs. On average, the number of sperm in the seminal receptacle declines at ~100-170 per day. By 48 h after mating, sperm storage in the seminal receptacle has declined by ~50%, while sperm stored in the spermathecae have decreased by only ~15% (Qazi, 2003 and references therein).

How does sperm storage occur? Sperm storage can be viewed as a series of steps: progression of sperm through the female reproductive tract after mating, entry of sperm into the storage organs, retention and maintenance of stored sperm, and release of sperm from storage up to the point of fertilization. Both female and male Drosophila play active roles in sperm storage, as shown by the nontransitivity of numbers of stored sperm 1 h after mating between different fly strains. Female-based mechanisms can include absorption of fluids from parts of her reproductive tract (called 'hydraulics' here), contractions of the uterus, contractions of the sperm storage organs, restriction of sperm to particular regions of the female reproductive tract, and/or factors secreted from within the female reproductive tract. Male-based mechanisms are also important and can continue even after copulation is complete. These include sperm motility and seminal proteins. Sperm are motile when they are transferred to the female and when they are in storage. Although it has been hypothesized that sperm motility is important for the release of sperm from storage, and it seems a likely contributor to that process, the role of sperm motility in sperm entry into storage in Drosophila is unknown. In contrast, studies have shown a profound effect on female sperm storage of secretions from the male's ejaculatory duct and accessory glands transferred to the female during mating. Matings between normal females and males transferring sperm, but no Acps, are infertile, suggesting that Acps play an essential role in sperm use once sperm are transferred to the female. When these females mate first with males transferring Acps, but no sperm, then mate with males transferring sperm, but no Acps, the second male's fertility is restored. Females mated to males that transferred sperm but greatly reduced (~1%) quantities of Acps store fewer than 10% as many sperm as do females mating to wild-type males (Qazi, 2003 and references therein).

What gets sperm into storage? Sperm may be drawn into storage by pressure changes within the female reproductive tract. In the Dipteran Culicoides melleus, and possibly in other lower Diptera, one component of sperm storage apparently involves fluid absorption from the spermathecae, which create a force that sucks sperm into storage. Although a similar mechanism could potentially function in Drosophila's seminal receptacle, ultrastructural studies argue against this hypothesis for sperm entry into the Drosophila spermathecae because substances from the surrounding cells accumulate within the spermathecae at the same time that sperm storage is occurring (Qazi, 2003 and references therein).

In many animals, female muscular contractions apparently push sperm from the uterus into storage. Consistent with this occurring in Drosophila, a Drosophila female CNS is required for sperm storage. This has been tested by manipulating expression of transformer (tra), whose product is required for normal female development. Various levels of ectopic expression of tra in either tra-deficient mutant XX flies or XY flies results in individuals possessing female genital morphology (phenotypic females), but either a masculinized CNS (evinced by male courtship behavior) or a (presumably) feminized CNS. Animals with the presumably female CNS store nearly 6.5 times more sperm within all storage organs (~550 sperm) but allocate proportionally fewer of the sperm to their seminal receptacles than do phenotypic females with a masculinized CNS. These results show that a feminized nervous system is necessary for sperm storage and that females actively distribute sperm among the storage organs. Results of experiments in which males mate with isolated female abdomens (that lack ganglia and therefore a CNS) support this hypothesis. It is possible that a female CNS is needed to trigger uterine muscle contractions that push sperm into storage. Alternatively, a female CNS could influence sperm storage by stimulating endocrine cells to release substances that attract sperm to the storage organs. Finally, the generous innervation of the seminal receptacle suggests that local contractions of the sperm storage organs might be important for sucking sperm into storage and/or for efficient arrangement of sperm within storage (Qazi, 2003 and references therein).

Sperm may be lured into storage by substances produced by the female or by male-derived substances activated once in the female. In sea urchins and other marine invertebrates, sperm-activating peptides (SAPs) secreted from the egg jelly stimulate sperm movements and orientation to eggs as well as activate other sperm-egg interactions (Suzuki 1995). Compounds within the female Drosophila reproductive tract could potentially act in a similar way, but none have been positively identified thus far (Qazi, 2003 and references therein).

Concentrating sperm to specific regions of the reproductive tract can increase the likelihood that they will encounter the openings to the storage organs. (1) Female anatomy, such as folds in the uterine wall, could channel sperm into the seminal receptacle. (2) In Drosophila females, a barrier of unknown composition exists at the base of the oviduct that keeps sperm in the uterus. At least one male seminal protein, the accessory gland protein Acp36DE, localizes at this barrier, but no chemical components required for barrier formation have been identified. In egg-less females, the barrier is mislocalized or does not form and sperm are found within the common and lateral oviducts. The presence of oocytes in the ovaries may help form the block by creating a back-pressure and/or causing the secretion of a substance that localizes near the base of the oviduct preventing sperm from premature access to oocytes within the ovary (Qazi, 2003 and references therein).

From within her reproductive tract, a female Drosophila secretes factors important for sperm storage. PubMed ID: Glucose dehydrogenase (Gld), an enzyme-producing reactive oxygen species, is secreted from the spermathecal stalks and the genital plates (located near the gonopore). gld-mutant females store fewer sperm within, and allocate sperm more unevenly between, the two spermathecae, but only when sperm storage is submaximal (<500 total sperm stored). Therefore, Gld may facilitate sperm storage, particularly when sperm are not plentiful, but is not essential for sperm storage to occur. The mechanism of Gld's sperm storage effects is unknown, but Gld is unlikely to serve as a chemoattractant since it is produced in more than one location within the female reproductive tract (Qazi, 2003 and references therein).

Corralling can also involve male contributions. The male accessory glands contain filaments composed of globular subunits. Similar looking filaments are observed in female storage organs interdigitated with stored sperm. If the filaments of similar appearance are indeed the same, those filaments might provide a scaffold along which sperm move or within which sperm are confined, to facilitate the efficient movement of sperm into storage. In an analogous way, the mating plug, contributed at least in part by the male, is thought to facilitate sperm storage by forming a physical barrier that prevents the loss of sperm from the uterus; sperm are confined above the plug, concentrating them near the entrances of the storage organs. The mating plug has also been proposed to provide a trellis to facilitate sperm movement toward storage. The mating plug contains several male seminal proteins, including PEB-me (its major component, derived from the ejaculatory bulb) and Acps, including the protein Acp36DE. Entering sperm traverse the mating plug and are limited to the anterior portion of the uterus. The mating plug is not detected more than 6 h after mating and, while its fate is unknown, it seems likely to be expelled when the first egg is laid or dissolved and reabsorbed (Qazi, 2003 and references therein).

By examining sperm storage in the presence of normal or very low amounts of Acps, it has been shown that Acps are required for sperm storage. One of these proteins, Acp36DE, is essential for proper sperm storage. Females mated to Acp36DE-deficient mutant males receive normal quantities of sperm during mating, but store far fewer sperm than females mated to wild-type males. Without Acp36DE, sperm start to enter storage at the normal time, but the subsequent accumulation of sperm into the seminal receptacle and spermathecae is less efficient. The rate at which sperm are released from spermathecal storage differs slightly in the presence or absence of Acp36DE. Within the first 24 h after mating, the rate at which sperm are released from the seminal receptacle is similar in the presence or absence of Acp36DE, but between 24-48 h after mating, proportionally more sperm are lost from the seminal receptacles of females receiving Acp36DE than females not receiving Acp36DE from their mates. It is not clear whether this phenomenon is a direct result of Acp36DE action or a secondary consequence of storing fewer sperm. The number of progeny produced in the absence of Acp36DE corresponds to the number of sperm in storage, indicating that those sperm that are stored without Acp36DE are fully viable. Thus, Acp36DE's primary role is in facilitating sperm storage. It may potentially have a secondary role in promoting sperm retention in the seminal receptacle, but there is no evidence that Acp36DE affects sperm viability (Qazi, 2003 and references therein).

Acp36DE is a novel 122-kDa glycoprotein that is transferred to females beginning within the first 5 min of mating. Within the female, it is processed to a 68-kDa product. Acp36DE is detectable within the female reproductive tract for as long as 3 h after mating. It localizes to the oviduct wall anterior to the sperm storage organ openings (at the barrier discussed above) and on the anterior end of the mating plug. Thus, it is present at the upper and lower areas of the 'corral' described above. In addition, Acp36DE enters the sperm storage organs. Finally, Acp36DE binds to sperm in vivo and in vitro. When the first egg is laid, the Acp36DE in the oviduct and mating plug are expelled. Although no longer detectable within the female, Acp36DE donated from one male facilitates the storage of a second male's sperm 24-48 h later (Qazi, 2003 and references therein).

The available data on Acp36DE support several, not mutually exclusive, models for its action. Acp36DE may interact with targets on female tissues stimulating females to push (from the uterus) or suck (from the sperm storage organs) sperm into storage via muscular contractions. It could potentially also stimulate the release of chemoattractant molecules. Alternatively, Acp36DE may limit sperm movements via its associations with the oviduct, mating plug, and sperm, thereby facilitating the rapid accumulation of sperm within storage. Finally, Acp36DE might help track sperm into storage by forming a scaffold or providing guidance cues along which the sperm move, or are moved, into storage (Qazi, 2003 and references therein).

What keeps sperm viable in storage? Once Drosophila sperm are in storage, they need to remain viable for up to 2 weeks. For example, even in cases of matings between genetically incompatible strains of Drosophila, sperm in the storage organs remain viable several days after mating (suggested by vital cell staining). Both female and male factors probably contribute to the maintainance of sperm in storage. Substances produced within the spermathecae may play roles in sperm viability and retention. The presence of the spermathecal capsule correlates with female fertility. The low and variable fertility of some female lz mutants suggests a similar model for Drosophila, since the lz mutant phenotype is proposed to be due to the presence/absence of the spermathecal capsule. Females with at least 1 spermathecal capsule produced an average of 216 progeny, 36 times as many progeny as mutant females lacking spermathecal capsules. Lower fertility is attributable to a shorter duration of progeny production among females lacking capsules compared with their normal sisters. Although the seminal receptacle appears normal, lz mutants store fewer sperm which lose motility earlier (less-than or equal to 5 days after mating) than do sperm in wild-type females (>11 days). These results suggest that presence of the secretory cells surrounding the spermathecal capsule and/or the spermathecal glands is important for the viability of stored sperm and for female fertility (Qazi, 2003 and references therein).

Stored sperm need to be protected from degradation. During storage, proteolysis of sperm surface proteins could destroy a sperm's ability to bind to eggs; alternatively, regulated proteolysis of the surface of stored sperm could be essential to activate or capacitate them. Indeed, in mice, mutations in seminal fluid protease inhibitors impair fertility, consistent with the hypothesis that protease inhibitors serve to protect sperm. Drosophila seminal fluid also contains regulators of proteolysis. Of ~83 predicted secreted male accessory gland proteins, 9 are predicted (or demonstrated) regulators of proteolysis. One, the trypsin inhibitor Acp62F, has been shown to enter the mated female's sperm storage organs, consistent with its playing a role in protecting sperm from degradation. Stored sperm are also potentially subject to untoward effects from microbes that might have entered the female's genital tract during mating. Perhaps to guard against this, sperm storage organs and seminal fluids contain antimicrobial peptides. The seminal receptacle and spermathecae also both secrete Drosomycin, a peptide with antifungal properties (Qazi, 2003 and references therein).

What releases sperm from storage? Sperm leave storage to two potential fates: one fate is to fertilize an ovulated egg, another is to leave storage but not to fertilize an egg either due to inefficient sperm use or to sperm displacement as a result of the female mating with another male (female sperm preference or sperm competition). Since sperm use in Drosophila is efficient, one (or very few) sperm leave(s) storage to fertilize an ovulated egg as the egg comes to rest in the uterus. The egg lodges there with its anterior end, containing its micropyle, close to the sperm storage organ entrances. It is not known whether sperm swim, are pushed, or are sucked through the micropyle. The entire sperm enters the egg. Its tail coils in the anterior end and persists within the embryo until shortly after hatching (Qazi, 2003 and references therein).

In Drosophila, conformational changes of the reproductive tract induced by ovulation might also effect sperm release. Single sperm have been observed near the opening of the seminal receptacle as an egg passes down the oviduct; it has been proposed that ovulation and sperm release are correlated. Muscular contractions of just the seminal receptacle could squeeze small numbers of sperm out of storage (Qazi, 2003 and references therein).

Motile sperm might motor their way out of storage. Sperm circulating within the storage organs have been observed and it has been speculated that, occasionally, a single sperm leaving storage would encounter an egg. This observation, coupled with the lack of detected sphincters at the base of the sperm storage organs, has led to the proposition that sperm motility aids their release (Qazi, 2003 and references therein).

At least one male-derived protein is suggested to play a role in sperm residence in storage in females: the carboxylesterase, Esterase-6 (Est-6). Est-6 is secreted from the male ejaculatory bulb and anterior ejaculatory duct, and is transferred to the female early during mating. Activity of male-derived Est-6 is detected for only 2 h after mating. Although the initial timing and storage of sperm into females that do or do not receive Est-6 from their mates is similar over time, more sperm are retained in females that do not receive Est-6. Est-6 therefore appears to play a role in the release of sperm from storage in the seminal receptacle; its role on spermathecal sperm is unclear. Est-6 might cause the release of sperm from the seminal receptacle by affecting sperm motility within the sperm storage organs or by catalyzing the production of molecules needed to sustain sperm motility. However, since Est-6 activity is also positively correlated with the rate of female oviposition as well as female latency to remating, and since Est-6 enters the female hemolymph, it may have more than one target or multiple interrelated effects (Qazi, 2003 and references therein).

If a female mates twice, the second male's ejaculate causes the release of previously stored sperm (last male sperm precedence). This effect is attributable to the removal of some sperm from storage and the inactivation of remaining sperm. The method of displacement depends on the time between matings. If a female remates within 2 days of an initial mating, the last-mating male's sperm plays a role in physical displacement of sperm, primarily from the seminal receptacle. With longer intervals between rematings, other seminal components, particularly Acps from the most recently mating male, functionally displace sperm by decreasing the use of previously stored sperm that remain after the second mating. If seminal proteins temporarily protected the male's sperm from displacement, then perhaps it is the inactivation of these 'protein companions' to sperm that leave the sperm vulnerable to displacement by future mating males. Males lacking Acp36DE are poor displacers of other males' sperm and often have their own sperm nearly completely displaced from storage (Chapman, 2000), but this is believed to be because they are poor at getting their own sperm moved into storage (Qazi, 2003 and references therein).

Sperm-storage defects and live birth in Drosophila females lacking spermathecal secretory cells

Male Drosophila flies secrete seminal-fluid proteins that mediate proper sperm storage and fertilization, and that induce changes in female behavior. Females also produce reproductive-tract secretions, yet their contributions to postmating physiology are poorly understood. Large secretory cells line the female's spermathecae, a pair of sperm-storage organs. This paper reports identification of the regulatory regions controlling transcription of two genes exclusively expressed in these spermathecal secretory cells (SSC): Spermathecal endopeptidase 1 (Send1), which is expressed in both unmated and mated females, and Spermathecal endopeptidase 2 (Send2), which is induced by mating. These regulatory sequences were used to perform precise genetic ablations of the SSC at distinct time points relative to mating. The SSC were shown to be required for recruiting sperm to the spermathecae, but not for retaining sperm there. The SSC also act at a distance in the reproductive tract, in that their ablation: (1) reduces sperm motility in the female's other sperm-storage organ, the seminal receptacle; and (2) causes ovoviviparity -- the retention and internal development of fertilized eggs. These results establish the reproductive functions of the SSC, shed light on the evolution of live birth, and open new avenues for studying and manipulating female fertility in insects (Schnakenberg, 2011).

Females of many animal species store sperm after mating, in specialized organs of the reproductive tract. In addition to sperm, seminal proteins are transferred to females during mating. In Drosophila, seminal proteins perform multiple functions that advance the male's reproductive interests. These functions include promoting sperm storage, decreasing the female's receptivity to subsequent courters, and stimulating egg production and ovulation (Schnakenberg, 2011).

Female reproductive interests do not necessarily coincide with those of their mates. A coevolutionary arms race can therefore ensue. In Drosophila, the coevolutionary pressure on female reproductive functions is apparently quite strong: seminal fluid decreases female lifespan and does so to a greater extent when females are experimentally prevented from coevolving with males. Of course, males and females do share some reproductive interests, such as successful production of offspring, and therefore evolutionary pressure also exists to coordinate their reproductive functions. Although it has been appreciated that molecular interactions -- both antagonistic and cooperative -- between male and female products are key to understanding insect fertility and its evolution, much more progress has been made in characterizing the composition and functions of seminal fluid than in characterizing female reproductive secretions (Schnakenberg, 2011).

A potentially major role of female secretions is in sperm storage. Insect females typically have multiple specialized sperm storage organs, to which sperm are recruited after copulation and in which sperm can be maintained for weeks or, in the case of queens of social insect species, years. Female Drosophila melanogaster have three such organs located at the anterior of the uterus: a long tubular seminal receptacle and a pair of spermathecae. Each spermatheca is mushroom shaped, with a duct that extends from the uterus to a cuticular cap. The seminal receptacle houses up to 80% of stored sperm, whereas the spermathecal caps house the remainder. Each spermathecal cap is lined with large glandular cells containing prominent secretory organelles that open into the lumen where sperm are stored. Despite considerable divergence in sperm-storage organ anatomy, such cells are found lining or adjacent to the spermathecae of a wide range of insects. The position of these spermathecal secretory cells (SSC) suggested they might have a role in sperm storage, yet direct in vivo evidence has been lacking. Indeed, the most direct evidence for a role in sperm storage comes from a 1975 study of boll weevils with surgically removed spermathecal glands: sperm did not enter the spermatheca in such females, although because sperm motility was greatly diminished it cannot be concluded whether the glands are necessary just for sperm viability or also for recruitment into storage (Schnakenberg, 2011).

The SSC might also contribute to sustained levels of egg production and fertilization, by secreting proteins that alter female reproductive physiology or by modulating the activities of male seminal proteins. Sex peptide, a seminal protein, binds to sperm tails and during storage is gradually cleaved to an active form that stimulates egg production. Sex peptide is also required for release of sperm from storage. In the female, the sex peptide receptor is expressed in neurons that mediate a decrease in courtship receptivity and an increase in egg laying after mating, and it is required for these changes. The sex peptide receptor is also expressed in the SSC, suggesting that sex peptide acts directly on these cells (Schnakenberg, 2011).

Other genes expressed specifically in the SSC have not been comprehensively identified, although some such genes have emerged from transcriptional profiling studies of: (1) somatic tissues of males versus females, (2) dissected whole spermathecae, and (3) virgin versus mated females. One notable class of genes revealed by these studies is those encoding proteases. Protease-encoding genes are over-represented among those induced in females by mating, and among those highly expressed in the spermathecae. Proteases are especially interesting due to their potential for interactions with seminal proteins. Such interactions could be antagonistic, for example by degradation of seminal proteins, or cooperative, for example by regulated cleavage of seminal proteins to their active forms. Male-female coevolution would be expected to lead to rapid divergence of female-expressed protease-encoding genes, and indeed such genes show elevated rates of coding-sequence evolution in several species (Schnakenberg, 2011).

To address how the SSC contribute to female postmating physiology, tools have been developed that allow manipulation of these cells in a precise spatiotemporal manner. To develop these tools, the regulatory regions were identified controlling transcription of two protease-encoding genes that are expressed exclusively in the SSC. The gene CG17012, which encodes a serine-type endopeptidase, is expressed exclusively in the SSC, in both unmated and mated females. CG17012 is refered as Spermathecal endopeptidase 1 (Send1). CG18125, which also encodes a serine-type endopeptidase, is expressed exclusively in the SSC and its transcription is upregulated 76-fold 3-6 h after mating. CG18125 is refered as Spermathecal endopeptidase 2 (Send2). Identifying these genes' regulatory regions allowed creation of drivers and reporters for manipulating and monitoring the SSC (Schnakenberg, 2011).

This study shows that the SSC are required to recruit sperm to the spermathecae, but not for maintaining them there. Moreover, the SSC act at a distance in the reproductive tract, in that they are required for maintaining sperm motility in the seminal receptacle. Action at a distance was also shown with respect to egg laying, in that females lacking SSC are ovoviviparous. Fertilized eggs develop, and indeed sometimes hatch into larvae, inside the uterus. This phenotype is reminiscent of two species of Drosophila that retain developing eggs, D. sechellia and D. yakuba. These results therefore not only reveal the functions of a poorly understood reproductive tissue, but also shed light on the evolution of live birth (Schnakenberg, 2011).

For each of the SSC-expressed genes, Send1 and Send2, a driver was created carrying ~4 kb of upstream sequence and ~4 kb of downstream sequence, flanking the coding sequence of the yeast transcriptional activator GAL4, which can activate transgene expression in Drosophila through its cognate UAS sequence. GAL4-independent reporters were also created by cloning a subfragment of the Send1 or Send2 upstream sequence in front of the coding sequence of a fast-maturing, nuclear-localized, red-fluorescent protein (Schnakenberg, 2011).

Send1-GAL4 drives expression of a membrane-bound green fluorescent protein (GFP) (UAS-mCD8-GFP) specifically in the SSC of virgin and mated females. GFP expression driven by Send1-GAL4 is visible by 20 h posteclosion and increases in intensity by day 4. Send2-GAL4 drives GFP expression in the SSC of mated females only, as early as 3 h postmating. Send1-nRFP and Send2-nRFP recapitulate expression of the respective endogenous genes as well (Schnakenberg, 2011).

The potential for redundancy among spermathecae-expressed serine proteases is high. Protease-encoding genes are over-represented among those induced in females by mating, and among those highly expressed in the spermathecae. Moreover, some spermathecae-expressed serine proteases are recently duplicated paralogs with high levels of amino acid identity. Consistent with redundancy, no effects were observed on female fecundity or fertility when Send1-GAL4 was used to drive RNAi efficiently targeting Send1 or Send2 transcripts (Schnakenberg, 2011).

To fully understand how SSC-expressed proteases contribute to female reproductive function might require the simultaneous knockdown or knockout of many genes. As an alternative approach, the Send1-GAL4 and Send2-GAL4 drivers were used to ablate the SSC at different times, thereafter eliminating their ability to secrete any products into the spermathecal lumen. As the SSC are terminally differentiated adult cells, the drivers were used to express a modified form of the apoptosis-promoting protein Hid (HidAla5) that is effective in postmitotic cells (Schnakenberg, 2011).

Sperm storage was examined in SSC-ablated and control females by individually mating them with males expressing protamine-GFP, which renders sperm heads fluorescent green. Males transfer between 3,000 and 4,000 sperm during mating, of which only ~25% are stored. Of the stored sperm, approximately 65% to 80% reside in the seminal receptacle and the rest reside in the spermathecae. Mortality of stored sperm remains quite low for about 2 wk. In control +/UAS-hidAla5; Sp/+; Send1-nRFP/+ females, sperm are stored in the spermathecae and seminal receptacle within 1 h of mating, although sperm are also found in the uterus and occasionally the oviduct. In their +/UAS-hidAla5; CyO, Send1-GAL4/+; Send1-nRFP/+ sisters, whose SSC were ablated prior to mating, sperm are also found in the seminal receptacle and uterus, and occasionally in the oviduct, but often are not present in the spermathecae. Indeed, out of 17 SSC-ablated females, 16 had at least one empty spermatheca, and eight of these had both spermathecae empty. By contrast, no control female had even one empty spermatheca, and in only one case did one of the spermathecae contain fewer than ten sperm. In cases of SSC-ablated females in which sperm were found in one of the spermathecae but not the other, the presence or absence of sperm correlated with the presence or absence of SSC in a mosaic female. These results imply that the SSC are required to recruit sperm to the spermathecae (Schnakenberg, 2011).

It was previously shown that glucose dehydrogenase, which is secreted from the proximal and distal ends of the spermathecal duct, promotes recruitment of sperm into the spermathecae. Some cases were observed in which sperm were present in the spermathecal duct leading to a spermathecal cap with no sperm, but in most cases the ducts were empty of sperm as well. This result suggests that recruitment of sperm by the duct cells is largely dependent on SSC function (Schnakenberg, 2011).

By 7 h postmating, nearly all remaining sperm in control females are in the seminal receptacle or spermathecae, as expected. By contrast, SSC-ablated females have sperm in their seminal receptacles, yet tend to lack sperm in the spermathecae. The lack of sperm in the spermathecae at 7 h suggests that sperm recruitment to the spermathecae is indeed impaired and not merely delayed (Schnakenberg, 2011).

At 24 h postmating, sperm storage in control females appears very similar to that observed at 7 h postmating. In SSC-ablated females, however, sperm dynamics in the seminal receptacle are aberrant. Whereas in control females, sperm are found throughout the tubular receptacle, in many SSC-ablated females, sperm have lost motility and clumped together in one part of the receptacle, leaving the rest of it largely empty. This result suggests that the products of the SSC travel to, and act in, the seminal receptacle. Consistent with this inference, females lacking entirely the spermathecae and female accessory glands lose fertility within a few days after mating. The loss of fertility is apparently caused by loss of motility of sperm stored in the females' seminal receptacles. These results localize the source of at least one motility-maintaining factor to the SSC (Schnakenberg, 2011).

At 6 to 9 d postmating, sperm storage in control females still appears very similar to that observed at 7 h or 24 h postmating. Likewise, sperm storage at 6 to 9 d postmating in SSC-ablated females appears very similar to that observed in SSC-ablated females at 24 h postmating. Although rare females contain a few sperm in their spermathecae, most do not . As at 24 h postmating, clumps of sperm in the seminal receptacle are also seen in some females (Schnakenberg, 2011).

The existence of a few sperm in the spermathecae of some SSC-ablated females approximately 1 wk postmating suggests that the SSC are not required to retain sperm in the spermathecae once they have been stored there. However, as described above, there is a correlation between sperm recruitment to the spermathecae and the existence of residual, nonablated SSC. It could be that the same residual SSC function that recruited the sperm to the spermathecae is sufficient to retain them there. To test definitively whether the SSC are required to retain sperm in the spermathecae, the SSC were eliminated after sperm were stored in the spermathecae. This was done by ablating the SSC after mating using Send2-GAL4 in combination with UAS-hidAla5 (Schnakenberg, 2011).

At 7 h postmating, sperm storage in control +/UAS-hidAla5; Send1-nRFP/+; MKRS/+ females does not appear different to that of their +/UAS-hidAla5; Send1-nRFP/+; Send2-GAL4/+ sisters. As noted above, SSC ablation is complete in the latter females within one more day. If the SSC are required for long-term sperm retention in the spermathecae, then SSC-ablated females should lose the sperm they had stored in the spermathecae. However, this is not the case. At 6-8 d postmating, SSC-ablated females retain as many sperm in their spermathecae as do their control sisters. Moreover, sperm clumping in the seminal receptacle is not observed in these SSC-ablated females, implying that whatever SSC products are required to maintain sperm motility need only be supplied up to or around the time of mating, not continually (Schnakenberg, 2011).

Because females whose SSC are ablated prior to mating do not store sperm in their spermathecae and lose sperm motility in their seminal receptacles, it was next asked whether this impaired sperm storage affects fecundity or fertility. Females with SSC ablated prior to mating lay as many eggs on days 1 to 3 postmating as their control sisters. However, after day 3, their egg laying is significantly reduced. Notably, after day 3 an individual SSC-ablated female tends to lay vastly different numbers of eggs on successive days. Indeed, 10 out of 18 SSC-ablated females had one day in which 0 or 1 egg was laid, followed immediately by a day with greater than ten eggs laid. By contrast, only one out of 15 control females had any day in which 0 or 1 was laid (Schnakenberg, 2011).

The alternation of low and normal levels of egg laying in SSC-ablated females suggests that the SSC play some role in either ovulation or oviposition. To determine which is the case, SSC-ablated females that had not laid an egg in the previous 24 h were dissected. Strikingly, SSC-ablated females are ovoviviparous: a large proportion of such females (eight out of 16) had a late-stage embryo or live first-instar larva stuck in the uterus. This result implies a defect in ejecting eggs from the uterus (oviposition) rather than egg production and release (ovulation). However, those females with stuck eggs did not appear to have a 'log jam' of eggs in the oviduct, suggesting either: (1) that inability to eject a fertilized egg signals back to the ovary to halt or slow ovulation; or (2) that ovulation is independently slowed by SSC loss (Schnakenberg, 2011).

A simple, mechanical explanation for the stuck-egg phenotype is that the SSC produce a lubricant that coats the uterus, allowing eggs to pass easily. An alternative explanation is that products of the SSC are required before or around the time of mating to trigger cellular and physiological changes in the reproductive tract that are required for full reproductive maturity. Consistent with the latter explanation, females whose SSC have been ablated postmating, using Send2-Gal4, do not show any difference from control sisters in the number of eggs laid on each of days 1 to 8 postmating. This result implies that proper egg laying after day 3 postmating requires SSC function earlier in adulthood. The earlier function could be the production of a secretion, such as a lubricant, that is long-lived. However, multiple lines of evidence support the existence of posteclosion and postmating developmental programs by which the female reproductive tract achieves full maturity. The triggering of such a program by one or more SSC gene products could explain not only the egg-laying defect but also the loss of sperm motility in the seminal receptacle in females with Send1-driven, but not Send2-driven, ablation of the SSC (Schnakenberg, 2011).

Work prior to this had suggested several possible functions for the secretory cells of the spermathecae, including recruitment and maintenance of sperm, yet direct in vivo evidence was lacking because of the absence of tools for precisely manipulating these cells. In D. melanogaster, it had been observed that females lacking the spermathecae and accessory glands lose fertility, despite having normal seminal receptacles, but this effect could not be ascribed to any particular cell population within the missing organs. These cell-specific drivers enabled determination that the SSC contribute to reproductive function in multiple ways, some expected and some not. The SSC do indeed produce one or more products required for recruiting sperm into storage, although they are not required to maintain sperm in the spermathecae once recruited. In contrast, the SSC are not required for sperm to reach the seminal receptacle, but they are required to maintain sperm motility there, consistent with the lost fertility of females lacking spermathecae and accessory glands. In addition to their action at a distance in the seminal receptacle, the SSC also act at a distance in sustaining egg laying. The impairment of egg laying in SSC-ablated females manifests as an unanticipated phenotype: ovoviviparity. SSC-ablated females retain fertilized eggs, which develop inside the uterus and, in some cases, hatch as larvae inside the mother (Schnakenberg, 2011).

These findings have relevance to two evolutionary patterns observed in the genus Drosophila. First, all Drosophila species that have been examined have a pair of spermathecae and a single seminal receptacle, yet there are at least 13 independent Drosophila lineages in which females use only the seminal receptacle to store sperm. In species that do not store sperm in the spermathecae, the spermathecal caps are small and weakly sclerotized, but are surrounded by large cells that are presumably their SSC. The finding that the SSC act at a distance in the female reproductive tract might explain why these species retain their spermathecae, despite not using them to store sperm (Schnakenberg, 2011).

Second, the ovoviviparity observed in SSC-ablated females suggests that transitions to live birth might require fewer evolutionary steps than once thought. In a surprising recent discovery it was found that two species of Drosophila are ovoviviparous: even when exposed to ample substrate for oviposition, females of D. sechellia and D. yakuba retain fertilized eggs, which develop internally, in contrast to those of all other examined Drosophila species, which are laid immediately after fertilization (Markow, 2009). SSC-ablation results suggest that the Drosophila uterus is 'preadapted' to support internal embryo development, in that eggs stuck there are capable of hatching into perfectly viable larvae (Schnakenberg, 2011).

The genetic tools that this study has developed will be useful for further dissection of the molecular and evolutionary mechanisms underlying female reproductive function. Insect reproductive secretions are of particular interest because of their relevance to the fertility of agricultural pests and human disease vectors. To date, attention has focused on male secretions, because of the far greater knowledge of male seminal proteins than of products of the female reproductive tract, but recent studies in malaria-vector mosquitoes have begun to counter this bias. Reproductive secretions are also highly relevant to the study of beneficial insects, as evidenced by recent work characterizing the seminal-fluid and spermathecal-fluid proteomes of honey bees. Increased knowledge of the regulation and functions of spermathecal secretions will add a new dimension both to insect-control efforts and to the maintenance of healthy breeding populations of agriculturally important insects (Schnakenberg, 2011).

Severe fertility effects of sheepish sperm caused by failure to enter female sperm storage organs in Drosophila melanogaster

In Drosophila, mature sperm are transferred from males to females during copulation, stored in the sperm storage organs of females, and then utilized for fertilization. This study reports a gene named sheepish (shps) of D. melanogaster that is essential for sperm storage in females. shps mutant males, although producing morphologically normal and motile sperm that are effectively transferred to females, produce very few offspring. Direct counts of sperm indicated that the primary defect was correlated to failure of shps sperm to migrate into the female sperm storage organs. Increased sperm motion parameters were seen in the control after transfer to females, whereas sperm from shps males have characteristics of the motion parameters different from the control. The few sperm that occasionally entered the female sperm storage organs showed no obvious defects in fertilization and early embryo development. The female post-mating responses after copulation with shps males appeared normal at least with respect to conformational changes of uterus, mating plug formation and female remating rates. The shps gene encodes a protein with homology to amine oxidases, including as observed in mammals, with a transmembrane region at the C-terminal end. The shps mutation was characterized by a nonsense replacement in the third exon of CG13611 and shps was rescued by transformants of the wild-type copy of CG13611 Thus, shps may define a new class of gene responsible for sperm storage (Tomaru, 2017).

Cleavage of the Drosophila seminal protein Acp36DE in mated females enhances its sperm storage activity

Sperm storage in the mated female reproductive tract (RT) is required for optimal fertility in numerous species with internal fertilization. In Drosophila melanogaster, sperm storage is dependent on female receipt of seminal fluid proteins (SFPs) during mating. The seminal fluid protein Acp36DE is necessary for the accumulation of sperm into storage. In the female RT, Acp36DE localizes to the anterior mating plug and also to a site in the common oviduct, potentially "corralling" sperm near the entry sites into the storage organs. Genetic studies showed that Acp36DE is also required for a series of conformational changes of the uterus that begin at the onset of mating and are hypothesized to move sperm towards the entry sites of the sperm storage organs. After Acp36DE is transferred to the female RT, the protein is cleaved by the astacin-metalloprotease Semp1. However, the effect of this cleavage on Acp36DE's function in sperm accumulation into storage is unknown. This study used mass spectrometry to identify the single cleavage site in Acp36DE. This site was then mutated and the effects on sperm storage were tested. Mutations of Acp36DE's cleavage site that slowed or prevented cleavage of the protein slowed the accumulation of sperm into storage, although they did not affect uterine conformational changes in mated females. Moreover, the N-terminal cleavage product of Acp36DE was sufficient to mediate sperm accumulation in storage, and it did so faster than versions of Acp36DE that could not be cleaved or were only cleaved slowly. These results suggest that cleavage of Acp36E may increase the number of bioactive molecules within the female RT, a mechanism similar to that hypothesized for Semp1's other substrate, the seminal fluid protein ovulin (Avila, 2017).

Dynamic Notch signaling specifies each cell fate in Drosophila spermathecal lineage

Spermathecae are glandular organs in insect female reproductive tract and play essential roles for insect reproduction; however, the molecular mechanism involved in their development is largely unknown. Drosophila spermathecae consist of class-III secretory units, in which each secretory cell discharges its products to the central lumen through an end-apparatus and a canal. Secretory unit formation in Drosophila spermathecae utilizes a fixed cell lineage, in which each secretory unit precursor (SUP) divides to produce one pIIb cell and one pIIa cell. The former differentiates into an apical cell (AC), whereas the latter divides again to produce a secretory cell (SC) and a basal cell (BC). It is unclear how each cell acquires its identity and contributes to secretory unit formation. This study demonstrates that Notch signaling is required and sufficient for the specification of lumen epithelial precursors (LEPs; vs. SUPs), pIIb (vs. pIIa), and SCs (vs. BCs) sequentially. Notch activation in LEPs and SCs apparently utilizes different ligand mechanism. In addition, Notch signaling both suppresses and activates transcription factors Hindsight (Hnt) and Cut during spermathecal lineage specification, supporting the notion that Notch signaling can have opposite biological outcomes in different cellular environment. Furthermore, LEP-derived epithelial cells (ECs) and ACs show distinct cellular morphology and are essential for securing secretory units to the epithelial lumen. These data demonstrate for the first time the dynamic role of Notch signaling in binary cell fate determination in Drosophila spermathecae and the role of ECs and ACs in secretory unit formation (Shen, 2017).

Mechanical stimulation by osmotic and hydrostatic pressure activates Drosophila oocytes in vitro in a calcium-dependent manner

Embryogenesis in vertebrates and marine invertebrates begins when a mature oocyte is fertilized, resulting in a rise in intracellular calcium (Ca2+) that activates development. Insect eggs activate without fertilization via an unknown signal imparted to the egg during ovulation or egg laying. One hypothesis for the activating signal is that deformation of eggs as they pass through a tight orifice provides a mechanical stimulus to trigger activation. Ovulation could produce two forms of mechanical stimulus: external pressure resulting from the passage of oocytes from the ovary into the narrow oviducts, and osmotic pressure caused by hydration-induced swelling of the oocyte within the oviducts. Ovulation could also trigger activation by placing the oocyte in a new environment that contains an activating substance, such as a particular ion. This study provide the first evidence that Drosophila oocytes require Ca2+ for activation, and that activation can be triggered in vitro by mechanical stimuli, specifically osmotic and hydrostatic pressure. The results suggest that activation in Drosophila is triggered by a mechanosensitive process that allows external Ca2+ to enter the oocyte and drive the events of activation. This will allow exploitation of Drosophila genetics to dissect molecular pathways involving Ca2+ and the activation of development (Horner, 2008).

Mature oocytes require an external signal to begin development. This signal, which differs among animals, 'activates' the oocyte to resume and complete meiosis, modify its outer coverings, reorganize its cytoskeleton, and translate or degrade certain maternal mRNAs. In most animals, activation is triggered by fertilization, but changes in the ionic environment, changes in pH, or mechanical deformation can initiate egg activation in some species. A frequent response to the activating trigger in vertebrates and marine invertebrates is a rise in free calcium within the egg. In these organisms calcium acts as a second messenger to drive the downstream processes of egg activation. In insects, the requirement for calcium during egg activation has never been directly tested; however, recent reports show that a calcium-responsive regulator, Sarah, is essential for egg activation in Drosophila melanogaster (Horner, 2006l; Takeo, 2006; Horner, 2008 and references therein).

Drosophila egg activation, as in other insects that have been examined, is independent of fertilization. Unfertilized laid eggs can complete meiosis, modify their vitelline membranes, and translate some maternal RNAs. Thus, Drosophila sperm trigger none of the traditional metrics of egg activation. Instead, activation initiates during ovulation but the activating signal itself remains unknown (Horner, 2008).

One hypothesis for the activating signal in Drosophila derives from studies of Hymenoptera in which embryo development is triggered by oviposition. It has been proposed that mechanical stress imparted upon the egg during passage through the ovipositor is the signal that starts development in Hymenoptera. For instance, in the haplodiploid wasp, Pimpla turionellae, the diameter of the ovipositor is about one-third of the width of the egg, suggesting that physical deformation during egg laying initiates development. Consistent with this hypothesis, when P. turionellae eggs are dissected from the ovary and squeezed through a narrow capillary tube, over 70% of eggs activate, as measured by their ability to develop into haploid male larvae. Pressure exerted on the egg can also activate eggs of another wasp, Nasonia vitripennis, since 23% of eggs dissected from the ovary and pressed with a needle were able to develop to larvae (Horner, 2008 and references therein).

The hypothesis that mechanical stimulation could also trigger Drosophila egg activation was initially suggested by two observations. First, it has been shown that application of hydrostatic pressure of an unspecified level or duration to Drosophila oocytes resulted in an increase in nuclear number in those oocytes. Whether such oocytes had properly completed meiosis and were undergoing haploid mitotic divisions was not reported. Second, it has been reported that pulling manually on the dorsal chorionic appendages of Drosophila oocytes triggered the resumption of meiosis in 3/3 cases. These intriguing observations suggested that mechanical stimulation might trigger the resumption and completion of meiosis, one aspect of egg activation. No other aspects of egg activation in response to mechanical stimulation were examined in those studies (Horner, 2008 and references therein).

Analogous to oviposition in wasps, a mechanical trigger might occur as Drosophila eggs move from the ovary into the narrow lateral oviduct during ovulation. Mechanical stimulation could rearrange egg contents, leading to new structural or molecular combinations. Alternatively, mechanical stimulation could stimulate a mechanically-gated (MG) process, such as the opening or closing of stretch-activated (SA) ion channels. Such alterations to ion channels could lead to ionic changes analogous to those that trigger egg activation in other metazoans (Horner, 2008).

Another potential activation trigger in Drosophila is hydration. Mature oocytes in the ovary appear desiccated, whereas laid eggs are taut and expanded. Some evidence suggests that the hydrated contents of the oviduct lumen are transferred to eggs during ovulation. Support for a hypothesis that hydration could lead to egg activation is that incubation in a hypotonic buffer in vitro causes oocytes to swell and activate. Such hypo-osmotic swelling in vivo or in vitro could serve as another form of mechanical stimulation, by altering membrane tension to trigger a MG process. Additionally, specific ion(s) in the hydrating medium could provide the activation signal (Horner, 2008).

To better understand the activating signal in Drosophila, whether pressure exerted on the egg effects activation was determined. It was found that external hydrostatic pressure accelerates activation, as assessed by vitelline membrane permeability changes and protein translation. In addition, an inhibitor of MG processes was able to inhibit hypo-osmotically induced activation, suggesting for the first time that the mechanism by which hydration leads to activation is through a MG response triggered by osmotic pressure. External calcium was shown to be necessary for both hypo-osmotic and pressure-accelerated activation. Therefore this study demonstrates that the phenomenon of calcium-dependent egg activation extends to a new and important class of metazoans: insects. Taken together, these results suggest that mechanical stimulation from hydration and/or physical deformation during ovulation triggers activation in Drosophila by causing an influx of calcium into the egg. Drosophila is now poised to join organisms traditionally used to study activation, with the advantage of valuable genetic resources to discover the likely conserved pathways that mediate egg activation (Horner, 2008).

Calcium waves occur as Drosophila oocytes activate

Egg activation is the process by which a mature oocyte becomes capable of supporting embryo development. In vertebrates and echinoderms, activation is induced by fertilization. Molecules introduced into the egg by the sperm trigger progressive release of intracellular calcium stores in the oocyte. Calcium wave(s) spread through the oocyte and induce completion of meiosis, new macromolecular synthesis, and modification of the vitelline envelope to prevent polyspermy. However, arthropod eggs activate without fertilization: in the insects examined, eggs activate as they move through the female's reproductive tract. This study shows that a calcium wave is, nevertheless, characteristic of egg activation in Drosophila. The calcium rise required influx of calcium from the external environment and was induced as the egg ovulated. Pressure on the oocyte (or swelling by the oocyte) could induce a calcium rise through the action of mechanosensitive ion channels. Visualization of calcium fluxes in activating eggs in oviducts showed a wave of increased calcium initiating at one or both oocyte poles and spreading across the oocyte. In vitro, waves also spread inward from oocyte pole(s). Wave propagation required the IP3 system. Thus, although a fertilizing sperm was not necessary for egg activation in Drosophila, the characteristic of increased cytosolic calcium levels spreading through the egg was conserved. Because many downstream signaling effectors are conserved in Drosophila, this system offers the unique perspective of egg activation events due solely to maternal components (Kaneuchi, 2015).

Egg activation is a conserved phenomenon that prepares an animal oocyte for successful embryogenesis through completion of meiosis, restructuring of the vitelline membrane, and changes to the existing protein and mRNA pools within the egg. The trigger for Drosophila (and other arthropod) egg activation differs from the better-known cases of vertebrate and echinoderm egg activation in that it is decoupled from fertilization. Despite this critical difference in egg activation trigger mechanisms, this study reports that a calcium wave occurs during egg activation in Drosophila, as in other animals. In vivo imaging of oocyte calcium levels indicates that the intracellular calcium rise is triggered by ovulation. In vitro imaging shows that this rise takes the form of wave(s) that initiate from egg pole(s) and move across the egg; the rise in cytosolic calcium is then followed by a decrease. It is proposed that this dynamic rise and fall in cytosolic calcium triggers the events of egg activation in Drosophila, as suggested for the calcium transients in other organisms such as mouse (Kaneuchi, 2015).

The calcium rise during Drosophila egg activation can occur only in the presence of calcium in the extracellular environment. It is proposed that the wave initiates when Ca2+ enters the oocyte through activation of mechanosensitive ion channels on the oocyte cell surface. These channels are proposed to be activated by either or both of the following mechanisms. First, the oocyte swells as it passes through the oviducts, presumably by taking up fluid: mature oocytes in the ovary are shriveled in appearance, but laid eggs are swollen and taut. In in vitro experiments, it was noted that the calcium wave does not initiate until after the egg has begun to swell. Additionally, it was possible to increase the speed of initiation by adding a few drops of water to the activating medium during imaging, thus increasing hypotonicity and causing faster egg swelling. It is postulated that swelling exerts a stretch tension force on the membrane, which triggers the opening of mechanosensitive Ca2+ channels. Second, it was shown that, independently of oocyte swelling, mechanical pressure exerted on the oocyte is capable of initiating the wave. It is proposed that oocytes may be subjected to both triggers during ovulation: pressure from the outside as they move out of the ovary and into the oviducts and swelling as they encounter the oviductal fluid. As a result, mechanosensitive ion channels open, and calcium levels rise in the oocyte. In vivo imaging of oocytes as they are ovulating supports the pressure hypothesis: the movement of the oocyte into the oviduct is not smooth and fluid; instead, the oocyte moves slowly at first and then rather suddenly pushes into the oviduct, as if it meets some resistance force as it begins ovulation (Kaneuchi, 2015).

Recent evidence from mice indicates that a requirement for external Ca2+ for egg activation is not unique to insects like Drosophila, although a requirement for calcium influx to initiate the first wave is. In mice, after the initial Ca2+ rise induced by sperm PLC, further calcium oscillations require Ca2+ uptake from the extracellular environment through a store-operated Ca2+ entry mechanism; when intracellular ER Ca2+ stores are depleted, plasma membrane channels open to allow Ca2+ back into the cell (Kaneuchi, 2015).

How could a wave be triggered from the egg pole(s)? It is possible that the mechanosensitive ion channels that mediate the calcium rise are localized at the poles, analogous to the localization of some of the embryo-polarity machinery including a terminal-group signaling cascade that marks the two ends of the embryo as similar to one another but different from the interior. In this model, mechanical cues applied to the egg would activate those channels, and because they are at the poles, the wave would initiate at the poles. It will be intriguing to test this hypothesis by determining which mechanosensitive ion channels are needed to trigger the calcium wave (and egg activation) and whether they show polar localization. Toward this end, the finding that the wave is inhibited by gadolinium and ACA suggests that the relevant channels might be members of the TRP family of calcium channels. The best candidates are three TRP family channels that are expressed in the ovary [painless (TRPA1), trpm (TRPM3), and trpml (TRPP1/Pkd2)]. Further experiments will be needed to determine the particular channel(s) that is needed to initiate the wave and its localization in the oocyte membrane. Alternatively, it is possible that the required channels are not localized but rather that the egg cytoskeleton is less rigid on the poles of the egg. Normally, uniform swelling of a prolate spheroid (such as a Drosophila oocyte) would exert greater tension along the center or waistline. However, different cytoskeletal makeup at the poles may cause tension to be experienced differently there, and in this way channels spread uniformly throughout the plasma membrane may open first at the poles. Further experiments will be required to determine why the wave initiates at the poles (Kaneuchi, 2015).

In other organisms in which the signaling pathway has been studied downstream of the Ca2+ influx, an increase in intracellular Ca2+ is thought to be the ultimate cause of the meiosis resumption that permits subsequent embryonic mitosis, and of changes in macromolecular synthesis or stability. However, the way in which these events are connected to the calcium wave is still unclear in any system. This study has shown that a calcium wave occurs during Drosophila egg activation and that the sensitivity of this wave to manipulations (pressure, swelling, chemical inhibitors) mirrors that for egg activation events. Given this finding, and the fact that many signaling pathways and events downstream of the calcium signal appear to be conserved between Drosophila other species, Drosophila will offer the unique perspective of isolating egg activation events from fertilization events, as well the possibility of genetic manipulation and larger-scale ‘omics' studies that will help to link a Ca2+ flux to downstream egg activation events (Kaneuchi, 2015).

The coevolutionary period of Wolbachia pipientis infecting Drosophila ananassae and its impact on the evolution of the host germline stem cell regulating genes

The endosymbiotic bacteria Wolbachia pipientis is known to infect a wide range of arthropod species yet less is known about the coevolutionary history it has with its hosts. Evidence of highly identical W. pipientis strains in evolutionary divergent hosts suggests horizontal transfer between hosts. For example, Drosophila ananassae is infected with a W. pipientis strain that is nearly identical in sequence to a strain that infects both D. simulans and D. suzukii, suggesting recent horizontal transfer among these three species. However, it is unknown whether the W. pipientis strain had recently invaded all three species or a more complex infectious dynamic underlies the horizontal transfers. This study examined the coevolutionary history of D. ananassae and its resident W. pipientis to infer its period of infection. Phylogenetic analysis of D. ananassae mitochondrial DNA and W. pipientis DNA sequence diversity revealed the current W. pipientis infection is not recent. In addition, the population genetics and molecular evolution of several Germline Stem Cell (GSC) regulating genes of D. ananassae were examined. These studies reveal significant evidence of recent and long-term positive selection at stonewall in D. ananassae, while pumillio showed patterns of variation consistent with only recent positive selection. Previous studies had found evidence for adaptive evolution of two key germline differentiation genes, bag of marbles (bam) and benign gonial cell neoplasm (bgcn), in D. melanogaster and D. simulans, and it was proposed that the adaptive evolution at these two genes was driven by arms race between the host GSC and W. pipientis. However, this study did not find any statistical departures from a neutral model of evolution for bam and bgcn in D. ananassae despite the new evidence that this species has been infected with W. pipientis for a period longer than the most recent infection in D. melanogaster. In the end analyzing the GSC regulating genes individually showed two out of the seven genes to have evidence of selection. However, combining the dataset and fitting a specific population genetic model, significant proportion of the nonsynonymous sites across the GSC regulating genes were driven to fixation by positive selection. Clearly the GSC system is under rapid evolution and potentially multiple drivers are causing the rapid evolution (Choi, 2014).

Populations of follicle cells

The Drosophila egg is an intricately patterned structure with distinct specializations and polarities. These features are critical to subsequent embryonic development because the polarities of the egg are transmitted to the embryo, establishing the initial pattern in a developing zygote. The pattern of the mature egg is established by complex cellular interactions among and between both somatic follicle cells and germline cells. Each egg begins as a 16-cell germline cyst, from which one cell will become the oocyte and the remainder will become the supporting nurse cells. In the germarium, the anterior structure in which oogenesis is initiated, the germline cyst, is surrounded by a monolayer of somatic follicle cell precursors. As the encapsulated cyst exits from the germarium, approximately 10-14 of the somatic cells cease proliferation and differentiate. This group of cells forms two distinct populations: two polar cells at the anterior and posterior poles of each chamber and approximately seven stalk cells that form a bridge between the consecutive cysts. As the cyst exits the germarium, the other somatic cells covering each chamber, the epithelial follicle cells, remain undifferentiated (Xi, 2003 and references therein).

After pinching off from the germarium, each germline cyst grows, while the epithelial follicle cells proliferate. During this time, the anterior-posterior polarity that will ultimately determine all of the epithelial follicular fates is established. Elegant experiments have shown that the underlying prepattern of the follicular epithelium displays mirror image symmetry at the termini in the anterior-posterior (A/P) axis. Cells adopt one of three anterior terminal fates [border, stretched, and centripetal cells (terminal to central)], depending on proximity to the poles. In the intervening region between the terminal domains, cells will adopt a default 'main body' identity, and the posterior terminal cells form nearest the posterior pole. The symmetry of the A/P pattern is broken by EGFR signaling at the posterior. Secreted Grk from the posteriorly localized oocyte activates EGFR on the overlying follicle cells, establishing posterior terminal fate. In the absence of EGFR signaling, the anterior pattern is repeated at the posterior (Xi, 2003 and references therein).

By stage 7, the epithelial follicle cells cease proliferation and enter an endocycle. Afterward, these cells begin to show morphological and molecular signs of differentiation into the five epithelial fates: border, stretched, centripetal, posterior, and main body cells. Each of these subpopulations of follicle cells has a specific function with respect to the production of a mature egg, such that the correct number and position of each type is critical to ultimate egg morphology. These functions inluence the production of structures that are essential to the egg, such as the dorsal respiratory appendages and the micropyle. These functions are also critical for proper anterior-posterior organization of the oocyte and, therefore, also for the resulting embryo (Xi, 2003 and references therein).

Epithelial rotation promotes the global alignment of contractile actin bundles during Drosophila egg chamber

Tissues use numerous mechanisms to change shape during development. The Drosophila egg chamber is an organ-like structure that elongates to form an elliptical egg. During elongation the follicular epithelial cells undergo a collective migration that causes the egg chamber to rotate within its surrounding basement membrane. Rotation coincides with the formation of a 'molecular corset', in which actin bundles in the epithelium and fibrils in the basement membrane are all aligned perpendicular to the elongation axis. This study shows that rotation plays a critical role in building the actin-based component of the corset. Rotation begins shortly after egg chamber formation and requires lamellipodial protrusions at each follicle cell's leading edge. During early stages, rotation is necessary for tissue-level actin bundle alignment, but it becomes dispensable after the basement membrane is polarized. This work highlights how collective cell migration can be used to build a polarized tissue organization for organ morphogenesis (Cetera, 2014).

Rab10-mediated secretion synergizes with tissue movement to build a polarized basement membrane architecture for organ morphogenesis

Basement membranes (BMs) are planar protein networks that support epithelial function. Regulated changes to BM architecture can also contribute to tissue morphogenesis, but how epithelia dynamically remodel their BMs is unknown. In Drosophila, elongation of the initially spherical egg chamber correlates with the generation of a polarized network of fibrils in its surrounding BM. This study used live imaging and genetic manipulations to determine how these fibrils form. BM fibrils are assembled from newly synthesized proteins in the pericellular spaces between the egg chamber's epithelial cells and undergo oriented insertion into the BM by directed epithelial migration. It was found that a Rab10-based secretion pathway promotes pericellular BM protein accumulation and fibril formation. Finally, by manipulating this pathway, it was shown that BM fibrillar structure influences egg chamber morphogenesis. This work highlights how regulated protein secretion can synergize with tissue movement to build a polarized BM architecture that controls tissue shape (Isabella, 2016).

Influence of ovarian muscle contraction and oocyte growth on egg chamber elongation in Drosophila

Organs are formed from multiple cell types that make distinct contributions to their shape. The Drosophila egg chamber provides a tractable model to dissect such contributions during morphogenesis. Egg chambers are comprised of 16 germ cells (GCs) surrounded by a somatic epithelium. Initially spherical, these structures elongate as they mature. This morphogenesis is thought to occur through a "molecular corset" mechanism, wherein structural elements within the epithelium become circumferentially organized perpendicular to the elongation axis and resist the expansive growth of the GCs to promote elongation. Whether this epithelial organization provides the hypothesized constraining force has been difficult to discern, however, and a role for GC growth has not been demonstrated. This study provides evidence for this mechanism by altering the contractile activity of the tubular muscle sheath that surrounds developing egg chambers. Muscle hypo-contraction indirectly reduces GC growth and shortens the egg, which demonstrates the necessity of GC growth for elongation. Conversely, muscle hyper-contraction enhances the elongation program. Although this is an abnormal function for this muscle, this observation suggests that a corset-like force from the egg chamber's exterior could promote its lengthening. These findings highlight how physical contributions from several cell types are integrated to shape an organ (Andersen, 2016).

Coupling of Hedgehog and Hippo pathways promotes follicle stem cell maintenance by stimulating proliferation

It is essential to define the mechanisms by which external signals regulate adult stem cell numbers, stem cell maintenance, and stem cell proliferation to guide regenerative stem cell therapies and to understand better how cancers originate in stem cells. This paper shows that Hedgehog (Hh) signaling in Drosophila melanogaster ovarian follicle stem cells (FSCs) induces the activity of Yorkie (Yki), the transcriptional coactivator of the Hippo pathway, by inducing yki transcription. Moreover, both Hh signaling and Yki positively regulate the rate of FSC proliferation, both are essential for FSC maintenance, and both promote increased FSC longevity and FSC duplication when in excess. It was also found that responses to activated Yki depend on Cyclin E induction while responses to excess Hh signaling depend on Yki induction, and excess Yki can compensate for defective Hh signaling. These causal connections provide the most rigorous evidence to date that a niche signal can promote stem cell maintenance principally by stimulating stem cell proliferation (Huang, 2014).

Gene amplification in follicle cells: Isolation of developmental amplicons

Gene amplification is known to be critical for upregulating gene expression in a few cases, but the extent to which amplification is utilized in the development of diverse organisms remains unknown. By quantifying genomic DNA hybridization to microarrays to assay gene copy number, two additional developmental amplicons, termed DAFC (Drosophila Amplicon in Follicle Cells)-30B and -62D were identified in the follicle cells of the Drosophila ovary. Both amplicons contain genes which, following their amplification, are expressed in the follicle cells, and the expression of three of these genes becomes restricted to specialized follicle cells late in differentiation. Genetic analysis establishes that at least one of these genes, yellow-g, is critical for follicle cell function, because mutations in yellow-g disrupt eggshell integrity. Thus, during follicle cell differentiation the entire genome is overreplicated as the cells become polyploid, and subsequently specific genomic intervals are overreplicated to facilitate gene expression (Claycomb, 2004).

The maximally amplified genes in DAFC-62D, yellow-g and yellow-g2, are members of the yellow gene family that are predicted to encode secreted proteins. The family shares homology with the Major Royal Jelly Protein Family in honeybees (Apis mellifera), involved in the specification of the queen bee. The founding member of the Yellow family, Yellow-y, is known to play a role in mating behavior and in the melanization and hardening of the adult cuticle. Other Yellow family members have been shown to act as dopachrome-conversion enzymes that catalyze a key reaction in the melanization process. Interestingly, a similar process is used in the hardening of the egg chorion in mosquitoes and suggests that Yellow-g and Yellow-g2 may play a catalytic role in the crosslinking of the chorion and/or underlying vitelline membrane proteins in Drosophila (Claycomb, 2004).

A second group of genes encodes proteins with chitin binding motifs that could function in egg production. Genes of this type are present in both amplicons, with DAFC-62D containing two such genes and DAFC-30B containing one. Chitin binding domains serve an antimicrobial function in a variety of plants and marine invertebrates. Homologs of marine invertebrate proteins, such as tachycitin, could provide the egg with protection against microbes. Alternatively, chitin, a structural polysaccharide found in many organisms, could also be a component of the eggshell, and interaction with the chitin binding proteins might contribute to eggshell integrity (Claycomb, 2004).

In both DAFC-30B and 62D, there are also a number of genes whose role in follicle cells is not yet clear. These include both genes encoding proteins without known sequence motifs and genes whose products are predicted to have the enzymatic activities of adenylate cyclases, membrane transporters, calcium-transporting ATPases, GTP dissociation inhibitors, and others (Claycomb, 2004).

The yellow-g gene is essential for a rigid eggshell, and the predicted gene products of the yellow-g and yellow-g2 genes suggest a molecular explanation for these mutant defects. The eggshell is composed of several layers, including the outermost exochorion, the endochorion, the inner chorion layer, and the vitelline membrane, which is the innermost structure that also contacts the oocyte. The collapsed embryos and disrupted vitelline membranes that result from mutation of yellow-g indicate that yellow-g is necessary for the structural integrity of the eggshell. At the level of the light microscope, the exochorion of embryos laid by mutant mothers appears normal. The collapsed embryos are reminiscent of vitelline membrane defects, leading to the hypothesis that yellow-g is necessary for proper vitelline membrane formation (Claycomb, 2004).

It is proposed that Yellow-g and Yellow-g2 act to crosslink the vitelline membrane, or perhaps the inner chorion layer. The Yellow family members, Yellow-f and Yellow-f2, are capable of catalyzing the conversion of dopachrome to dihydroxyindole, a limiting step in the melanization pathway, during larval, pupal, and adult stages. The enzymatic events leading to the crosslinking of the vitelline membrane are not well understood, but seem to involve one phase of disulfide bond formation and a subsequent disulfide bond-independent phase. Additionally, the α methyl dopa resistant (amd) gene product, which acts in the conversion of dopamine during the polymerization of the adult cuticle, is required in the follicle cells for proper vitelline membrane crosslinking. This suggests that a similar set of dopamine conversion reactions catalyzed by Yellow-g and Yellow-g2 may be necessary for the crosslinking of the vitelline membrane just prior to egg laying. Consistent with this hypothesis, it is observed that eggs laid by homozygous yellow-g mutant females are highly sensitive to sodium hypochlorite (bleach), and the majority of these embryos burst upon brief exposure. Of the remaining, intact embryos, 100% were permeable to the dye neutral red, which has been used to assay vitelline membrane defects. These results are indicative of a failure to crosslink the vitelline membrane and further implicate yellow-g in the crosslinking process. However, this hypothesis does not explain the specific expression of the yellow-g and yellow-g2 genes in the follicle cells producing the micropyle late in egg chamber development. It is possible that crosslinking of the vitelline membrane or inner chorion layer within this specialized structure requires distinct regulation or timing. A more detailed analysis of the eggshell defect and biochemical studies of Yellow-g and Yellow-g2 will help gain a better understanding of the steps necessary for vitelline membrane crosslinking and will uncover any specialized micropyle functions (Claycomb, 2004).

DAFC-30B and DAFC-62D provide insights into the use of amplification as a developmental strategy. All of the previously characterized amplified genes play a purely structural role in eggshell formation; no enzymes necessary for proper eggshell formation have been examined. None of the genes of DAFC-30B and DAFC-62D encode known structural components of the eggshell. However, several of the amplified genes that are highly expressed in follicle cells, including CG18419 and the yellow-g genes, encode products predicted to possess enzymatic, signal transduction, or transporting activities. Furthermore, at least yellow-g is essential for proper egg formation, thus revealing an additional function of amplification: to increase the levels of enzymes needed to catalyze developmentally important reactions. Thus the identification of additional amplicons highlights genes likely to be crucial in developmental events and opens the possibility that other tissues employ amplification to maximize gene expression during differentiation. It is surprising that a 4- to 6-fold increase in gene copy number would affect gene product levels in a developmentally significant manner. It is possible, however, that copy number increases are considerably higher in subsets of follicle cells, or that the replication process itself facilitates transcription (Claycomb, 2004).

The follicle cell amplicons serve as superb model metazoan replicons, permitting delineation of cis-regulatory elements, identification of replication proteins, and clarifying the developmental control of the initiation and elongation. Developmental distinctions between DAFC-62D and the previously studied DAFCs provide clues into how origin firing can be linked to developmental signals. It has been shown by real-time PCR that replication initiates at DAFC-66D and -7F, coupled with replication fork movement, during egg chamber stages 10B and 11. Subsequently (stages 12 and 13), origins cease firing and only existing replication forks move bidirectionally to produce a gradient of copy number that extends over 100 kb. Furthermore, the replication initiation factor ORC2 localizes to amplification origins only during the initiation phase and dissociates at the onset of the elongation phase. Replication factors involved in multiple steps of DNA replication, such as MCM2-7 and PCNA, colocalize with BrdU throughout amplification (Claycomb, 2004).

DAFC-62D behaves differently from these amplicons and from DAFC-30B. There is a final increase in copy number at a very precise region of the amplicon, about 1.5 kb downstream of yellow-g2, during stage 13. As it is the peak of amplification, this region is likely to possess a replication origin. Understanding how DAFC-62D can undergo a final initiation hours after ORC is no longer detectable at origins by immunofluorescence will provide insights into the control of replication initiation. The additional replication in stage 13 may occur in only subsets of follicle cells, and ORC could persist specifically at DAFC-62D in these cells. For example, additional gene copies could permit optimal levels of expression of the yellow-g genes in the follicle cells building the micropyle (Claycomb, 2004).

These studies were initiated to devise a systematic approach for finding developmental amplicons. The microarray assay is sensitive and can detect low levels of gene amplification, and amplification levels as low as 4-fold can be developmentally important. Thus, this approach will be invaluable in surveying for gene amplification in a number of tissues and in a variety of organisms where amplification has not been detected. Not only has the microarray strategy identified additional amplicons, but when coupled with the power of a genetic organism, it has proven to be a functional genomics approach for highlighting genes involved in specific developmental pathways (Claycomb, 2004).

DNA sequence templates adjacent nucleosome and ORC sites at gene amplification origins in Drosophila

Eukaryotic origins of DNA replication are bound by the origin recognition complex (ORC), which scaffolds assembly of a pre-replicative complex (pre-RC) that is then activated to initiate replication. Both pre-RC assembly and activation are strongly influenced by developmental changes to the epigenome, but molecular mechanisms remain incompletely defined. The activation of origins responsible for developmental gene amplification was examined in Drosophila. At a specific time in oogenesis, somatic follicle cells transition from genomic replication to a locus-specific replication from six amplicon origins. Previous evidence indicated that these amplicon origins are activated by nucleosome acetylation, but how this affects origin chromatin is unknown. This study examine nucleosome position in follicle cells using micrococcal nuclease digestion with Ilumina sequencing. The results indicate that ORC binding sites and other essential origin sequences are nucleosome-depleted regions (NDRs). Nucleosome position at the amplicons was highly similar among developmental stages during which ORC is or is not bound, indicating that being an NDR is not sufficient to specify ORC binding. Importantly, the data suggest that nucleosomes and ORC have opposite preferences for DNA sequence and structure. It is proposed that nucleosome hyperacetylation promotes pre-RC assembly onto adjacent DNA sequences that are disfavored by nucleosomes but favored by ORC (Liu, 2015).

Replication fork progression during re-replication requires the DNA damage checkpoint and double-strand break repair

Replication origins are under tight regulation to ensure activation occurs only once per cell cycle. Origin re-firing in a single S phase leads to the generation of DNA double-strand breaks (DSBs) and activation of the DNA damage checkpoint. If the checkpoint is blocked, cells enter mitosis with partially re-replicated DNA that generates chromosome breaks and fusions. It has been proposed that fork instability and DSBs formed during re-replication are the result of head-to-tail collisions and collapse of adjacent replication forks. This study utilized the Drosophila ovarian follicle cells, which exhibit re-replication under precise developmental control, to model the consequences of re-replication at actively elongating forks. Re-replication occurs from specific replication origins at six genomic loci, termed Drosophila amplicons in follicle cells (DAFCs). Precise developmental timing of DAFC origin firing permits identification of forks at defined points after origin initiation. This study shows that DAFC re-replication causes fork instability and generates DSBs at sites of potential fork collisions. Immunofluorescence and ChIP-seq demonstrate the DSB marker γH2Av is enriched at elongating forks. Fork progression is reduced in the absence of DNA damage checkpoint components and nonhomologous end-joining (NHEJ), but not homologous recombination. NHEJ appears to continually repair forks during re-replication to maintain elongation (Alexander, 2015).

Development of the dorsal appendages

Dorsal appendage morphogenesis in Drosophila oogenesis has been used as a model system for studying the relationship between patterning and morphogenesis. Each of the two dorsal respiratory appendages of the Drosophila egg chamber is formed by secretion of eggshell proteins into a tube of follicle cells. This tube is generated by cell shape changes and rearrangements within an epithelial sheet. Dorsal appendage formation is therefore similar to more complicated examples of organogenesis. In addition, the study of dorsal appendage formation provides several advantages that make it an excellent system for investigating the regulation of epithelial morphogenesis. For example, the signaling events that determine two populations of dorsal follicle cells are well understood. This understanding facilitates an ability to uncouple effects on patterning from morphogenesis. Further, powerful genetic tools, including mutations that disrupt dorsal appendage formation, have allowed for an unraveling of the genetic circuitry underlying the regulation of epithelial morphogenesis (French, 2003).

The Drosophila egg chamber contains 16 interconnected germline cells, consisting of 1 oocyte nourished by 15 highly polyploid nurse cells; these germline cells are surrounded by a monolayer of ~1000 somatic follicle cells. The follicle cells secrete the chorion that makes up the three layers of the eggshell: the vitelline envelope, the endochorion, and the exochorion. A subset of these follicle cells undergoes morphogenesis to generate the dorsal appendages, specialized structures that facilitate gas exchange in the developing embryo (French, 2003).

At stage 10 of oogenesis, the oocyte occupies the posterior half of the egg chamber, the nurse cells the anterior half, and the oocyte nucleus is positioned at the dorsal anterior corner of the oocyte. The majority of follicle cells forms a columnar layer over the oocyte, while a few follicle cells are stretched out over the nurse cells. During stage 10B, those follicle cells closest to the nurse cell/oocyte boundary begin to migrate centripetally, between nurse cells and oocyte. The centripetal cells secrete the operculum (a thin layer of chorion that functions as an escape hatch for the larva), the collar (a hinge on which the operculum swings), and the micropyle, a coneshaped structure through which the sperm enters (French, 2003).

Shortly after centripetal migration (stage 10B), the nurse cells rapidly transfer their contents into the oocyte (stage 11) then begin to degenerate and undergo apoptosis (stages 12-14). At the same time, two groups of approximately 65-80 anterior, dorsal follicle cells, one on each side of the dorsal midline of the egg chamber, migrate over the nurse cells, laying down the chorion of the two dorsal appendages. Extensive studies have defined the signaling events that determine two populations of dorsal follicle cells. Dorsal follicle-cell fate determination begins when transcripts encoding the TGFalpha-like signaling molecule Gurken (Grk) become localized in a cap above the oocyte nucleus. Grk signals via the epidermal growth factor receptor homolog (Egfr) to the follicle cells, activating a signal transduction cascade involving the Ras/Raf/MAPK pathway. This initial signaling event defines a set of dorsal anterior follicle cells and induces a second signaling cascade involving three additional Egfr ligands. This second cascade amplifies and refines the initial Grk signal, leading to the definition of two separate populations of dorsal follicle cells. These events are required for the production of two separate dorsal appendages. Disruptions of this process result in dorsalization or ventralization of the follicular epithelium and the eggshell. Partial ventralization generally results in failure to determine two separate populations of cells, leading to the production of a single dorsal appendage at the dorsal midline. Complete ventralization results in the absence of dorsal cell fates and the concomitant loss of dorsal appendages (French, 2003).

Information along the anterior-posterior axis also contributes to cell-fate determination within the dorsal appendage primordia. The BMP2/4 homolog encoded by dpp is expressed in the stretch cells and a single row of centripetally migrating cells. This morphogen radiates posteriorly and alters columnar cell fates. High levels of Dpp repress dorsal identities and specify operculum; moderate levels synergize with Grk to define dorsal, while low levels of Dpp are insufficient to allow cells to respond to Egfr signaling. Thus, loss-of-function mutations generate short, often paddleless appendages, while overexpression either expands the operculum at the expense of appendage material or creates multiple, often antler-shaped dorsal structures. The subsequent events underlying dorsal appendage morphogenesis are only beginning to be understood. Analyses of cultured wild-type egg chambers have revealed several phases of dorsal appendage morphogenesis. From stages 10B to 12, two groups of dorsal anterior follicle cells move out from the follicular epithelium to form short tubes. Each tube extends forward over the nurse cells, secreting chorion proteins that make up the cylindrical stalk of the dorsal appendage. Cells at the anterior end of the tube change shape to produce the flattened paddle of the distal dorsal appendage. Finally, upon oviposition, the entire follicular epithelium sloughs off, leaving behind the chorionic structures (French, 2003).

Proteomics analysis identifies orthologs of human chitinase-like proteins as inducers of tube-morphogenesis defects in Drosophila

Elevated levels of human chitinase-like proteins (CLPs) are associated with numerous chronic inflammatory diseases and several cancers and can promote disease progression by remodeling tissue, activating signaling cascades, stimulating proliferation and migration, and by regulating adhesion. This study has identified Imaginal disc growth factors (Idgfs), orthologs of human CLPs CHI3L1, CHI3L2, and OVGP1, in a proteomics analysis designed to discover factors that regulate tube morphogenesis. The approach used magnetic beads to isolate a small population of specialized ovarian cells, cells that non-autonomously regulate morphogenesis of epithelial tubes that form and secrete eggshell structures called dorsal appendages. Elevated levels were detected of four of the six Idgf family members (Idgf1, Idgf2, Idgf4, and Idgf6) in flies mutant for Bullwinkle, which encodes a transcription factor and is a known regulator of dorsal-appendage tube morphogenesis. During oogenesis, dysregulation of Idgfs (either gain or loss of function) disrupts the formation of the dorsal-appendage tubes. Previous studies demonstrate roles for Drosophila Idgfs in innate immunity, wound healing, and cell proliferation and motility in cell culture. This study identified a novel role for Idgfs in both normal and aberrant tubulogenesis processes (Zimmerman, 2017).

Regulation of epithelial stem cell replacement and follicle formation in the Drosophila ovary

Though much has been learned about the process of ovarian follicle maturation through studies of oogenesis in both vertebrate and invertebrate systems, less is known about how follicles form initially. In Drosophila, two somatic follicle stem cells (FSCs) in each ovariole give rise to all polar cells, stalk cells, and main body cells needed to form each follicle. One daughter from each FSC founds most follicles but that cell type specification is independent of cell lineage, in contrast to previous claims of an early polar/stalk lineage restriction. Instead, key intercellular signals begin early and guide cell behavior. An initial Notch signal from germ cells is required for FSC daughters to migrate across the ovariole and on occasion to replace the opposite stem cell. Both anterior and posterior polar cells arise in region 2b at a time when approximately 16 cells surround the cyst. Later, during budding, stalk cells and additional polar cells are specified in a process that frequently transfers posterior follicle cells onto the anterior surface of the next older follicle. These studies provide new insight into the mechanisms that underlie stem cell replacement and follicle formation during Drosophila oogenesis (Nystul, 2010).

The Drosophila ovary is a highly favorable system for studying epithelial cell differentiation downstream from a stem cell. New follicles consisting of 16 interconnected germ cells surrounded by an epithelial (follicle cell) monolayer are continuously produced during adult life and develop sequentially within ovarioles (see Prefollicle cells associate with cysts in an ordered fashion downstream from follicle stem cells). Follicle formation begins in the germarium, a structure at the tip of each ovariole that houses 2-3 germline stem cells (GSCs) and 2 follicle stem cells (FSCs) within stable niches. Successive GSC daughters known as cystoblasts are enclosed by a thin covering of squamous escort cells and divide asymmetrically four times in sucession to produce 16-cell germline cysts, comprising 15 presumptive nurse cells and a presumptive oocyte. At the junction between region 2a and region 2b, cysts are forced into single file as they encounter the FSCs, lose their escort cell covering, and begin to acquire a follicular layer. Follicle cells derived from both FSCs soon mold them into a 'lens shape' characteristic of region 2b. Under the influence of continued somatic cell growth, cysts and their surrounding cells round up, enter region 3 (also known as stage 1), and bud from the germarium as new follicles that remain connected to their neighbors by short cellular stalks (Nystul, 2010).

A complex sequence of signaling and adhesive interactions between follicular and germline cells is required for follicle budding, oocyte development, and patterning. However, the mechanisms orchestrating the initial association between follicle cells and cysts within the germarium are less well understood. While lineage analysis indicates the presence of two FSCs, low fasciclin III (FasIII) expression has been claimed to specifically mark FSCs, leading to the conclusion that more FSCs are present under some conditions (Nystul, 2010).

The differentiation of polar cells at both their anterior and posterior ends is required for normal follicle production, and depends on Notch signals received from the germline. Subsequently, anterior polar cells send JAK-STAT and Notch signals that specify stalk cells. While the source of these signals and their effects are clear, the timing of polar cell specification and its dependence on cell lineage are not. Some anterior and posterior polar cells (but not stalk cells) were inferred by lineage analysis to arise and cease division within region 2b. In contrast, on the basis of marker gene expression it was concluded that anterior polar cells are specified later, in stage 1, and posterior polar cells in stage 2. Up to four polar cells may eventually form, but apoptosis reduces their number to a single pair at each end by stage 5. Moreover, polar and stalk are believed to arise exclusively from 'polar/stalk' precursors that separate from the rest of the FSC lineage and these cells were proposed to invade between the last region 2b cyst to affect follicle budding (Nystul, 2010).

This study analyzes the detailed behavior of FSCs and their daughters in the germarium. No evidence of polar/stalk precursors was observed, and it was shown that the first anterior and posterior polar cells are specified in region 2b, prior to the previously accepted time of follicle cell specialization. Additional polar cells are also formed later during stages 1 and 2. Follicle cell differentiation appears to be independent of cell lineage, but is orchestrated by sequential cell interactions, and in particular by Notch signaling. These results reveal the sophisticated, self-correcting behavior of an epithelial stem cell lineage at close to single-cell resolution (Nystul, 2010).

The data provide a much clearer picture of the follicle stem cell lineage than was previously available. They suggest that key aspects of FSC regulation depend on mechanisms that move cysts into a single file and program the loss of their escort cells precisely as they encounter FSCs and enter region 2b. Contact with incoming region 2a cysts likely induces FSC divisions, ensuring that cysts acquire a daughter cell from each stem cell as they stretch out to span the width of the germarium at the region 2a/2b junction. The asymmetry in cyst organization exposes FSC daughter cells to different cyst faces and, therefore, potentially to different signals. The FSC and daughter located on the same side as the entering cyst are exposed to the posterior face of the cyst while it is still in region 2a, covered by escort cells. In contrast, the opposite FSC and daughter contact the anterior face of the cyst as it migrates into 2b, at a time when the cyst is shedding its escort cell layer and exposing the Delta signal on the germ cell surface. Since region 2a cysts tend to interdigitate in forming a single file, cyst entry will usually alternate sides as successive cysts pass, causing FSC daughters arising from the same side to alternate migration paths. An advantage to this system may be its flexibility, allowing follicles to form normally even if multiple cysts enter from the same side in succession (Nystul, 2010).

Notch signaling in early FSC daughters promotes a 'prefollicle' state by blocking follicle cell differentiation. Consistent with this, it was observed that FSCs and their early daughters have much lower levels of differentiation markers such as FasIII and IMP-GFP. This developmental delay may prevent prefollicle cells from immediately incorporating into the differentiated follicular epithelium, allowing them to instead retain a more mesenchymal character conducive to cross-migration, and may also contribute to their ability to compete with the resident FSC for niche occupancy. Notch mutant daughters did not replace wild-type FSCs, most likely because they were unable to migrate into proximity. A role for Notch in suppressing differentiation downstream from the FSC might also explain why cells expressing activated Notch failed to migrate posteriorly (Nystul, 2010).

Follicle cell fates are specified by intercellular signals rather than lineage: The two FSC daughters and their descendants, with few exceptions, continue to associate with the cyst they first contact at the 2a/2b boundary throughout subsequent development. Their division rate increases briefly, because daughters divide four times in the time it takes to generate three new cysts. Despite their growing number, however, all the cells retain the ability to produce main body, stalk, and polar cells for at least the first two to three divisions (8- to 16-cell stage). In contrast to previous reports, no evidence was found that polar and stalk cells derive from a lineage-restricted polar/stalk precursor population. Claims of a polar/stalk fate were based on experiments using higher rates of clone induction than in the experiments reported in this study. While many clones were also observed in these studies that contained both polar and follicle cells or both stalk and follicle cells, they were discounted as double clones (Nystul, 2010).

By examining clones induced at low frequency (more than threefold lower than in previous studies) it was possible to minimize the need for statistical correction for double clones. Furthermore, by studying clones induced at multiple times downstream from the FSC, overweighting small clones induced just as the polar and stalk cell fates are being determined by signaling within small cell groups was avoided. This has the effect of increasing the proportion of clones containing only one or two cell types even in the absence of any lineage restriction. At early, intermediate, and late times in somatic cell development in the germarium, clones that included all combinations of cell fates were always observed, indicating that follicle cells are multipotent prior to polar or stalk specification. This fits well with recent studies showing that many additional cells in the germarium can be induced to take on a polar cell fate by strong Notch signaling, while high levels of JAK-STAT signaling can induce more stalk cells. In contrast, no mechanism, time, or location where putative polar/stalk precursor cells are specified has ever been documented. Previous models also did not explain how these cells would preferentially arrive in the zone of cells separating regions 2b and 3 or what would become of the many extra cells that can sometimes be found in this region beyond the number needed for these fates (Nystul, 2010).

The finding that polar cells are initially specified in region 2b suggests that more spatial information is available within region 2b follicles than has been detected in earlier experiments. It was found that the first anterior and posterior polar cells are specified when cysts are associated with 8- to 16-cell follicle cells, in mid-to-late region 2b. This agrees closely with previous studies, which found that polar cells were first specified at the 14-cell stage. The early polar cells are detected by lineage because they cease dividing; however, no gene expression markers specific for these cells have been identified. Consequently, it remains uncertain where they are located at the time of induction or whether they function while remaining in region 2b. Since evidence was observed of Notch signal reception within individual follicle cells located at the anterior and posterior regions of stage 2b cysts, the simplest model is that these polar cells are induced by Delta signaling from the germline in a normal anterior/posterior (A/P) orientation. Although no Upd expression was detected at this time, these cells may nonetheless signal to the surrounding somatic cells to establish the graded levels of cadherin that define the initial anterior/posterior axis of the cyst (Nystul, 2010).

Where does the information come from that allows a small number of polar cells to be specified at this time? One possibility is a 'signal relay' from more posterior follicles. Highly accurate timing of polar cell formation relative to the signaling events during follicle budding might help to further test this model. However, the observation of localized Notch signal reception and polar cell specification in region 2b follicles suggests that the germline at this stage is already sufficiently polarized to signal in a limited manner along the future A/P axis. Some of this information may come from the inherent asymmetry within the germline cyst whose cells differ systematically in their fusome content, organelle content, and microtubule organization. The future oocyte and its sister four-ring canal cell are always located in the center of the region 2b cysts and hence might be one source for this inductive signal. Alternatively, there may be additional differences within this region of the germarium that have yet to be detected and that may contribute (Nystul, 2010).

These studies confirm previous conclusions that additional polar cells are formed during the process of budding and provide new insight into the budding process itself. Anterior-biased clones were almost always confined to a single follicle, but a significant fraction of posterior-biased clones (~33%) extended onto the next older follicle where they encompassed both an anterior polar cell and 2-30 anterior follicle cells. This suggests that cells at the posterior of the nascent follicle outgrow their cyst as it rounds up and are forced into the space between the posterior 2b cyst and the budding cyst. The origin of these cells has long been a mystery. A fraction of the interstitial cells likely contact and move onto the anterior of the downstream cyst where those that happen to lie adjacent to the existing polar cells are induced as new polar cells and stalk cells. Any remaining interstitial cells likely rejoin the main body of follicle cells as budding is completed or are eliminated by apoptosis as the stalk resolves to its final size (Nystul, 2010).

This study of early follicle cell development provides a rare opportunity to analyze how epithelial cells behave downstream from a stem cell. Most characterized Drosophila stem cell daughters receive information asymmetrically from their mother stem cell and differentiate rapidly. Germline stem cells and their niches ensure that cystoblasts receive an asymmetric fusome segment as well as differential environmental signals that program exactly four stereotyped divisions prior to entering meiosis. Under nonstressed conditions, intestinal stem cells utilize Notch signals to specify their daughters as either enterocytes or enteroendocrine cells and to terminate subsequent division. Neuroblasts program a stereotyped sequence of daughter cell fates by differential division and signaling. In contrast, FSC daughters undergo eight to nine divisions and differentiate independently of lineage over the course of several divisions and are capable of producing normal follicles even when the usual pattern of cellular interactions is altered. The increased resolution of follicle cell behavior afforded by these studies provides a valuable opportunity to study how epithelial cells are able to robustly bring about defined outcomes in the absence of the precise early programming (Nystul, 2010).

Several mechanisms are likely to contribute to successful follicle formation. First, genes characteristic of a polarized epithelium turn on slowly downstream of the FSC. The cross-migrating cell and several other cells frequently lacked such gene expression, but instead expressed genes characteristic of escort cells, suggesting that follicles are able to maintain some germline-soma interactions while completely replacing their somatic coverings. The early differentiation of polar cells may help guide subsequent cell behavior. In conjunction with the intrinsic asymmetric structure of germline cysts, differential adhesive interactions between germ and somatic cells across the follicle, differential pressures resulting from cell growth, and the resistive forces of the ovariolar wall, signals from these cells may be sufficient to ensure that the oocyte moves to the posterior and that cysts begin to round (Nystul, 2010).

These characteristics of the FSC lineage, although unique among well-studied stem cells in Drosophila, may be closer to those governing the epithelial lineages within many mammalian tissues. Thus, the mechanisms that give FSCs and their daughters their developmental flexibility and robustness are likely to be both widespread and medically relevant (Nystul, 2010).

Systematic analysis of the transcriptional switch inducing migration of border cells

Cell migration within a natural context is tightly controlled, often by specific transcription factors. However, the switch from stationary to migratory behavior is poorly understood. Border cells perform a spatially and temporally controlled invasive migration during Drosophila oogenesis. Slbo, a C/EBP family transcriptional activator, is required for them to become migratory. Wild-type and slbo mutant border cells as well as nonmigratory follicle cells were purified and comparative whole-genome expression profiling was performed, followed by functional tests of the contributions of identified targets to migration. About 300 genes were significantly upregulated in border cells, many dependent on Slbo. Among these, the microtubule regulator Stathmin was strongly upregulated and was required for normal migration. Actin cytoskeleton regulators were also induced, including, surprisingly, a large cluster of 'muscle-specific' genes. It is concluded that Slbo induces multiple cytoskeletal effectors, and that each contributes to the behavioral changes in border cells (Borghese, 2006).

Only one of the identified cytoskeletal regulators is known to affect microtubules, namely, Stathmin. Mammalian Stathmin/Op18 protein is well characterized. It binds to microtubules and promotes depolymerization by sequestration of tubulin dimers or direct action at microtubule ends. Interestingly, the activity of Stathmin can be regulated by phosphorylation in response to signaling or cell cycle phases. Drosophila Stathmin appears to have similar biochemical features. The availability of an antibody directed against Drosophila Stathmin allowed analysis of protein levels in situ. As expected, the level of Stathmin was higher in border cells than follicle cells. When analyzing slbo mutant border cells, a clear difference was observed between the inner polar cells and the outer border cells. The outer border cells are the migratory cells and require Slbo expression. In these cells, Stathmin expression was undetectable in the absence of Slbo, indicating a very strong dependence on Slbo. In contrast, Stathmin was still expressed in mutant polar cells, explaining why only a moderate reduction of stathmin mRNA levels was seen in whole border cell clusters (Borghese, 2006).

To analyze the function of Stathmin in border cells, stathmin mutants were generated. This was done by imprecise excision of a P element located immediately upstream of the stathmin C transcript. A mutant deleting only the stathmin C isoform (stathminexC), leaving stathmin A and B intact, was homozygous viable and had no effect on border cell migration. A mutant deleting the complete stathmin locus (stathminL27) and four adjacent genes (including Arc-p20, a component of the Arp2/3 complex) was homozygous lethal, and clones of stathminL27 mutant border cells were unable to migrate. Both the lethality and the migration block were rescued by reintroducing ubiquitously expressed stathmin and Arc-p20 at the same time. Reintroducing Arc-p20 alone did not rescue border cell migration, indicating that stathmin is essential for this process. To interfere with stathmin upregulation at the time of migration, a functional stathmin “hairpin”-RNAi construct was expressed in the sensitized stathminexC/stathminL27 background. By using the slbo-GAL4 driver, stathmin RNAi expression could be could specifically targeted to outer border cells right before and during migration. This strongly decreased the amount of Stathmin protein in border cells and caused significant delays in migration. The delays in migration could be reversed by driving higher levels of stathmin expression from a UAS construct. These results identify Stathmin as an important regulator downstream of Slbo. To test whether lack of Stathmin was solely responsible for the slbo phenotype, Stathmin was overexpressed in the slbo mutant background. Migration was not restored, indicating that additional genes downstream of Slbo must also be important (Borghese, 2006).

Singed is an actin-bundling protein related to Fascin, highly expressed in border cells. Fascin is important for the formation of cell protrusions and has been implicated in the control of cell migration, also in vivo. It was confirmed by clonal analysis that Singed protein levels are regulated by Slbo. Despite the strong and regulated expression, migration is normal in border cells mutant for singed. The strongest allele of singed available was used, but it retained a low level of protein expression. In addition, functional overlap may exist between actin regulators. Quail is an actin binding protein of the villin family, and its function in the germline of the ovary genetically overlaps with that of Singed. quail mRNA is also upregulated in border cells relative to follicle cells, and Quail protein is detected in border cells. Quail is structurally similar to Gelsolin, which was also upregulated in border cells, as well as the Gelsolin-related FliI, which was not detectably expressed. However, Gelsolin is enriched in polar cells rather than the migratory outer border cells. As for singed, no migration defects were observed in quail mutant border cells, nor in cells mutant for quail and only one functional copy of singed or vice versa. It was not possible to recover clones of border cells simultaneously mutant for both singed and quail, which is likely to reflect a functional overlap between the two genes at an earlier stage. The simultaneous upregulation of redundant actin regulators may reflect a genetically robust approach to changing the actin cytoskeleton in border cells (Borghese, 2006).

A rather surprising finding of this global expression analysis was that the remaining genes encoding cytoskeleton-associated proteins and upregulated in border cells in a slbo-dependent manner were all “muscle specific”. This included a complete palate of structural genes: muscle actin (57B), muscle myosin heavy chain and light chains, tropomyosin 2 (tm2), troponins, and the calponin-related mp20. The muscle-specific expression has been shown for this group of genes in Drosophila embryos as well as mature muscles. For tropomyosin 2, a GFP gene trap allele was available and, and this allele confirmed expression in border cells as well as in the muscle sheath. The expression profiling indicated that border cells also express the corresponding non-muscle forms such as actin42A, zipper (myosin heavy chain), and sqh (myosin light chain), but at the same level as in follicle cells. The nonmuscle proteins are generally required for many cellular processes, including, where tested, migration of border cells. This raised the question of why this large cluster of muscle-specific structural genes would be turned on in border cells as well. To address this, migration was analyzed of border cells mutant for individual muscle genes for which mutants were available (mhc, mlc2, upheld=troponinT and tm2). Since mhc and mlc2 are essential genes, this was done by clonal analysis. No defects were seen in border cells mutant for mlc2, upheld, or tm2, but clear migration defects were observed in border cells mutant for mhc (mhc1 or mhc3). Thus, while not all of the muscle structural genes are required for border cell migration, at least muscle Mhc expression contributes to effective migration (Borghese, 2006).

Given that both muscle and nonmuscle forms of the same cytoskeletal proteins have a role in border cell migration, their functions are likely to be different. In agreement with this, no genetic interactions were observed between mutants affecting muscle and nonmuscle forms of myosin heavy or light chains. There is precedence for such nonoverlapping functions. For example, Zipper has a unique role in developing muscle cells, which contain plenty of muscle myosin heavy chain. In mammalian cells, different myosin heavy chain isoforms can have distinct subcellular localization. Also, the actin proteins, despite having few amino acid differences, are functionally distinct in vivo (Borghese, 2006).

The muscle gene expression program activated in migratory border cells extended beyond structural genes to regulatory genes. One such gene was bent, encoding a very large titin-like molecule with a myosin light chain kinase domain. Being essential but on the fourth chromosome, bent was not amenable to standard clonal analysis. Genes required for myoblast fusion were also identified, namely, rols/antisocial and rost. mbc, which encodes a DOCK180 family Rac GEF and is required for myoblast fusion, has a role in border cell migration downstream of the PVR guidance receptor. Mbc protein interacts physically with the presumed adaptor protein Rols. Clonal analysis with a strong (likely complete loss-of-function) allele of rols showed defects in border cell migration, suggesting that Mbc and Rols might act together during migration as well. The defect was milder than for mbc, implying that Mbc activity might not be completely dependent on Rols. For the small transmembrane protein Rost, no useful mutants were available. It was also noted that a very closely related and adjacent gene, CG13101, was similarly regulated in border cells and might overlap rost function. Thus, at least mbc and rols function in border cells as well as in muscle (myoblast fusion). Activation of a broad “muscle-specific” gene expression program in border cells may reflect a requirement for a specific subset of the genes within this program (Borghese, 2006).

Previous unbiased genetic approaches to identify genes important for border cell migration have largely identified transcription factors or inducing signals. Changes in cell fate can alter cell behavior dramatically without affecting cell survival, thus still allowing analysis of the mutant cells. The transcription factors themselves often show differential expression. In addition to Slbo, the posttranslationally regulated transcription factor STAT, which is important for border cell migration, was also upregulated in border cells (1.6-fold). The transcription factors that were upregulated in border cells and had mutants available for effects on border cell migration were also tested. aop/yan transcripts were increased 1.9-fold in border cells. In a PiggyBac transposon-based clonal screen for border cell migration defects, an insertion was identified in aop. Complementation analysis confirmed the gene assignment, and quantification of the phenotype showed a clear effect of aop on border cell migration. As expected, border cell migration was strongly affected, but, in addition, clones were rare and morphological abnormalities were seen in other follicle cells as well as in germline cells. Thus, aop may affect the behavior of multiple cell types in the ovary. Another transcription factor, vrille, was also upregulated (over 2-fold). vrille has been implicated in signaling, circadian rhythm, and cellular morphogenesis, but border cells mutant for vrille were largely unaffected and experienced only subtle delays (Borghese, 2006).

The most border cell-enriched RNA encoding a transcription factor, apart from Slbo, was Six4 (4.5-fold). Six4 expression in border cells was confirmed by in situ analysis. Drosophila Six4 is the homeodomain transcription factor most related to mammalian Six4 and Six5. Six family proteins act in complex with proteins of the Eya (Eyes absent) family. eya transcripts were also 2.7-fold enriched in border cells relative to follicle cells of the same stage, and Eya was expressed in a pattern similar to that of six4. Both six4 and eya were expressed in earlier-stage follicle cells as well, and eya has been shown to function at these stages to repress polar cell fate. Follicle cells mutant for six4 expressed a polar cell marker (Fas3) and were functional polar cells, as determined by the ability to induce surrounding anterior follicle cells to become Slbo-positive, migratory border cells. This suggested that Six4 cooperates with Eya in repressing polar cell fate. It had been indicated that Six proteins affect nuclear localization of their Eya partner. The six4 mutant allowed testing this in an in vivo context. Although six4 mutant cells were transformed to functional polar cells, Eya protein was not absent as in the endogenous polar cells, showing that Eya accumulation was independently regulated. However, Eya protein was partially relocalized to the cytoplasm of six4 mutant cells, supporting the hypothesis that Six4 and Eya interact in vivo. Since six4 and eya are both upregulated in outer border cells when they migrate, they are likely to act together in this process as well. However, their earlier roles precludes straightforward loss-of-function analyses in border cells, since “border cell clusters” consisting only of six4 or eya mutant cells are not functional simply because polar cells do not migrate on their own. Overexpression of HA-tagged six4 in border cells interfered with migration, as found for transcription factors required in border cells slbo (Borghese, 2006).

The expression of Six4 in border cells may contribute to activation of the muscle gene program described above. The conserved muscle transcription factor Mef2, an activator of muscle actin and myosin expression, was not detected in border cells by expression profiling or by antibody staining, nor were Twist and Nautilus/MyoD. Six4 is required for development of muscle and other mesodermal tissues in Drosophila. Mutants of C. elegans Unc-39, belonging to the Six4/5 family, also affect muscle/mesodermal differentiation as well as directed cell migration. Mammalian Six5, also called myotonic dystrophy-associated homeodomain protein (DMAHP), has been analyzed due to its contribution to DM1, and Six4/5 affect normal muscle development. Another transcription factor complex that might contribute to the activation of the muscle program is that of MAL-D (or MRTF) and SRF (serum response factor). The MRTF/SRF complex plays an important role in muscle development in mammals and directly regulates muscle (structural) genes. MAL-D/SRF plays a crucial role in border cell migration and this complex acts to strengthen the cytoskeleton of invasive border cells in response to perceived tension. This mode of regulation makes MAL-D/SRF activity in border cells indirectly dependent on Slbo, which could be responsible for the apparent regulation of the muscle gene cluster by Slbo. The possibility cannot be excluded that Slbo might affect muscle genes directly; the mammalian C/EBP transcription factors are known to regulate different differentiation-specific genes in different contexts (Borghese, 2006).

This study analyzed overall gene expression changes resulting from a transcriptional switch that induces invasive migratory behavior in vivo. The major goal of the analysis was to identify transcriptional changes that directly affect cell behavior and make the cells move. The results indicate that regulation of both the actin cytoskeleton and the microtubule cytoskeleton, likely coordinated regulation, is important for this transition. Identifying Stathmin as an important regulator downstream of Slbo in border cells indicates that microtubule dynamics are critical for border cell migration. Key questions are now how microtubule dynamics affect the process, and whether Stathmin activity is regulated. Two recent findings suggest that Stathmin may be a more general regulator of cell migration: Stathmin-microtubule interactions are spatially regulated in migrating cells in culture, and Stathmin upregulation may promote migration and metastasis of sarcoma cells. The actin cytoskeleton is clearly crucial for cell migration and is controlled by many regulators. The upregulated modulators identified in this study were different from those identified in a whole-genome study of tumor cells selected, in vivo, to be highly motile. There are obviously many differences between these studies; for one, a normal transition to migratory behavior may differ from unrestrained, high motility. The activation of a “muscle-specific” program in migratory border cells was unexpected and provides an intriguing connection between these cells that move and the specialized cells that move an animal (muscle). Overall, the analysis of actin regulators indicates that this is a robust system, with many effectors coregulated, even by one transcription factor. Genetically, this is reflected by minor defects in individual “effector” mutants despite absolute dependence on the transcriptional switch. Further analysis in other systems, and subsequent comparisons, will reveal to what extent the gene expression program employed by border cells to become migratory is a general one (Borghese, 2006).

Tousled-like kinase regulates cytokine-mediated communication between cooperating cell types during collective border cell migration

Collective cell migration is emerging as a major contributor to normal development and disease. Collective movement of border cells in the Drosophila ovary requires cooperation between two distinct cell types: 4-6 migratory cells surrounding two immotile cells called polar cells. Polar cells secrete a cytokine, Unpaired (Upd), which activates JAK/STAT signaling in neighboring cells, stimulating their motility. Without Upd, migration fails, causing sterility. Ectopic Upd expression is sufficient to stimulate motility in otherwise immobile cells. Thus regulation of Upd is key. This study reports a limited RNAi screen for nuclear proteins required for border cell migration, which revealed that the gene encoding Tousled-like kinase (Tlk) is required in polar cells for Upd expression without affecting polar cell fate. In the absence of Tlk, fewer border cells are recruited and motility is impaired, similar to inhibition of JAK/STAT signaling. It was further shown Tlk in polar cells is required for JAK/STAT activation in border cells. Genetic interactions further confirmed Tlk as a new regulator of Upd/JAK/STAT signaling. These findings shed light on the molecular mechanisms regulating the cooperation of motile and non-motile cells during collective invasion, a phenomenon that may also drive metastatic cancer (Xiang, 2015).

The Hippo pathway controls border cell migration through distinct mechanisms in outer border cells and polar cells of the Drosophila ovary

The Hippo pathway is a key signaling cascade in controlling organ size. The core components of this pathway are two kinases, Hippo (Hpo) and Warts (Wts), and a transcriptional coactivator Yorkie (Yki). YAP (a Yki homolog in mammals) promotes epithelial-mesenchymal transition and cell migration in vitro. This study used border cells in the Drosophila ovary as a model to study Hippo pathway functions in cell migration in vivo. During oogenesis, polar cells secrete Unpaired (Upd), which activates JAK/STAT signaling of neighboring cells and specifies them into outer border cells. The outer border cells form a cluster with polar cells and undergo migration. This study found that hpo and wts are required for migration of the border cell cluster. In outer border cells, over-expression of hpo disrupts polarization of the actin cytoskeleton and attenuates migration. In polar cells, knockdown of hpo, wts, or over-expression of yki impairs border cell induction and disrupts migration. These manipulations in polar cells reduce JAK/STAT activity in outer border cells. Expression of upd-lacZ is increased and decreased in yki and hpo mutant polar cells, respectively. Furthermore, forced-expression of upd in polar cells rescues defects of border cell induction and migration caused by wts knockdown. These results suggest that Yki negatively regulates border cell induction by inhibiting JAK/STAT signaling. Together, these data elucidate two distinct mechanisms of the Hippo pathway in controlling border cell migration: 1) in outer border cells, it regulates polarized distribution of the actin cytoskeleton; 2) in polar cells, it regulates upd expression to control border cell induction and migration (Lin, 2014).

An atypical tropomyosin in Drosophila with intermediate filament-like properties
A longstanding mystery has been the absence of cytoplasmic intermediate filaments (IFs) from Drosophila despite their importance in other organisms. In the course of characterizing the in vivo expression and functions of Drosophila Tropomyosin (Tm) isoforms, this study discovered an essential but unusual product of the Tm1 locus, Tm1-I/C, which resembles an IF protein in some respects. Like IFs, Tm1-I/C spontaneously forms filaments in vitro that are intermediate in diameter between F-actin and microtubules. Like IFs but unlike canonical Tms, Tm1-I/C contains N- and C-terminal low-complexity domains flanking a central coiled coil. In vivo, Tm1-I/C forms cytoplasmic filaments that do not associate with F-actin or canonical Tms. Tm1-I/C is essential for collective border cell migration, in epithelial cells for proper cytoarchitecture, and in the germline for the formation of germ plasm. These results suggest that flies have evolved a distinctive type of cytoskeletal filament from Tm (Cho, 2016).

DRP1-dependent mitochondrial fission initiates follicle cell differentiation during Drosophila oogenesis

Exit from the cell cycle is essential for cells to initiate a terminal differentiation program during development, but what controls this transition is incompletely understood. This paper demonstrates a regulatory link between mitochondrial fission activity and cell cycle exit in follicle cell layer development during Drosophila melanogaster oogenesis. Posterior-localized clonal cells in the follicle cell layer of developing ovarioles with down-regulated expression of the major mitochondrial fission protein DRP1 had mitochondrial elements extensively fused instead of being dispersed. These cells did not exit the cell cycle. Instead, they excessively proliferated, failed to activate Notch for differentiation, and exhibited downstream developmental defects. Reintroduction of mitochondrial fission activity or inhibition of the mitochondrial fusion protein Marf-1 in posterior-localized DRP1-null clones reversed the block in Notch-dependent differentiation. When DRP1-driven mitochondrial fission activity was unopposed by fusion activity in Marf-1–depleted clones, premature cell differentiation of follicle cells occurred in mitotic stages. Thus, DRP1-dependent mitochondrial fission activity is a novel regulator of the onset of follicle cell differentiation during Drosophila oogenesis (Matra, 2012).

The Drosophila follicle cell layer encapsulates egg chambers containing 15 nurse cells and one oocyte. The follicle cells comprising this cell layer progress through different developmental stages. During stages 1-5 (S1-5), most follicle cells undergo mitotic divisions, with a few cells exiting the mitotic cycle under Notch activation to form stalk cells separating consecutive egg chambers. During S6-8, all follicle cells exit the mitotic cycle in response to Notch activation and differentiate into an endocycling, polarized epithelium patterned into posterior follicle cells (PFCs), main body cells (MBCs), and anterior follicle cells (AFCs). To examine the effect of inhibiting mitochondrial fission activity in this system, Drosophila follicle cell clones where generated mozygous for a functionally null allele of DRP1 called drp1KG. Clones were identified by lack of a ubiquitin promoter-GFP (UbiGFP) label in their nucleus. The potentiometric dye tetramethylrhodamine ethyl ester (TMRE), which incorporates into the mitochondrial matrix, was used to label mitochondria (Mitra, 2009).

In an S10 egg chamber, nonclonal cells containing a nuclear UbiGFP label have mitochondrial elements widely distributed. Microirradiation at a single point within mitochondria of these cells triggers depolarization (i.e., loss of fluroescent TMRE signal) only at the irradiated site, with little loss of TMRE outside the microirradiated site. This suggested the mitochondrial network of these cells is discontinuous. In drp1KG clones (no UbiGFP label), mitochondria were tightly clustered in a small region of each cell. Single-point microirradiation of mitochondria in a drp1KG clone depolarizes the cell's entire mitochondrial cluster, with complete loss of TMRE signal in 5 s. This indicated that mitochondria in drp1KG clones are highly fused. Reduced mitochondrial fission in drp1KG clones, therefore, causes normally fragmented mitochondrial elements in follicle cells to hyperfuse into a tight cluster (Matra, 2012).

Next, weather presence of drp1KG clones affects follicle epithelial layer organization was examined. In S6-8 egg chambers, follicle cells normally form a single epithelial monolayer. The presence of drp1KG clones, however, disrupts this monolayer arrangement. The effect is most striking in the PFC region, in which drp1KG clones massively overproliferate. The overpopulated clones undergo mitotic cycling even at S10 or later: they incorporate BrdU, demonstrating that they synthesize DNA, and stain with pH3 antibody, indicating that they transit through mitosis. Surrounding heterozygous tissue and drp1KG MBC clones, in contrast, are postmitotic: they neither incorporate BrdU nor stain for pH3. DRP1 depletion thus prevents cell cycle exit primarily in drp1KG PFC clones, leading to their overpopulation in postmitotic egg chambers (Matra, 2012).

As cell cycle exit is a prerequisite for initiating differentiation, whether the drp1KG PFCs are prevented from differentiating was examined. Follicle cells in S6-8 egg chambers normally undergo cell cycle exit to differentiate under the influence of the homeodomain gene Hindsight (Hnt). Notably, clones of drp1KG in the PFC region marked by CD8GFP fail to express Hnt, unlike surrounding nonclonal cells. 95% of drp1KG PFC clones show this phenotype, whereas no drp1KG MBC clonal cells do. Thus, drp1KG PFC clones fail to differentiate (Matra, 2012).

Hnt expression is rescued in all drp1KG PFC clones generated in the background of HA-DRP1 and in 43% of drp1KG PFC clones with DRP1 reintroduced into them. In both conditions, DRP1 expression prevented the clustered mitochondrial phenotype. Lack of differentiation in drp1KG PFC clones, therefore, results from loss of DRP1 activity (Matra, 2012).

Down-regulation of Marf-1, the Drosophila homologue of mitofusins (Deng, 2008), combined with DRP1 down-regulation in drp1KG PFC clones causes 22% of the clones to now partially express Hnt. Because Marf-1 RNAi expression causes mitochondrial fragmentation when expressed alone or in drp1KG PFC clones, it was concluded that fragmentation of mitochondria reverses the differentiation block in drp1KG PFCs. Therefore, DRP1-driven mitochondrial fission is required for PFCs to differentiate. Loss of function of the inner mitochondrial membrane fusion protein OPA1 caused cell death in this system (Matra, 2012).

Differentiation of Drosophila follicle cells requires Notch receptor activation. Upon ligand binding, the Notch receptor is cleaved to release the Notch intracellular domain (NICD), which redistributes into the nucleus to activate genes required for differentiation. To investigate whether DRP1-driven mitochondrial fission activity acts upstream or downstream of Notch activation in driving PFC differentiation, whether NICD is cleaved and released from the plasma membrane was examined in drp1KG PFC clones. Significant NICD levels are retained on the plasma membrane in drp1KG PFC clones marked by CD8GFP relative to nonclonal cells in S6-8 egg chambers. The Notch extracellular domain (NECD) is also retained on the plasma membrane in these clones, confirming that Notch is inactive. In addition, Cut down-regulation, which occurs in response to Notch activation, does not occur in drp1KG PFC clones. DRP1-driven mitochondrial fission activity thus acts upstream of Notch activation to drive PFC differentiation (Matra, 2012).

NICD loss from the membrane (indicative of Notch activation) increases by 28.2% in drp1KG PFC clones after Marf-1 down-regulation. This suggested that Notch inactivation in drp1KG PFC clones is related to mitochondria being highly fused, with mitochondrial fission a prerequisite for Notch receptor activation in the PFCs. Importantly, expression of an activated Notch (N-Act) domain in drp1KG PFC clones partially overrides the differentiation block in 53% of drp1KG PFC clones, resulting in Hnt expression in these clones. As this occurs without the fused mitochondrial morphology of drp1KG PFC clones changing, the data confirmed that DRP1's role in triggering PFC differentiation is upstream of Notch (Matra, 2012).

Why is DRP1's role in triggering follicle cell differentiation specific to PFCs? Indeed, drp1KG MBC clones show no differentiation block, as Notch activation still occurs in drp1KG MBC clones. Higher levels of bound DRP1 was found in PFCs compared with MBCs after cell permeabilization with digitonin, which may reflect different mitochondrial morphology between PFCs and MBCs. Supporting this, in S6-8 ovarioles it was found that mitochondria in PFCs exist as dispersed fragments both apically and basolaterally, whereas mitochondria in MBCs are tightly clustered at the lateral side of the nucleus. After S9, no observable differences were seen in mitochondrial morphology (Matra, 2012).

Fluorescence loss in photobleaching (FLIP) experiments in follicle cells of S6-8 egg chambers revealed that the dispersed mitochondria of PFCs have less matrix continuity relative to the fused mitochondrial cluster of MBCs. Furthermore, single-point microirradiation caused a 44% loss in TMRE mitochondrial signal per MBC compared with a 12% loss per PFC. The rapid loss of mitochondrial TMRE signal in MBCs was similar to drp1KG clonal cells, with mitochondrial morphology in wild-type MBCs indistinguishable from that of drp1KG MBC clones. Together, the observed differences in mitochondrial organization and bound DRP1 levels in PFCs and MBCs suggested greater DRP1-driven mitochondrial fission activity occurs in PFCs relative to MBCs. This corroborates findings that PFCs, unlike MBCs, differentiate under the influence of DRP1 (Matra, 2012).

PFCs are known to be specified by EGF receptor (EGFR) signaling. In egfrt1/egfrt1 egg chambers (hypomorphic allele of EGFR), mitochondria in PFCs are primarily clustered to one side of the nucleus, in contrast to those in wild-type or egfrt1/+ egg chambers, in which mitochondria are dispersed throughout cells. A similar clustering of mitochondria occurs when a dominant-negative (DN) form of EGFR (EGFR-DN) is clonally expressed in the PFC population. Because PFC mitochondria cluster/fuse in the absence of EGFR signaling, the data suggest that EGFR activation in PFCs promotes mitochondria fragmentation in these cells. This could explain why MBCs, which do not receive the EGFR signal, have fused mitochondria. The underlying basis for how EGFR signaling influences mitochondrial dynamics (by altering fission or fusion components) requires further investigation (Matra, 2012).

Interestingly, PFCs expressing EGFR-DN did not escape differentiation in spite of having clustered mitochondria. This may imply that a highly fused mitochondrial cluster may only allow escape from differentiation in the context of activated EGFR signaling. Indeed, EGFR-DN expression in drp1KG PFC clones (with fused mitochondria) partially induces differentiation (i.e., Hnt expression) in 40% of the clonal cells compared with no Hnt expression in drp1KG PFC clone. Expression of an activated form of EGFR (EGFR-Act) did not induce differentiation in drp1KG PFC clones. This explains why MBCs, which are not exposed to the EGFR ligand, do not proliferate under DRP1 down-regulation. Thus, cross talk exists between mitochondria and the EGFR signaling pathway in postmitotic PFCs, which helps cells decide whether to differentiate or continue in the mitotic cycle (Matra, 2012).

Whether DRP1 activity is important for regulation of cell cycle exit of mitotic follicle cells to allow onset of differentiation was investigated. The majority of follicle cells in S1-5 (during which all cells are mitotic) have fragmented mitochondria, suggesting that DRP1-dependent fission activity is high. drp1KG follicle cell clones introduced into the mitotic follicle cell layer and lacking UbiGFP harbor characteristic mitochondrial clusters. Clones also contain more pH3-positive cells and have qualitatively greater incorporation of BrdU relative to nonclonal tissue, with Cut expression unaltered. Without DRP1, therefore, S1-5 follicle cells undergo faster mitotic cycling (Matra, 2012).

To test whether DRP1 activity is necessary for mitotic cells to differentiate, Marf-1 RNAi was expressed to allow unopposed DRP1 activity in S1-5 egg chambers. Strikingly, Marf-1 RNAi expressing follicle cell clones (marked by CD8GFP) show premature expression of Hnt, whereas neighboring nonclonal mitotic follicle cells do not. The effect is not restricted to any stage or region of the mitotic follicle cell layer. The Marf-1 RNAi follicle cell clones exhibit increased mitochondrial mass as assessed by HSP-60 staining and MitoTracker loading, similar to that reported previously from mitofusin knockout mice . Importantly, drp1KG follicle cell clones expressing Marf-1 RNAi do not show premature differentiation; Hnt and HSP-60 expression levels are comparable with wild-type cells. Therefore, the premature differentiation of Marf-1 RNAi clones is dependent on DRP1. This indicates that DRP1-driven mitochondrial fission activity is required for mitotic follicle cells to exit the cell cycle and initiate their differentiation regimen (Matra, 2012).

Because of DRP1's role in differentiation, lack of DRP1 should generate developmental defects. Consistently, DRP1 down-regulation in early follicle cells in the germarium inhibits stalk cell formation, required to separate consecutive egg chambers. The missing stalk cells in egg chambers, encapsulated by early drp1KG follicle cell clones, leads to fused egg chambers containing pH3-labeled drp1KG clonal cells that lack UbiGFP. FasIII-enriched polar cells, known to induce stalk cells, are seen in wild-type ovarioles but are absent in the drp1KG clonal follicle cell population. Lack of polar cells is not the basis of cell proliferation of drp1KG PFCs because FasIII-positive polar cells appear in the surrounding heterozygous tissue. In addition, compound egg chambers with drp1KG follicle stem cell clones frequently arise, including egg chambers with 30 nurse cells and two oocytes (Matra, 2012).

Down-regulation of DRP1 also causes developmental defects in the postmitotic follicle cell layer. There, in 22% of the cases, drp1KG PFC clones fail to trigger migration of the oocyte nucleus toward the anterior. The postmitotic stage drp1KG phenotypes resemble loss of function of the Hippo-Salvador-Warts pathway, which has tumor suppressor effects in higher organisms, including mice (Matra, 2012).

The observed link between cell differentiation and mitochondrial fission state during oogenesis could relate to cyclin E, which controls S-phase entry. Indeed, inhibition of mitochondrial ATP synthesis in a cytochrome oxidase mutant promotes specific degradation of cyclin E (but not other cyclins) and blocks S-phase entry in Drosophila. In fibroblasts, cyclin E levels increase under conditions of DRP1 inhibition. In Drosophila follicle cells cyclin E levels were found to increase when DRP1 is down-regulated and decrease when Marf-1 is down-regulated. This suggests that DRP1-driven mitochondrial fission activity may cause cell cycle exit by lowering cyclin E levels to allow differentiation (Matra, 2012).

The results support a model in which mitochondrial fission/fusion dynamics regulates cell differentiation across the follicle cell layer of the Drosophila ovariole (see A model for mitochondria's role in cell fate determination). In mitotic stages, increased DRP1-driven mitochondrial fission is required for cell cycle exit as noted in premature DRP1-dependent differentiation of Marf-1 RNAi clones and enhanced proliferation of drp1KG clones. During postmitotic transition, activation of EGFR in the posterior region causes mitochondrial fragmentation. This, in turn, permits cell cycle exit and Notch activation, which drives PFC differentiation. In drp1KG PFC clones with fused mitochondria, therefore, Notch remains inactive, and cells proliferate. In the main body region, not exposed to the EGFR ligand, postmitotic differentiation and patterning occur in the absence of DRP1. Thus, cell proliferation/differentiation mechanisms have an intimate relationship to mitochondrial morphology and function during follicle layer development (Matra, 2012).

Lamellipodin and the Scar/WAVE complex cooperate to promote cell migration in vivo

Cell migration is essential for development, but its deregulation causes metastasis. The Scar/WAVE complex is absolutely required for lamellipodia and is a key effector in cell migration, but its regulation in vivo is enigmatic. Lamellipodin (Lpd) controls lamellipodium formation through an unknown mechanism. This study reports that Lpd directly binds active Rac, which regulates a direct interaction between Lpd and the Scar/WAVE complex via Abi. Consequently, Lpd controls lamellipodium size, cell migration speed, and persistence via Scar/WAVE in vitro. Moreover, Lpd knockout mice display defective pigmentation because fewer migrating neural crest-derived melanoblasts reach their target during development. Consistently, Lpd regulates mesenchymal neural crest cell migration cell autonomously in Xenopus laevis via the Scar/WAVE complex. Further, Lpd's Drosophila melanogaster orthologue Pico binds Scar, and both regulate collective epithelial border cell migration. Pico also controls directed cell protrusions of border cell clusters in a Scar-dependent manner. Taken together, Lpd is an essential, evolutionary conserved regulator of the Scar/WAVE complex during cell migration in vivo (Law, 2013).

This study reveals that Lpd colocalizes with the Scar/WAVE complex at the very edge of lamellipodia and directly interacts with this complex by binding to the Abi-SH3 domain. Active Rac directly binds Lpd, thereby regulating the interaction between Lpd and the Scar/WAVE complex. It is therefore postulated that Lpd acts as a platform to link active Rac and the Scar/WAVE complex at the leading edge of cells to regulate Scar/WAVE-Arp2/3 activity and thereby lamellipodium formation and cell migration (Law, 2013).

Knockdown of Lpd expression or KO of Lpd highly impaired lamellipodium formation, phenocopying the effect of Scar/WAVE complex knockdown on lamellipodium formation. Conversely, it was observed that overexpression of Lpd increased lamellipodia size in Xenopus NC cells, and this was dependent on the interaction with Abi, linking it to the Scar/WAVE complex. Overexpression of Pico, the Lpd fly orthologue, aberrantly increased the number and frequency of cellular protrusions at the rear of border cell clusters in a Scar-dependent manner, which suggests that the regulation of Scar/WAVE by Lpd is evolutionary conserved. Collectively, these data suggest that Lpd functions to generate lamellipodia via the Scar/WAVE complex (Law, 2013).

Lpd or Pico knockdown or Lpd KO impaired cell migration in vitro and in vivo in Drosophila, Xenopus, and mice. Lpd KO or knockdown cells were unable to migrate via lamellipodia but instead migrated very slowly by extending filopodia. The same residual migration mode had been observed for Arp2/3 knockdown cells. Arp2/3 is activated by the Scar/WAVE complex to regulate cell migration. It was also observed that both Lpd and Abi knockdown impaired NC migration in vivo. Consistently, it was found that Lpd and Abi-Scar/WAVE are in the same pathway regulating cell migration. This is consistent with recent studies suggesting that the Lpd orthologue in C. elegans, mig-10, genetically interacts with abi-1 to regulate axon guidance, synaptic vesicle clustering, and excretory canal outgrowth in C. elegans (Stavoe, 2012; Xu, 2012; McShea, 2013). Collectively, these results suggest that Lpd functions in cell migration via the Scar/WAVE complex in mammalian cells, Xenopus NC cells, and Drosophila border cells (Law, 2013).

Lpd not only interacts with the Scar/WAVE complex but also directly binds to Ena/VASP proteins. Ena/VASP proteins regulate actin filament length by temporarily preventing capping of barbed ends and by recruiting profilin-actin to the growing end of actin filaments. In contrast, the Scar/WAVE-Arp2/3 complexes increase branching of actin filaments. Lamellipodia with a highly branched actin network protrude more slowly but are more persistent, whereas lamellipodia with longer, less branched actin filaments protrude faster but are less stable and quickly turn into ruffles. It was observed that Lpd overexpression increases cell migration in a Scar/WAVE- and not Ena/VASP-dependent manner. This is consistent with a predominant function of Scar/WAVE downstream of Lpd to regulate a highly branched actin network supporting persistent lamellipodia protrusion and cell migration. Other actin-dependent cell protrusions such as axon extension or dorsal ruffles of fibroblasts require Lpd-Ena/VASP-mediated F-actin structures (Law, 2013).

Collective cell migration describes a group of cells that moves together and affect each other, and various types of collective cell migration exists during development and cancer invasion. Xenopus NC cells migrate as loose streams, whereas Drosophila border cells migrate as a cluster of cells with close cell-cell contacts. This study found that Rac regulates Lpd and Scar/WAVE interaction and that both are required for Xenopus NC migration, which is consistent with previous work in which Rac activity mediates this type of migration. Similarly, NC-derived melanoblast migration in the mouse depends on Rac-Scar/WAVE-Arp2/3, and it was found that Lpd functions in this process as well (Law, 2013).

Drosophila border cell clusters migrate through the fly egg chamber in two phases: an early part characterized by large and persistent front extensions, which are regulated predominantly by PVR (the fly PDGF receptor); and a late part characterized by dynamic collective 'tumbling' behavior. Surprisingly, Pico overexpression resulted in the appearance of a higher proportion of rear facing extensions, a phenotype previously observed with dominant-negative PVR, causing premature tumbling of the border cell cluster. This suggests that Pico function is normally tightly controlled to stabilize specific extensions and functions also in guidance of collective cell migration. Because Lpd-Scar/WAVE control single cell migration as well as collective cell migration, this suggests that they function as general regulators of cell migration (Law, 2013).

Collectively, this study has identified a novel pathway in which Lpd functions as an essential, evolutionary conserved regulator of the Scar/WAVE complex during cell migration in vivo (Law, 2013).

Drosophila eggshell production: identification of new genes and coordination by Pxt

Drosophila ovarian follicles complete development using a spatially and temporally controlled maturation process in which they resume meiosis and secrete a multi-layered, protective eggshell before undergoing arrest and/or ovulation. Microarray analysis revealed more than 150 genes that are expressed in a stage-specific manner during the last 24 hours of follicle development. These include all 30 previously known eggshell genes, as well as 19 new candidate chorion genes and 100 other genes likely to participate in maturation. Mutations in pxt, encoding a putative Drosophila cyclooxygenase, cause many transcripts to begin expression prematurely, and are associated with eggshell defects. Somatic activity of Pxt is required, as RNAi knockdown of pxt in the follicle cells recapitulates both the temporal expression and eggshell defects. One of the temporally regulated genes, cyp18a1, which encodes a cytochrome P450 protein mediating ecdysone turnover, is downregulated in pxt mutant follicles, and cyp18a1 mutation itself alters eggshell gene expression. These studies further define the molecular program of Drosophila follicle maturation and support the idea that it is coordinated by lipid and steroid hormonal signals (Tootle, 2011).

These studies show that a large fraction of the genes involved in eggshell production can be identified by simply scoring for stage-specific changes in transcript levels during late oogenesis. Not only were virtually all of the previously known structural genes involved in producing yolk, the vitelline membrane and the chorion identified, but we also discovered at least 19 new candidate eggshell proteins. Despite the simplicity of the protocol, the expression profiles revealed by these experiments agreed closely with previous studies, including the temporal programs reported by Fakhouri (2006) for 10 minor chorion proteins as determined by whole mount in situ hybridization. Many of the genes identified as eggshell genes are included among 81 genes reported as candidate targets of Egfr signaling by Yakoby (2008). Additionally, the expression of many genes involved in follicle maturation are spatially regulated within the folliclar epithelium, and the method used in this study could serve as an efficient pre-screen before undertaking such studies (Tootle, 2011).

It is interesting to compare these results with studies of eggshell proteins isolated from whole ovaries and analyzed by mass spectrometry (Fakhouri, 2006). The current analyses confirm many of the 22 genes identified in that study, including CG11381 (FBgn0029568), CG13083 (FBgn0032789), CG13084, CG13114, CG14796 (FBgn0025390), CG15570, CG4009, and CG31928. CG3074 (FBgn0034709) and CG13992 (FBgn0031756) are expressed at lower levels during stage 10 and may encode vitelline membrane proteins. The value of using temporal regulation as a criterion to identify eggshell components is highlighted by the fact that transcripts corresponding to the minor basement membrane components identified by Fakhouri (2006) remain constant during eggshell formation, suggesting that they derive from matrix material adherent to the purified eggshells, rather than representing true eggshell constituents (Tootle, 2011).

Like the known eggshell genes, many of the 19 new candidate eggshell genes reside within existing or new gene clusters. For example, within the vitelline membrane gene cluster at 26A, it is predicted that CG13998 encodes a novel Vm protein. CG13998 is expressed with the same stage specificity as other Vm proteins encoded within the 26A cluster, but is much less abundant. It may encode a rare or spatially restricted component of the vitelline membrane (Tootle, 2011).

A number of the putative eggshell genes encode for proteins with mucin-like domains (CG32774/Muc4B, CG32642/Mur11D, CG32602/Muc12Ea). All three were scored as ovary-specific in FlyAtlas. Mucin domains are heavily glycosylated and proteins with such domains often create a gel-like secretion. Mucins are known to function as components of chicken eggshells and coat the reproductive tract in some animals. CG32774/Muc4B is expressed mainly at S12. Based on the functions of mucin proteins and the timing of gene expression, it is postulated the Muc4B may be a component of the wax layer that is located between the vitelline membrane and the chorion. The other mucin-like domain proteins are expressed at S14 and may mediate chorion hardening, protection against infection, or serve as a coating necessary for passage through the oviduct (Tootle, 2011).

Another class of predicted eggshell genes encode for proteases and protease inhibitors. Such proteins have recently been shown to be a major class of chicken eggshell components. CG31928, CG31926, and CG31661 are aspartic proteases. Proteases may act to process other chorion proteins into their mature form, and/or contribute to eggshell hardening. Previously, the protease CG31928 has been shown by in situ hybridization to exhibit a posterior restriction. This raises the idea that proteases may be spatially restricted to alter chorion structure at specific regions that are important for subsequent function. It seems likely that other proteases may be restricted anteriorly, to the operculum area, to alter the eggshell structure to subsequently mediate larval hatching. In addition to proteases, it was found that a number of protease inhibitors are eggshell candidates, including CG15721 (S14), CG12716 (S14), CG1077 (S12), and CG15418 (S12; FBgn0031554). Stage 12-expressed protease inhibitors may regulate eggshell proteases, while stage 14-expressed inhibitors may perform anti-microbial activities. Both the proteases and their inhibitors may also contribute to the embryo's ability to reutilize eggshell components for its development (Tootle, 2011).

The nature of the peroxidase that crosslinks the eggshell has been controversial. Various proteins have been suggested to function as the crosslinking peroxidase, including Pxd (FBgn0004577) and Pxt. This study found pxt, the COX-like enzyme, is expressed and pxd is absent throughout all stages of egg maturation. In contrast, the eggshell protein and putative peroxidase CG4009 is very highly expressed during S12. It is proposed that CG4009 is the peroxidase that crosslinks the eggshell (Tootle, 2011).

In addition to known and novel eggshell components, many temporally regulated genes expressed during late follicle development were identified. A number of putative lipid-processing genes exhibit stage specific expression, suggesting a role in yolk or pheromone production. CG9747 (FBgn0039754) encodes for an acyl-CoA Δ11-desaturase, which is likely to desaturate palmitate; such enzymes are important for pheromone biosynthesis in other insects. CG8303 (FBgn0034143) is highly expressed as stages 9/10A and 10B, and encodes for an acyl-CoA reductase, which activates fatty acids by adding CoA. Additionally two Elvol (elongation of very long chaing fatty acids) encoding genes, bond (CG6921; FBgn0260942) and CG2781 (FBgn0260942), are expressed at S10A and S10B, respectively. Bond has previously been shown to be required for both male and female fertility, and by in situ hybridization appears to be expressed during oogenesis in the main body follicle cells at stages 9 and 10. These genes may contribution in a manner that is not currently understood to mediate the production of lipid yolk, or they may function in the production of lipid-based signals that contribute to egg maturation (Tootle, 2011).

Pxt mutations partially uncouple morphological development and gene expression. Yolk protein genes turn off normally in pxt mutant follicles, but vitelline membrane genes continue to be expressed longer than normal. Some chorion genes turn on earlier than normal, while the expression of others is delayed or prolonged. Many possible mechanisms may underlie these changes. However, the possibility that Pxt coordinates the production of Prostaglandins (PGs) that interact with other mechanisms to precisely control egg maturation is particularly interesting (Tootle, 2011).

In all sexually reproducing organisms the growth and development of the somatic and germ cells are mutually dependent and must be coordinated. Such coordination requires bi-directional communication. Historically, somatic cells were thought to regulate follicle development, including maintaining meiotic arrest, promoting meiotic resumption, and suppressing oocyte transcription prior to nuclear maturation. It has more recently been shown that the oocyte also signals to the soma. Oocyte signaling is necessary for follicular formation, and regulating the proliferation and differentiation of the somatic cells. It is generally thought that the oocyte has a greater influence on the soma early in follicular development and this is reversed during the later stages (Tootle, 2011).

There is emerging evidence that PG signaling coordinates germline and somatic development within mammalian follicles. While both oocyte and somatic maturation are delayed in COX2 knockout mice, it has been shown that the PGs are required in the soma for fertility. Specifically, COX2 is required in the somatic cells for cumulus (somatic) cell expansion and survival. However, meiotic resumption is not controlled by PGs from the soma. These germline and somatic events must be coordinated for the follicle to be competent for fertilization. This study found that PG signaling is required for both germline and somatic development during Drosophila follicle development. Fertility requires both of these signals. Specifically, PG signaling within the germline is necessary for mediating nurse cell dumping, the contractile process by which the oocyte is supplied with materials required for embryonic development, while PG signaling within the follicle cells is needed to regulate the timing of eggshell gene expression and subsequent eggshell structure. Thus PG signals, from insects to mammals, maintain the synchronized development of the germline and somatic cells within the individual follicle (Tootle, 2011).

Female reproduction is regulated by a complement of hormones that are cyclically produced and secreted. One such hormone that interacts with PG signaling in mammals is oxytocin. Oxytocin plays critical roles in regulating the function of the corpus luteum, a transient endocrine organ that secretes hormones to regulate the menstrual cycle and the early stages of pregnancy. In the absence of pregnancy, PGF2alpha stimulates the release of oxytocin to mediate luteolysis or the regression of the corpus luteum. During parturition, oxytocin and PGF2alpha also play critical roles. Oxytocin initiates labor, inducing PGF2alpha, which maintains labor and dilates the cervix (Tootle, 2011).

PGs and estrogen co-regulate each other in multiple cells types, including breast cancer cells. Breast tissue is the largest producer of estrogen in post-menopausal women; aromatase, Cyp19, leads to the production of estradiol. There is a high correlation between aromatase and COX2 expression in human breast cancer samples. Specifically, PGE2 signals via cAMP and PKA to stimulate a promoter upstream of cyp19, leading to increased aromatase expression. Autocrine and paracrine feedback loops via estradiol subsequently increase PGE2 secretion. Therefore, in breast cancer cells, PG and estrogen signaling are intimately linked (Tootle, 2011).

PGs and estrogen also interact in endometriotic tissue. Both PGE2 and PGF2alpha are excessively produced in uterine and endometriotic tissues of women with endometriosis. In the endometriotic stromal cells, PGE2 stimulates the expression of all the steroidogenic genes needed to synthesis estradiol from cholesterol. This occurs via PGE2 activation of cAMP/PKA signaling which upregulates of the expression of steroidogenic acute regulatory gene (StAR) and cyp19 . The expression of these steroidogenic genes is regulated by Steroidogenic Factor 1 (SF1), a nuclear hormone receptor. PGE2 signaling leads to SF1 out competing other transcription factors, Chicken Ovalbumin Upstream Promoter Transcription Factor (COUP-TF) and Wilms' tumor-1 (WT-1), for binding to steroidogenic gene promoters. Thus, PG signaling coordinates the expression of all steroidogenic genes (Tootle, 2011).

These results encourage future efforts to further establish the roles for PG signaling during Drosophila egg maturation and specifically, to learn how PGs are connected to steroid hormones. The Drosophila hormone ecdysone plays several critical roles during oogenesis. The loss of ecdysone signaling arrests follicle development at stage 8. Additionally, ecdysone signaling is needed to control the onset of chorion gene amplification, and to activate eggshell gene expression via transcriptional regulation. Temporally programmed changes in ecdysone levels may contribute to the timed control of eggshell gene expression. These studies provide a foundation for further dissecting the roles of Pxt and ecdysone-mediated signaling during late follicle development. If important aspects of these interactions have been conserved during evolution, the Drosophila ovary may emerge as a model for understanding the cellular and molecular changes underlying mammalian follicular maturation, endometriosis and infertility (Tootle, 2011).

Simple expression domains are regulated by discrete CRMs during Drosophila oogenesis

Eggshell patterning has been extensively studied in Drosophila melanogaster. However, the cis-regulatory modules (CRMs), which control spatiotemporal expression of these patterns, are vastly unexplored. The FlyLight collection contains over 7,000 intergenic and intronic DNA fragments that, if containing CRMs, can drive the transcription factor GAL4. The 84 genes known to be expressed during D. melanogaster oogenesis were cross-listed with the ~1200 listed genes of the FlyLight collection, and 22 common genes were found that are represented by 281 FlyLight fly lines. Of these lines, 54 show expression patterns during oogenesis when crossed to an UAS-GFP reporter. Of the 54 lines, 16 recapitulate the full or partial pattern of the associated gene pattern. Interestingly, while the average DNA fragment size is ~3kb in length, the vast majority of fragments show one type of a spatiotemporal pattern in oogenesis. Mapping the distribution of all 54 lines, a significant enrichment of CRMs was found in the first intron of the associated genes' model. In addition, the use was demonstrated of different anteriorly active FlyLight lines as tools to disrupt eggshell patterning in a targeted manner. This screen provides further evidence that complex gene-patterns are assembled combinatorially by different CRMs controlling the expression of genes in simple domains (Revaitis, 2017).

Three-dimensional epithelial morphogenesis in the developing Drosophila egg

Morphogenesis of the respiratory appendages on eggshells of Drosophila species provides a powerful experimental system for studying how cell sheets give rise to complex three-dimensional structures. In Drosophila, each of the two tubular eggshell appendages is derived from a primordium comprising two distinct cell types. Using live imaging and three-dimensional image reconstruction, it was demonstrated that the transformation of this two-dimensional primordium into a tube involves out-of-plane bending followed by a sequence of spatially ordered cell intercalations. These morphological transformations correlate with the appearance of complementary distributions of myosin and Bazooka in the primordium. These distributions suggest that a two-dimensional pattern of line tensions along cell-cell edges on the apical side of the epithelium is sufficient to produce the observed changes in morphology. Computational modeling shows that this mechanism could explain the main features of tissue deformation and cell rearrangements observed during three-dimensional morphogenesis (Osterfield, 2013).

The formation of 3D structures from epithelial sheets is a key feature of embryonic development. The Drosophila egg chamber provides a powerful model for studying these processes. This study analyzed how the dorsal appendage tubes emerge from the follicular epithelium. It was found that tube formation in this system preserves the integrity of the follicular epithelium and proceeds through a combination of sheet bending and lateral cell rearrangements. Based on the localization patterns of myosin and Baz, it is hypothesized that these events are caused by forces within the apical surface of the sheet. The special feature of this model is that it results in tissue transformations similar to those observed experimentally utilizing tensions generated exclusively in the 2D apical surface. Note that previous 3D extensions to the vertex model have modeled cells as 3D prisms. In contrast, the current approach involves allowing an essentially 2D object, the apical surface of the epithelial sheet, to move and deform in 3D space. The morphological changes in this model are driven by apical processes, without consideration of other cellular features such as volume constraints and active processes on the basal surface. At this point, the feasibility of this model is supported mainly by the computational studies that demonstrate how a pattern of tensions within a sheet can first bend the sheet and then initiate ordered intercalations, forming the seam of the tube. The patterns of apical tension predicted by this model agree qualitatively with the localization patterns of myosin in the appendage primordium at different stages of tube formation. In the future, however, this model should be tested by direct measurements of tensions, for example by laser ablation, and extended to account for processes on the basolateral cell surfaces as well as the processes associated with tube elongation (Osterfield, 2013).

Several mathematical models have been proposed for bending of cell sheets. One mechanism, working in both plants and animals, relies on spatial differences in cell proliferation, which causes tissue deformations. Since the follicle cells do not divide during the stages analyzed in this work, this mechanism does not apply to dorsal appendage morphogenesis. Other mechanisms, such as those put forward for vertebrate neurulation and ventral furrow formation in Drosophila, work through apical constriction, which occurs in the current system as well. However, one additional element common to these models is that bending is generated by a difference in apical versus basal properties. This clearly does not drive dynamics in the current model, which considers only the apical surfaces. Instead, out-of-plane displacements of the appendage primordium can be understood as a manifestation of buckling, whereby mechanical forces within the sheet give rise to states that can be either flat or bent, with the bent state having a lower energy. It will be interesting to explore whether similar models can predict out-of-plane deformations in other systems, such as those seen during eversion of imaginal discs (Osterfield, 2013).

In the current model, patterned apical tension is sufficient to explain not only buckling but also ordered intercalation. Although cell intercalation in the simulations is spatially ordered in a manner reminiscent to that seen in live imaging, there are some differences that should be interesting to explore in the future. In the imaging data, the floor cells eventually form two rows of floor cells separated by a relatively straight seam, while in the simulations the seam is more uneven and sometimes disrupted by the presence of one or more floor cells between these row. This suggests the possible existence of additional mechanisms for highly ordered intercalation, beyond those included in the model. One potential mechanism to explore further both experimentally and computationally is the possible formation of rosettes, since recent studies in other systems indicate that the use of rosettes in addition to T1 transitions may increase the efficiency of intercalation-mediated processes such as migration and tissue elongation (Osterfield, 2013).

In the current model of dorsal appendage formation, patterned line tension plays a key role. Future work will be needed to address the molecular mechanisms by which patterns of tension are established. Included among the genes with patterned expression in the late follicular epithelium are several that encode proteins involved in cytoskeleton regulation or cell-cell adhesion. Mutations in some of these genes result in dorsal appendage defects, but whether these genes work through regulating tension or through some other process has been largely unexplored (Osterfield, 2013).

Tube formation is a common outcome of epithelial morphogenesis. Sealing or closure of the tube is one of the least understood aspects in systems where tubes form by wrapping, as in the vertebrate neural tube or the Drosophila ventral furrow. The dorsal appendage tube appears to be sealed by spatially ordered lateral cell rearrangements. This suggests that lateral rearrangements may play a role in seam sealing in other cases of wrapping as well. Lateral rearrangements alone cannot be sufficient to drive morphogenesis in cases where the tube becomes discontinuous from its parental sheet, but future studies may reveal whether lateral rearrangements nevertheless play a key role in such systems (Osterfield, 2013).

A dynamic population of stromal cells contributes to the follicle stem cell niche in the Drosophila ovary

Epithelial stem cells are maintained within niches that promote self-renewal by providing signals that specify the stem cell fate. In the Drosophila ovary, epithelial follicle stem cells (FSCs) reside in niches at the anterior tip of the tissue and support continuous growth of the ovarian follicle epithelium. This study demonstrates that a neighboring dynamic population of stromal cells, called escort cells, are FSC niche cells. Escort cells produce both Wingless and Hedgehog ligands for the FSC lineage, and Wingless signaling is specific for the FSC niche whereas Hedgehog signaling is active in both FSCs and daughter cells. In addition, this study shows that multiple escort cells simultaneously encapsulate germ cell cysts and contact FSCs. Thus, FSCs are maintained in a dynamic niche by a non-dedicated population of niche cells (Sahai-Hernandez, 2013).

Taken together, the results of this study challenge the notion that the FSC niche is maintained by gradients of ligands produced solely at distant sites. Instead, the data indicate that the FSC niche has a more canonical architecture in which at least some key niche signals are produced locally, although the FSC niche might also differ from other well-characterized niches in some ways, such as the extent to which it remodels during adulthood. Notably, the results do not contradict the observation that Hh protein relocalizes from apical cells to the FSC niche during changes from a poor to a rich diet, as the flies were consistently maintained on nutrient-rich media. It will be interesting to investigate how such distantly produced ligands interact with locally produced niche signals to control FSC behavior during normal homeostasis and in response to stresses (Sahai-Hernandez, 2013).

In addition, the results confirm and extend the conclusion that Wg acts specifically on FSCs and ISCs, thus highlighting the role of Wg as a specific epithelial stem cell niche factor. As in other types of stem cell niches, this specificity could be achieved through multiple mechanisms, including local delivery of the Wg ligand to the niche and crosstalk with other pathways such as Notch and Hh, which are known to interact with the Wg pathway. Although the precise function(s) of Wg signaling in FSCs is unclear, the observation that a reduction in Wg ligand results in a backup of cysts near the FSC niche at the region 2a/2b border and fused cysts downstream from the FSC niche suggests that one role is to promote FSC proliferation. In addition, the finding that FSC daughter cells with ectopic Wg signaling fail to form into a polarized follicle epithelium suggests that Wg signaling might also promote self-renewal in FSCs by suppressing the follicle cell differentiation program. By contrast, observations and published studies indicate that Hh signaling is not specific for the FSC niche but instead constitutes a more general signal that derives from multiple sources and regulates proliferation and differentiation in both FSCs and prefollicle cells. Consistent with this conclusion, Hh signaling is active throughout the germarium and is required both in FSCs to promote self-renewal and in prefollicle cells to promote development toward the stalk and polar lineages (Sahai-Hernandez, 2013).

Finally, multicolor labeling of somatic cells in the germarium indicated that multiple densely packed escort cell membranes surround region 2a cysts and contact the FSC niche. Although the possibility cannot be ruled out that one or more cells in this region are dedicated FSC niche cells, observations strongly suggest that at least some escort cells contribute to both germ cell development and the FSC niche. Since these escort cells are dynamic, constantly changing their shape and position to facilitate the passage of germ cell cysts, it is perhaps somewhat surprising that the FSCs are so stable in the tissue. Indeed, the rate of FSC turnover is comparable to that of female GSCs, which are maintained by a dedicated and more static niche cell population. It will be interesting to investigate how this dynamic population of escort cells is able to maintain such a stable microenvironment for the FSCs. One possibility is that redundant sources of niche signals may allow niches of this type to partially break down and reform as needed to rapidly accommodate the changing demands of the tissue (Sahai-Hernandez, 2013).

The current observations reinforce several themes that are emerging from recent studies of stem cell niches in different epithelial tissues. First, as in the FSC niche, the Wnt/Wg signaling pathway is a key stem cell niche signal in many Drosophila and mammalian epithelial tissues. Second, in several epithelial tissues, the stem cell self-renewal signals are also known to be produced by differentiated cells rather than a dedicated niche cell population. For example, Drosophila ISCs of the gut receive self-renewal signals from both nearby enterocytes and the surrounding visceral muscle. Likewise, mammalian ISCs at the base of the crypt receive self-renewal signals from Paneth cells, which are adjacent secretory cells with antimicrobial functions. Lastly, several epithelial niches have recently been shown to have a transitory capacity that may resemble the dynamic nature of the FSC niche. For example, stem cell niches can form de novo in the Drosophila intestine to accommodate increased food availability, and in the mammalian skin in response to hyperactive Wnt signaling. In addition, mammalian ISCs produce niche cells in vivo and can spontaneously reform a niche in culture. In all of these examples, it seems likely that the relationship between the epithelial stem cell and its niche is not static, but instead flexible and dynamic. Further studies of the Drosophila FSC niche and these other experimental models will continue to provide insights into the mechanism by which a dynamic epithelial stem cell niche functions (Sahai-Hernandez, 2013).

A genome-scale in vivo RNAi analysis of epithelial development in Drosophila identifies new proliferation domains outside of the stem cell niche

The Drosophila oogenesis system provides an excellent model to study the development of epithelial tissues. This study reports the first genome-scale in vivo RNAi screen for genes controlling epithelial development. By directly analysing cell and tissue architecture, 1125 genes were identified that were assigned to seven different functions in epithelial formation and homeostasis. The significance of the screen was validated by generating mutants for Vps60, a component of the ESCRT machinery. This analysis provided new insights into spatiotemporal control of cell proliferation in the follicular epithelium. Previous studies identified signals controlling divisions in the follicle stem cell niche. However, 99% of cell divisions occur outside of the niche and it is unclear how these divisions are controlled. The data distinguish two new domains with differential proliferation control outside of the stem cell niche. One domain abuts the niche and is characterised by ESCRT, Notch and JAK/STAT mediated proliferation control. Adjacently, another domain is defined by loss of ESCRT impact on cell division. Thus, during development epithelial cells pass through different modes of proliferation control. The switch between these modes might reflect regressing stemness of epithelial cells over time (Berns, 2014).

Coordinated niche-associated signals promote germline homeostasis in the Drosophila ovary

Stem cell niches provide localized signaling molecules to promote stem cell fate and to suppress differentiation. The Drosophila melanogaster ovarian niche is established by several types of stromal cells, including terminal filament cells, cap cells, and escort cells (ECs). This study shows that, in addition to its well-known function as a niche factor expressed in cap cells, the Drosophila transforming growth factor β molecule Decapentaplegic (Dpp) is expressed at a low level in ECs to maintain a pool of partially differentiated germline cells that may dedifferentiate to replenish germline stem cells upon their depletion under normal and stress conditions. This study further reveals that the Dpp level in ECs is modulated by Hedgehog (Hh) ligands, which originate from both cap cells and ECs. Hh signaling exerts its function by suppressing Janus kinase/signal transducer activity, which promotes Dpp expression in ECs. Collectively, these data suggest a complex interplay of niche-associated signals that controls the development of a stem cell lineage (Liu, 2015).

Stage-specific plasticity in ovary size is regulated by Insulin/Insulin-Like growth factor and Ecdysone signalling in Drosophila

Animals from flies to humans adjust their development in response to environmental conditions through a series of developmental checkpoints, which alter the sensitivity of organs to environmental perturbation. Despite their importance, little is known about the molecular mechanisms through which this change in sensitivity occurs. This study has identified two phases of sensitivity to larval nutrition that contribute to plasticity in ovariole number, an important determinant of fecundity, in Drosophila melanogaster. These two phases of sensitivity are separated by the developmental checkpoint called critical weight; poor nutrition has greater effects on ovariole number in larvae before critical weight than afterwards. This switch in sensitivity results from distinct developmental processes. In pre-critical weight larvae, poor nutrition delays the onset of terminal filament cell differentiation, the starting point for ovariole development, and strongly suppresses the rate of terminal filament addition and the rate of increase in ovary volume. Conversely, in post-critical weight larvae, poor nutrition only affects the rate of increase in ovary volume. These results further indicate that two hormonal pathways, the insulin/insulin-like growth factor and the ecdysone signalling pathways, modulate the timing and rates of all three developmental processes. The change in sensitivity in the ovary results from changes in the relative contribution of each pathway to the rates of TF addition and increase in ovary volume before and after critical weight. This work deepens the understanding of how hormones act to modify the sensitivity of organs to environmental conditions, thereby affecting their plasticity (Mendes, 2015).

Adipocyte metabolic pathways regulated by diet control the female germline stem cell lineage in Drosophila

Nutrients affect adult stem cells through complex mechanisms involving multiple organs. Adipocytes are highly sensitive to diet and have key metabolic roles, and obesity increases the risk for many cancers. How diet-regulated adipocyte metabolic pathways influence normal stem cell lineages, however, remains unclear. Drosophila melanogaster has highly conserved adipocyte metabolism and a well-characterized female germline stem cell (GSC) lineage response to diet. This study conducted an isobaric tags for relative and absolute quantification (iTRAQ) proteomic analysis to identify diet-regulated adipocyte metabolic pathways that control the female GSC lineage. On a rich (relative to poor) diet, adipocyte Hexokinase-C and metabolic enzymes involved in pyruvate/acetyl-coA production are upregulated, promoting a shift of glucose metabolism towards macromolecule biosynthesis. Adipocyte-specific knockdown shows that these enzymes support early GSC progeny survival. Further, enzymes catalyzing fatty acid oxidation and phosphatidylethanolamine synthesis in adipocytes promote GSC maintenance, whereas lipid and iron transport from adipocytes controls vitellogenesis and GSC number, respectively. These results show a functional relationship between specific metabolic pathways in adipocytes and distinct processes in the GSC lineage, suggesting the adipocyte metabolism-stem cell link as an important area of investigation in other stem cell systems (Matsuoka, 2017).

Impact of gut microbiota on the fly's germ line

Unlike vertically transmitted endosymbionts, which have broad effects on their host's germ line, the extracellular gut microbiota is transmitted horizontally and is not known to influence the germ line. This study provides evidence supporting the influence of these gut bacteria on the germ line of Drosophila melanogaster. Removal of the gut bacteria represses oogenesis, expedites maternal-to-zygotic-transition in the offspring and unmasks hidden phenotypic variation in mutants. It was further shown that the main impact on oogenesis is linked to the lack of gut Acetobacter species, and the Drosophila Aldehyde dehydrogenase (Aldh) gene was identified as an apparent mediator of repressed oogenesis in Acetobacter-depleted flies. The finding of interactions between the gut microbiota and the germ line has implications for reproduction, developmental robustness and adaptation (Elgart, 2016). 

Phantom, a cytochrome P450 enzyme essential for ecdysone biosynthesis, plays a critical role in the control of border cell migration in Drosophila

The border cells of Drosophila are a model system for coordinated cell migration. Ecdysone signaling has been shown to act as the timing signal to initiate the migration process. This study found that mutations in phantom (phm), encoding an enzyme in the ecdysone biosynthesis pathway, block border cell migration when the entire follicular epithelium of an egg chamber is mutant, even when the associated germline cells (nurse cells and oocyte) are wildtype. Conversely, mutant germline cells survive and do not affect border cell migration, as long as the surrounding follicle cells are wildtype. Interestingly, even small patches of wildtype follicle cells in a mosaic epithelium are sufficient to allow the production of above-threshold levels of ecdysone to promote border cell migration. The same phenotype is observed with mutations in shade (shd), encoding the last enzyme in the pathway that converts ecdysone to the active 20-hydroxyecdysone. Administration of high 20-hydroxyecdysone titers in the medium can also rescue the border cell migration phenotype in cultured egg chambers with an entirely phm mutant follicular epithelium. These results indicate that in normal oogenesis, the follicle cell epithelium of each individual egg chamber must supply sufficient ecdysone precursors, leading ultimately to high enough levels of mature 20-hydroxyecdysone to the border cells to initiate their migration. Neither the germline, nor the neighboring egg chambers, nor the surrounding hemolymph appear to provide threshold amounts of 20-hydroxyecdysone to do so. This 'egg chamber autonomous' ecdysone synthesis constitutes a useful way to regulate the individual maturation of the asynchronous egg chambers present in the Drosophila ovary (Domanitskaya, 2013).

Ecdysone response gene E78 controls ovarian germline stem cell niche formation and follicle survival in Drosophila.

Nuclear hormone receptors have emerged as important regulators of mammalian and Drosophila adult physiology, affecting such seemingly diverse processes as adipogenesis, carbohydrate metabolism, circadian rhythm, stem cell function, and gamete production. Although nuclear hormone receptors Ecdysone Receptor (EcR) and Ultraspiracle (Usp) have multiple known roles in Drosophila development and regulate key processes during oogenesis, the adult function of the majority of nuclear hormone receptors remains largely undescribed. Ecdysone-induced protein 78C (E78), a nuclear hormone receptor closely related to Drosophila E75 and to mammalian Rev-Erb and Peroxisome Proliferator Activated Receptors, was originally identified as an early ecdysone target; however, it has remained unclear whether E78 significantly contributes to adult physiology or reproductive function. To further explore the biological function of E78 in oogenesis, this study used available E78 reporters and created a new E78 loss-of-function allele. E78 was found to be expressed throughout the germline during oogenesis, and was important for proper egg production and for the maternal control of early embryogenesis. E78 was required during development to establish the somatic germline stem cell (GSC) niche; E78 function in the germline promoted the survival of developing follicles. Consistent with its initial discovery as an ecdysone-induced target, there were significant genetic interactions between E78 and components of the ecdysone signaling pathway. Taken together with the previously described roles of EcR, Usp, and E75, these results suggest that nuclear hormone receptors are critical for the broad transcriptional control of a wide variety of cellular processes during oogenesis (Ables, 2015).

Although nuclear hormone receptors are known to play important roles in a wide variety of biological processes, it remains largely unknown whether or how most of the Drosophila nuclear hormone receptors function during oogenesis. This study adds to a growing body of literature demonstrating that nuclear hormone receptors are integral to reproductive function at multiple levels, including reproductive organ development, stem cell function, and gamete development and survival. While it remains unclear how E78 contributes mechanistically to the ecdysone signaling network in the ovary, these studies also highlight the intricate connections between Drosophila nuclear hormone receptors and ecdysone signaling. Given the level of structural and functional conservation between Drosophila and mammalian hormonal signaling pathways, it is proposed that similar connections may exist among diverse mammalian nuclear hormone receptor subtypes. Further studies will be necessary to fully elucidate the molecular networks that tie these pathways together to achieve such important biological regulation (Ables, 2015).

It is interesting to note that each of the ecdysone early response genes studied in the ovary to date (EcR, E74, E75, E78) encode at least two different mRNA isoforms: one long mRNA isoform resulting from splicing of a very long intron separating conserved DNA- and ligand-binding domains, and a shorter isoform that may or may not produce a distinct protein isoform. Previous studies have indicated that Ftz-f1, another ecdysone-regulated nuclear hormone receptor, is also encoded by two different mRNA isoforms: ftz-f1-RA (short isoform) is maternally deposited and required for embryogenesis, while ftz-f1-RB (long isoform) is required at other developmental stages. Future studies investigating whether the various isoforms of ecdysone early-response genes differentially control oogenesis versus embryogenesis will help refine understanding of how a steroid hormone may induce temporal-, developmental-, and cell type-specific effects (Ables, 2015).

While these studies demonstrate a specific requirement for E78 in promoting the survival of germline cysts, the mechanisms by which E78 controls cyst survival remain a topic for further exploration. Indeed, mutants of several ecdysone early-response genes, including EcR, E74, and E75, display similar cyst death near the onset of oocyte vitellogenesis, suggesting that ecdysone signaling promotes a maturation or survival cue during follicle development. Very little is known, however, about the targets of ecdysone signaling during earlier previtellogenic stages. Two recent large-scale screens for regulators of ecdysone-regulated cell death in a haemocyte cell line and in salivary glands may prove useful for identifying targets involved in the decision between cell death and survival. Interestingly, the stage 4/5 cyst death observed in E78δ31 mutants is phenocopied by mutations in insulin and target of rapamycin (TOR) signaling pathway components, including InR, chico, TOR, and S6 kinase Since EcR and E78 appear to functionally cooperate, future studies should test whether EcR and E78 regulate members of the insulin/TOR signaling pathways (or vice versa) to control cyst viability. These basic studies not only will help elucidate the mechanisms by which nuclear hormone receptors control biological processes, but may also add to general understanding of how nuclear hormone receptor signaling is integrated into other endocrine networks to coordinate cell-specific responses with whole-animal physiology (Ables, 2015).

A visual screen for diet-regulated proteins in the Drosophila ovary using GFP protein trap lines

The effect of diet on reproduction is well documented in a large number of organisms; however, much remains to be learned about the molecular mechanisms underlying this connection. The Drosophila ovary has a well described, fast and largely reversible response to diet. Ovarian stem cells and their progeny proliferate and grow faster on a yeast-rich diet than on a yeast-free (poor) diet, and death of early germline cysts, degeneration of early vitellogenic follicles and partial block in ovulation further contribute to the approximately 60-fold decrease in egg laying observed on a poor diet. Multiple diet-dependent factors, including insulin-like peptides, the steroid ecdysone, the nutrient sensor Target of Rapamycin, AMP-dependent kinase, and adipocyte factors mediate this complex response. This describe the results of a visual screen using a collection of green fluorescent protein (GFP) protein trap lines to identify additional factors potentially involved in this response. In each GFP protein trap line, an artificial GFP exon is fused in frame to an endogenous protein, such that the GFP fusion pattern parallels the levels and subcellular localization of the corresponding native protein. Fifty-three GFP-tagged proteins were identified that exhibit changes in levels and/or subcellular localization in the ovary at 12-16 hours after switching females from rich to poor diets, suggesting them as potential candidates for future functional studies (Hsu, 2017).

The genetic architecture of ovariole number in Drosophila melanogaster: Genes with major, quantitative, and pleiotropic effects

Ovariole number has a direct role in the number of eggs produced by an insect, suggesting that it is a key morphological fitness trait. Many studies have documented the variability of ovariole number and its relationship to other fitness and life-history traits in natural populations of Drosophila. However, the genes contributing to this variability are largely unknown. A genome-wide association study of ovariole number was conducted in a natural population of flies. Using mutations and RNAi-mediated knockdown, the effects of twenty-four candidate genes on ovariole number was confirmed, including a novel gene, anneboleyn (formerly CG32000), that impacts both ovariole morphology and numbers of offspring produced. Pleiotropic genes were identified that regulated ovariole number traits and sleep and activity behavior. While few polymorphisms overlapped between sleep parameters and ovariole number, thirty-nine candidate genes were nevertheless in common. The effects of seven genes on both ovariole number and sleep were verified: bin3, blot, CG42389, kirre, slim, VAChT, and zfh1. Linkage disequilibrium among the polymorphisms in these common genes was low, suggesting that these polymorphisms may evolve independently (Lobell, 2017).

DIP1 modulates stem cell homeostasis in Drosophila through regulation of sisR-1

Stable intronic sequence RNAs (sisRNAs) are by-products of splicing and regulate gene expression. How sisRNAs are regulated is unclear. This study reports that a double-stranded RNA binding protein, Disco-interacting protein 1 (DIP1) regulates sisRNAs in Drosophila. DIP1 negatively regulates the abundance of sisR-1 and INE-1 sisRNAs. Fine-tuning of sisR-1 by DIP1 is important to maintain female germline stem cell homeostasis by modulating germline stem cell differentiation and niche adhesion. Drosophila DIP1 localizes to a nuclear body (satellite body) and associates with the fourth chromosome, which contains a very high density of INE-1 transposable element sequences that are processed into sisRNAs. DIP1 presumably acts outside the satellite bodies to regulate sisR-1, which is not on the fourth chromosome. Thus, this study identifies DIP1 as a sisRNA regulatory protein that controls germline stem cell self-renewal in Drosophila. Stable intronic sequence RNAs (sisRNAs) are by-products of splicing from introns with roles in embryonic development in Drosophila. The study shows that the RNA binding protein DIP1 regulates sisRNAs in Drosophila, which is necessary for germline stem cell homeostasis (Wong, 2017).

Recent studies have uncovered a class of stable intronic sequence RNAs (sisRNAs) that are derived from the introns post splicing. sisRNAs are present in various organisms such as viruses, yeast, Drosophila, Xenopus, and mammals. Studies in Drosophila and mammalian cells suggest that sisRNAs function in regulating the expression of their parental genes (host genes where they are derived from) via positive or negative feedback loops. In yeast, sisRNAs are involved in promoting robustness in response to stress, while in Drosophila, sisRNAs have been shown to be important for embryonic development. However, very little is understood about the biological functions of sisRNAs in terms of regulating cellular processes such as differentiation, proliferation, and cell death (Wong, 2017).

The Drosophila genome encodes for several double-stranded RNA (dsRNA) binding proteins that localize to the nucleus. Most of them have been found to regulate specific RNA-mediated processes such as RNA editing, X chromosome activation, and miRNA biogenesis. The Disco-interacting protein 1 (DIP1) is a relatively less characterized dsRNA binding protein that has been implicated in anti-viral defense and localizes to the nucleus as speckles. Otherwise, not much is known about the biological processes regulated by DIP1 (Wong, 2017 and references therein).

This paper show that the regulation of a Drosophila sisRNA sisR-1 by DIP1 is important for keeping female germline stem cell homeostasis in place. DIP1 is shown to regulate INE-1 sisRNAs and localizes to a previously undescribed nuclear body around the fourth chromosomes, called the satellite body. The regulation of sisR-1, which is not on the fourth chromosome, by DIP1 presumably does not occur in the satellite bodies (Wong, 2017).

The results reveal the importance of the regulation of sisRNA activity/expression in GSC-niche occupancy. It is proposed that the sisR-1 axis maintains GSCs in the niche, however, uncontrolled accumulation of sisR-1 due to its unusual stability can lead to increase number of GSCs at the niche. DIP1 in turn limits the build-up of sisR-1 to maintain ~2 GSCs per niche. GSC-niche occupancy is highly regulated by homeostatic mechanisms via negative feedback loops at the cellular and molecular levels. Misregulation of the niche may pose a problem as it allows for a greater chance of GSCs to accumulate mutations that may lead to tumor formation. On the other hand, mechanisms that promote GSC-niche occupancy may be important to facilitate the replenishment of GSCs during aging. Understanding the control of stem cell-niche occupancy will provide important insights to reproduction, cancer, and regenerative medicine (Wong, 2017).

In a large-scale RNAi screen for genes that regulate GSC self-renewal and differentiation, rga was identified as a gene required for GSC differentiation. How rga regulates GSC self-renewal is currently unknown but the current data suggest that GSC-niche adhesion and Mei-P26 are involved. The rga gene encodes for the NOT2 protein in the CCR4 deadenylase complex. Surprisingly, studies have shown that other components of the CCR4 complex such as CCR4, Not1, and Not3 function in promoting GSC maintenance. Interestingly, Twin has been proposed to function with distinct partners to mediate different effects on GSC fates. This suggests that other components such as CCR4 can also have additional functions outside the CCR4-NOT deadenylase complex in mediating GSC maintenance, thus affecting GSCs in opposite ways to Rga (Wong, 2017).

This study puts forward a proposed model for sisR-1-mediated silencing. It is hypothesized that folded sisR-1 harboring a 3' tail may form a ribonucleoprotein complex, which confers its stability, and allows scanning for its target via its 3' tail. Binding of the 3' tail to the target may promote local unwinding of sisR-1 as the 3' end of ASTR invades to form a more stable 76 nt duplex. This study shows that, in principle, it is possible to design a chimeric sisRNA to target a long ncRNA of interest such as rox1. In future, sisRNA can be potentially developed as tools to regulate nuclear RNAs of interest. Clearly, the efficiency and specificity of sisRNA-mediated silencing need to be optimized. Because sisRNA-mediated target degradation requires a more extensive base-pairing between sisRNA and the target, the chances of off-target effects ought to be lower than siRNAs and antisense oligonucleotides. In broader terms, this study provides a paradigm, which encourages exploration of whether other sisRNAs or ncRNAs utilize a similar silencing strategy as sisR-1 (Wong, 2017).

This study describes a nuclear body (named satellite body) that associates with the fourth chromosomes. Satellite body adds to an existing group of nuclear bodies (nucleolus, HLB, and pearl) that associate with specific genomic loci. It is generally believed that formation of such nuclear bodies correlates with a high concentration of RNA transcribed from the tandemly repeated gene loci. The formation of satellite bodies around the fourth chromosomes probably reflects a high concentration of DIP1 in regulating INE-1 sisRNAs transcribed there. The formation of satellite bodies may be promoted by the high concentration of INE-1 sisRNAs transcribed on the fourth chromosomes, and may facilitate the decay of INE-1 sisRNAs . It is speculated that in the nucleoplasm, DIP1 that does not form observable satellite bodies is sufficient to regulate sisRNAs such as sisR-1 transcribed from other chromosomes. Since DIP1 is a dsRNA binding protein, it may bind to mature sisRNAs to destabilize them. It may do so by recruiting RNA degradation factors (such as nuclear exosomes) or introducing RNA modification to 'mark' sisRNAs for degradation. In future, it will be important to identify more components of the satellite bodies and their dynamics during differentiation and in response to stimuli in order to better understand the molecular mechanism of sisRNA metabolism (Wong, 2017).

Cyclin B3 deficiency impairs germline stem cell maintenance and its overexpression delays cystoblast differentiation in Drosophila ovary

It is well known that cyclinB3 (cycB3) plays a key role in the control of cell cycle progression. However, whether cycB3 is involved in stem cell fate determination remains unknown. The Drosophila ovary provides an exclusive model for studying the intrinsic and extrinsic factors that modulate the fate of germline stem cells (GSCs). Here, using this model, Drosophila cycB3 was shown to plays a new role in controlling the fate of germline stem cells (GSC). Results from cycB3 genetic analyses demonstrate that cycB3 is intrinsically required for GSC maintenance. Results from green fluorescent protein (GFP)-transgene reporter assays show that cycB3 is not involved in Dad-mediated regulation of Bmp signaling, or required for dpp-induced bam transcriptional silencing. Double mutants of bam and cycB3 phenocopied bam single mutants, suggesting that cycB3 functions in a bam-dependent manner in GSCs. Deficiency of cycB3 fails to cause apoptosis in GSCs or influence cystoblast (CB) differentiation into oocytes. Furthermore, overexpression of cycB3 dramatically increases the CB number in Drosophila ovaries, suggesting that an excess of cycB3 function delays CB differentiation. Given that the cycB3 gene is evolutionarily conserved, from insects to humans, cycB3 may also be involved in controlling the fate of GSCs in humans (Chen, 2018).

Quantitative microscopy of the Drosophila ovary shows multiple niche signals specify progenitor cell fate

Adult stem cells commonly give rise to transit-amplifying progenitors, whose progeny differentiate into distinct cell types. It is unclear if stem cell niche signals coordinate fate decisions within the progenitor pool. This study used quantitative analysis of Wnt, Hh, and Notch signalling reporters and the cell fate markers Eyes Absent (Eya) and Castor (Cas) to study the effects of hyper-activation and loss of niche signals on progenitor development in the Drosophila ovary. Follicle stem cell (FSC) progeny adopt distinct polar, stalk, and main body cell fates. Wnt signalling transiently inhibits expression of the main body cell fate determinant Eya, and Wnt hyperactivity strongly biases cells towards polar and stalk fates. Hh signalling independently controls the proliferation to differentiation transition. Notch is permissive but not instructive for differentiation of multiple cell types. These findings reveal that multiple niche signals coordinate cell fates and differentiation of progenitor cells (Dai, 2017).

A targeted RNAi screen reveals Drosophila female-sterile genes that control the size of germline stem cell niche during development

Adult stem cells maintain tissue homeostasis. This unique capability largely depends on the stem cell niche, a specialized microenvironment, which preserves stem cell identity through physical contacts and secreted factors. In many cancers, latent tumor cell niches are thought to house stem cells and aid tumor initiation. However, in developing tissue and cancer it is unclear how the niche is established. The well-characterized germline stem cells (GSCs) and niches in the Drosophila melanogaster ovary provide an excellent model to address this fundamental issue. As such, this study conducted a small-scale RNAi screen of 560 individually expressed UAS-RNAi lines with targets implicated in female fertility. RNAi was expressed in the soma of larval gonads, and screening for reduced egg production and abnormal ovarian morphology was performed in adults. Twenty candidates that affect ovarian development were identified and subsequently knocked down in the soma only during niche formation. Feminization factors (Transformer, Sex lethal, and Virilizer), a histone methyltransferase (Enhancer of Zeste), a transcriptional machinery component (Enhancer of yellow 1), a chromatin remodeling complex member (Enhancer of yellow 3) and a chromosome passenger complex constituent (Incenp) were identified as potentially functioning in the control of niche size. The identification of these molecules highlights specific molecular events that are critical for niche formation and will provide a basis for future studies to fully understand the mechanisms of GSC recruitment and maintenance (Cho, 2018).

Stonewall and Brickwall: Two partially redundant determinants required for the maintenance of female germline in Drosophila

Proper specification of germline stem cells (GSCs) in Drosophila ovaries depends on niche derived non-autonomous signaling and cell autonomous components of transcriptional machinery. Stonewall (Stwl), a MADF-BESS family protein, is one of the cell intrinsic transcriptional regulators involved in the establishment and/or maintenance of GSC fate in Drosophila ovaries. This stufy reports identification and functional characterization of another member of the same protein family, CG3838/Brickwall (Brwl) with analogous functions. Loss of function alleles of brwl exhibit age dependent progressive degeneration of the developing ovarioles and loss of GSCs. Supporting the conclusion that the structural deterioration of mutant egg chambers is a result of apoptotic cell death, activated caspase levels are considerably elevated in brwl(-) ovaries. Moreover, as in the case of stwl mutants, on several instances, loss of brwl activity results in fusion of egg chambers and misspecification of the oocyte. Importantly, brwl phenotypes can be partially rescued by germline specific over-expression of stwl arguing for overlapping yet distinct functional capabilities of the two proteins. Taken together with the phylogenetic analysis, these data suggest that brwl and stwl likely share a common MADF-BESS ancestor and they are expressed in overlapping spatiotemporal domains to ensure robust development of the female germline (Shukla, 2018).

GTP exchange factor Vav regulates guided cell migration by coupling guidance receptor signalling to local Rac activation

Guided cell migration is a key mechanism for cell positioning in morphogenesis. The current model suggests that the spatially controlled activation of receptor tyrosine kinases (RTKs) by guidance cues limits Rac activity at the leading edge, which is crucial for establishing and maintaining polarized cell protrusions at the front. However, little is known about the mechanisms by which RTKs control the local activation of Rac. Using a multidisciplinary approach, this study identified the GTP exchange factor (GEF) Vav as a key regulator of Rac activity downstream of RTKs in a developmentally regulated cell migration event, that of the Drosophila border cells (BCs). Elimination of the vav gene impairs BC migration. Live imaging analysis reveals that vav is required for the stabilization and maintenance of protrusions at the front of the BC cluster. In addition, activation of the PDGF/VEGF-related receptor (PVR) by its ligand the PDGF/PVF1 factor brings about activation of Vav protein by direct interaction with the intracellular domain of PVR. Finally, FRET analyses demonstrate that Vav is required in BCs for the asymmetric distribution of Rac activity at the front. These results unravel an important role for the Vav proteins as signal transducers that couple signalling downstream of RTKs with local Rac activation during morphogenetic movements (Fernandez-Espartero, 2013).

Directed cell migration plays a crucial role in many normal and pathological processes such as embryo development, immune response, wound healing and tumor metastasis. During development, cells migrate to their final position in response to extracellular stimuli in the microenvironment. To migrate towards or away from a stimulus, individual cells or groups of cells must first achieve direction of migration through the establishment of cell polarity. Guidance cues, such as growth factors, control cell polarization through the regulated recruitment and activation of receptor tyrosine kinases (RTKs) to the leading edge. A key event downstream of RTK signalling in cell migration is the localization of activated Rac at the leading edge. However, little is known about the mechanisms by which external cues regulate Rac activity during cell migration. Rac is activated by GTP exchange factors (GEFs), which facilitate the transition of these GTPases from their inactive (GDP-bound) to their active (GTP-bound) states. Thus, GEFs appear as excellent candidates to regulate the cellular response to extracellular cues during cell migration (Fernandez-Espartero, 2013).

Among the different Rac GEF families characterized so far, the Vav proteins are the only ones known to combine in the same molecule the canonical Dbl (DH) and pleckstrin homology (PH) domains of Rac GEFs and the structural hallmark of tyrosine phosphorylation pathways, the SH2 domain. In addition, Vav activity is regulated by tyrosine phosphorylation in response to stimulation by transmembrane receptors with intrinsic or associated tyrosine kinase activity. These features make Vav proteins ideal candidates to act as signalling transducer molecules coupling growth factor receptors to Rac GTPase activation during cell migration. In fact, a number of cell culture experiments have suggested a role for the Vav proteins in cell migration downstream of growth factor signalling. Thus, the ubiquitously expressed mammalian Vav2 is tyrosine phosphorylated in response to different growth factors, including epidermal (EGF) and platelet-derived (PDGF) growth factors, and its phosphorylation correlates with enhanced Rac activity and migration in some cell types. However, the biological relevance for many of these interactions and the cellular mechanisms by which Vav regulates in vivo cell migration remains to be determined (Fernandez-Espartero, 2013).

The Vav proteins are present in all animal metazoans but not in unicellular organisms. There is a single representative in multicellular invertebrates and urochordata species (such as C. elegans, Drosophila melanogaster and Ciona intestinalis) and usually three representatives in vertebrates. The single Drosophila vav ortholog possesses the same catalytic and regulatory properties as its mammalian counterparts. In addition, the Drosophila Vav is tyrosine phosphorylated in response to EGF stimulation in S2 cells. Furthermore, a yeast two hybrid analysis has shown that the SH2-SH3 region of Vav can bind the epidermal growth factor receptor (EGFR) and the intracellular domain of PVR, PVRi, but not a kinase-dead version of PVRi, suggesting that Vav SH2-SH3-HA::PVRi interactions depend on PVR autophosphorylation. Altogether, these results suggest that the role of mammalian Vavs as transducer proteins coupling signalling from growth factors to Rho GTPase activation has been conserved in Drosophila. Thus, this study took advantage of Drosophila to analyse vav contribution to growth factor-induced cell migration in the physiological setting of a multicellular organism (Fernandez-Espartero, 2013).

The migration of the border cells (BCs) in the Drosophila egg chamber represents an excellent model system to study guided cell migration downstream of PVR/EGFR signalling in vivo. Each egg chamber contains one oocyte and 15 nurse cells surrounded by a monolayer of follicle cells (FCs), known as follicular epithelium (FE). The BC cluster is determined at the anterior pole of the FE and it comprises 6-8 outer cells and two anterior polar cells in a central position. BCs delaminate from the anterior FE and migrate posteriorly between the nurse cells until they contact the anterior membrane of the oocyte. BCs use the PVR and the EGFR to read guidance cues, the PDGF-related Pvf1 and the TGFβ-related Gurken, secreted by the oocyte. The Rho GTPase Rac is required for BC migration. The current model proposes that higher levels of Rac activity present in the leading cell determine the direction of migration and that this asymmetric distribution of Rac activity requires guidance receptor input. The unconventional Rac GEF Myoblast city, Mbc, is the only identified downstream signalling effector in this context. However, although genetic analysis have led to propose that the unconventional GEF for Rac, Mbc/DOCK 180, could activate Rac downstream of PVR during BC migration, this has not been formally proven. In addition, Mbc is unlikely to be the only Rac GEF actin downstream of guidance receptors in BCs as the migration phenotype due to complete removal of mbc is not as severe as the loss of both Pvr and Egfr. Thus, other effectors are likely to contribute to the complicated task of guiding BC migration. Many candidate molecules have been tested for their requirement in BC migration, MAPK pathway, PI3K, PLC-gamma, as well as RTK adaptors, such as DOCK, Trio, and Pak, but none of these is individually required (Fernandez-Espartero, 2013).

Vav proteins were initially involved in lymphocyte ontology. Only recently, cell culture experiments have implicated these proteins in cell migration events downstream of guidance factors. Interestingly, Vav proteins can either promote or inhibit cell migration. In macrophages, Vav is required for macrophage colony-stimulating factor-induced chemotaxis. In human peripheral blood lymphocytes, Vav is involved in the migratory response to the chemokine stromal cell-derived factor-1. Conversely, in Schwann cells, Vav2 is required to inhibit cell migration downstream of the brain-derived neurotrophic factor and ephrinA5. In spite of the knowledge gained from cell culture experiments, the biological relevance for many of the above interactions has remained elusive. In recent years, Vav proteins have started to emerge as critical Rho GEFs acting downstream of RTKs in diverse biological processes. Analysis of Vav2-/- Vav3-/- mice revealed retinogeniculate axonal projection defects and impaired ephrin-A1-induced migration during angiogenesis, suggesting a role for Vav in axonal targeting and angiogenesis downstream of Eph receptors in vivo. This study has shown that Vav can act downstream of growth factors receptors to promote BC migration in the developing Drosophila ovary, supporting a role of this family of GEFs in transducing signals from RTKs to regulate cell migration during development (Fernandez-Espartero, 2013).

Analysis of the cellular mechanisms by which Vav regulates cell migration in vertebrates is hampered by the inaccessibility of the cells and the difficulty of visualizing them in their natural environment within the embryo. Thus, it is not yet clear how Vav proteins regulate cell migration downstream of RTKs during development. In this study, by analysing cell movement in their physiological environment, it has been possible to show that Vav is required to control the length, stabilization and life of front cellular protrusions. In addition, disruption of Vav function in vivo was found to result in a decrease in Rac activity at the leading edge. Defective signalling downstream of EGFR/PVR results in defects in the dynamics of cellular protrusion and Rac activation, which are very similar to those observed in vav-/- BCs. In addition, this study found that ectopic activation of Vav in BCs, as it is the case for PVR/EGFR and Rac, causes non-polarized massive F-actin accumulation. Thus, it is suggested that one of the roles of Vav in directed cell migration downstream of EGF/PVF signals is to remodel the actin cytoskeleton via Rac activation, hence promoting the formation and stabilization of cellular protrusions in the direction of migration. Studies in cultured neurons, have shown that the main role for mouse Vav2 during axonal repulsion is to mediate a Rac-dependent endocytosis of ephrin-Eph. Although endocytosis has been normally shown to be involved in attenuation of RTKs signalling, in BCs it has been proposed to ensure RTKs recycling to regions of higher signalling, thus promoting directed BC movement. This is based on the fact that elimination in BCs of the ubiquitin ligase Cbl, which has been shown to regulate RTK endocytosis, leads to delocalized RTK signal and migration defects. In this context, another possible role for Vav downstream of EGFR/PVR could be to mediate RTK endocytosis, as it is the case during axonal repulsion. Further analysis will be needed to fully explore the molecular and cellular mechanisms by which Vav proteins regulate cell migration in vivo in other developmental contexts (Fernandez-Espartero, 2013).

BC migration is a complex event and activation of EGF/PDGF receptors will most likely engage different GEFs to affect the distinct cytoskeletal changes necessary to accomplish it. In fact, the migration phenotype of BCs mutant for vav is less severe than that of BCs double mutant for both EGFR and PVR. In addition, although reducing Vav function decreases the asymmetry in Rac activity between front and back present in wild-type clusters, it does not eliminate it, as it happens when the function of both guidance receptors is compromised. All these results suggest that there are other GEFs besides Vav that could act downstream of EGFR and PVR to activate Rac. Previous analysis have implicated the Rac exchange factor Mbc/DOCK180 and its cofactor ELMO on BC migration. In this context, Vav and the Mbc/ELMO complex could act synergistically as GEFs to mediate Rac activation to a precise level and/or to a precise location. This awaits the validation of the Mbc/ELMO complex as a GEF for Rac in BCs. In the future, it will be important to determine how the different GEFs contribute to Rac activation, which specific downstream effectors of Rac they activate, and ultimately what cellular aspects of the migration process they control (Fernandez-Espartero, 2013).

In summary, this work demonstrates that Vav functions downstream of RTKs to control directed cell migration during development. Furthermore, this study has unravelled the cellular and molecular mechanism by which Vav regulates cell migration in the developing Drosophila egg chamber: binding of PDGF/EGF to their receptors would induce Vav activation through tyrosine phosphorylation and its association with the activated receptors. This would lead to an increase in Rac activity at the leading edge of migrating cells, which promotes the stabilization and growth of the cellular front extensions, thus controlling directed cell migration (Fernandez-Espartero, 2013).

Regulation of Vav signalling downstream of RTKs can participate not only in development or normal physiology but also in tumorigenesis. Vav1 is mis-expressed in a high percentage of pancreatic ductular adenocarcinomas and lung cancer patients. Thus, understanding the mechanisms by which Vav controls cellular processes downstream of RTKs is likely to be relevant for both developmental and tumor biology (Fernandez-Espartero, 2013).

The Octopamine receptor Octβ2R regulates ovulation in Drosophila melanogaster

Oviposition is induced upon mating in most insects. Ovulation is a primary step in oviposition, representing an important target to control insect pests and vectors, but limited information is available on the underlying mechanism. This study reports that the beta adrenergic-like octopamine receptor Octβ2R serves as a key signaling molecule for ovulation and recruits Protein kinase A and Ca2+/calmodulin-sensitive kinase II as downstream effectors for this activity. The octβ2r homozygous mutant females are sterile. They displayed normal courtship, copulation, sperm storage and post-mating rejection behavior but are unable to lay eggs. It has been shown previously that octopamine neurons in the abdominal ganglion innervate the oviduct epithelium. Consistently, restored expression of Octβ2R in oviduct epithelial cells is sufficient to reinstate ovulation and full fecundity in the octβ2r mutant females, demonstrating that the oviduct epithelium is a major site of Octβ2R's function in oviposition. It was also found that overexpression of the protein kinase A catalytic subunit or Ca2+/calmodulin-sensitive protein kinase II leads to partial rescue of octβ2r's sterility. This suggests that Octβ2R activates cAMP as well as additional effectors including Ca2+/calmodulin-sensitive protein kinase II for oviposition. All three known β adrenergic-like octopamine receptors stimulate cAMP production in vitro. Octβ1R, when ectopically expressed in the octβ2r's oviduct epithelium, fully reinstated ovulation and fecundity. Ectopically expressed Octβ3R, on the other hand, partly restores ovulation and fecundity while OAMB-K3 and OAMB-AS that increase Ca2+ levels yielded partial rescue of ovulation but not fecundity deficit. These observations suggest that Octβ2R have distinct signaling capacities in vivo and activate multiple signaling pathways to induce egg laying. The findings reported in this study narrow the knowledge gap and offer insight into novel strategies for insect control (Lim, 2014; PubMed).

Adipocyte amino acid sensing controls adult germline stem cell number via the amino acid response pathway and independently of Target of Rapamycin signaling in Drosophila

How adipocytes contribute to the physiological control of stem cells is a critical question towards understanding the link between obesity and multiple diseases, including cancers. Previous studies have revealed that adult stem cells are influenced by whole-body physiology through multiple diet-dependent factors. For example, nutrient-dependent pathways acting within the Drosophila ovary control the number and proliferation of germline stem cells (GSCs). The potential role of nutrient sensing by adipocytes in modulating stem cells in other organs, however, remains largely unexplored. This study report that amino acid sensing by adult adipocytes specifically modulates the maintenance of GSCs through a Target of Rapamycin-independent mechanism. Instead, reduced amino acid levels and the consequent increase in uncoupled tRNAs trigger activation of the GCN2-dependent amino acid response pathway within adipocytes, causing increased rates of GSC loss. These studies reveal a new step in adipocyte-stem cell crosstalk (Armstrong, 2014).

Stem cell lineages are inextricably linked to whole-body physiology and nutrient availability in multiple organisms. For example, diet influences wound healing, hematopoietic transplants and cancer risk in humans, and evidence ranging from human epidemiological to model organism experimental data suggests that diet-dependent pathways impact a variety of adult stem cells. As intact living organisms vary their dietary input, multiple tissues and organs sense and respond to diet; however, knowledge of how inter-organ communication contributes to the dietary control of adult stem cells remains limited (Armstrong, 2014).

The obesity epidemic has brought to light the crucial importance of normal adipocyte function in maintaining a healthy physiology. Adipocytes are highly sensitive to diet and produce long-range factors with key roles in metabolism, reproduction and other physiological processes. Conversely, dysfunctional adipocytes underlie the link between obesity and several diseases, including cancers. Whether sensing of dietary inputs by adipocytes leads to specific effects on adult stem cells in other organs, however, remains largely unexplored (Armstrong, 2014).

Drosophila female germline stem cells (GSCs) sense and respond to diet through complex endocrine mechanisms. Two or three GSCs reside within a well-defined niche in the germarium, the anterior region of the ovariole. Each asymmetric GSC division yields another GSC and a cystoblast that forms a 16-cell cyst, which is enveloped by follicle cells to generate a follicle that develops through oogenesis to form a mature oocyte. On a yeast-rich diet, GSCs and their progeny grow and proliferate faster than on a yeast-free diet, and this response is mediated by diet-dependent factors that act on or within the ovary. For example, optimal levels of Target of Rapamycin (TOR) activity likely controlled by circulating amino acids are intrinsically required in GSCs for their proliferation and maintenance. Insulin-like peptides produced by median neurosecretory cells in the brain act directly on GSCs to modulate how fast they proliferate to generate new cystoblasts. In parallel, insulin-like peptides act directly on cap cells, the major cellular components of the niche, to control GSC maintenance via two mechanisms. Insulin-like peptides promote the response of cap cells to Notch ligands, which are required for proper cap cell numbers, and also GSC-cap cell attachment via E-cadherin. These previous studies, however, did not address whether or how nutrient sensing by adipocytes influences the dietary response of GSCs and their descendants (Armstrong, 2014 and references therein).

Drosophila adipocytes, together with hepatocyte-like oenocytes, compose the fat body, a nutrient-sensing organ with endocrine roles. In the larval fat body, TOR activation downstream of amino acid sensing results in the production of unknown factors that modulate overall growth of the organism. In both the larval and adult fat body, sensing of sugars and lipids leads to the production of a leptin-like cytokine, Unpaired 2 (Upd2), which controls the secretion of brain insulin-like peptides. This study reports that partially inhibiting amino acid transport in adult adipocytes results in a specific reduction in the number of ovarian GSCs and that, surprisingly, this effect is independent of TOR signaling. Instead, reduced amino acid levels and the consequent increase in uncoupled tRNAs trigger activation of the GCN2-dependent amino acid response (AAR) pathway within adipocytes, causing increased rates of GSC loss. These results indicate that amino acid sensing by adipocytes through a TOR-independent mechanism is communicated to GSCs to control their maintenance, thereby contributing to their response to diet. These findings bring to light the importance of elucidating how adipocytes contribute to the regulation of various adult stem cell types by diet, and how these mechanisms might be adversely affected in obese individuals (Armstrong, 2014).

Tight coordination of growth and differentiation between germline and soma provides robustness for Drosophila egg development

Organs often need to coordinate the growth of distinct tissues during their development. This study analyzed the coordination between germline cysts and the surrounding follicular epithelium during Drosophila oogenesis. Genetic manipulations of the growth rate of both germline and somatic cells influence the growth of the other tissue accordingly. Growth coordination is therefore ensured by a precise, two-way, intrinsic communication. This coordination tends to maintain constant epithelial cell shape, ensuring tissue homeostasis. Moreover, this intrinsic growth coordination mechanism also provides cell differentiation synchronization. Among growth regulators, PI3-kinase and TORC1 also influence differentiation timing cell-autonomously. However, these two pathways are not regulated by the growth of the adjacent tissue, indicating that their function reflects an extrinsic and systemic influence. Altogether, these results reveal an integrated and particularly robust mechanism ensuring the spatial and temporal coordination of tissue size, cell size, and cell differentiation for the proper development of two adjacent tissues (Vachias, 2014: PubMed).

Several main conclusions can be drawn from this work. First, in each follicle, growth is intrinsically coordinated between the two tissues. Second, this growth control tends to optimize cell shape in the epithelium. This is likely to be representative of the development of many epithelia where cell shape must be maintained because it is essential for the function of the tissue. In the third place, growth control has a very important impact on differentiation timing in each tissue. Furthermore, several growth pathways can cell-autonomously influence differentiation rate but are not regulated by the adjacent tissue, indicating that they only respond to extrinsic cues. Finally, as a whole, this study reveals the robustness of the spatiotemporal pattern allowing the production of mature eggs with a normal shape and a normal size. At least two examples based on Pten somatic clones can illustrate this robustness. WT border cells migrate perfectly 'on time' in a follicle in which mutant somatic cells have induced a faster germline development. Second, a WT looking mature egg can be found in the middle of an ovariole, suggesting that all developmental steps have been faster but correctly orchestrated. This robustness probably reflects the fact that final egg size is constant, that most of the developmental steps have to occur at a specific size, and that differentiation is able to block growth when the definitive egg size is reached. These observations raise the question as to how the differentiation program regulates growth and especially growth arrest in each follicle (Vachias, 2014).

These results indicate a two-way communication between the germline and the soma to ensure their coordination. It was also observed that somatic cells can influence other somatic cells but, importantly, that such an effect depends on the relay of the germ cells. This result suggests that coordination is achieved by different signals depending on the tissue. The soma and germline could communicate via the secretion of growth factors controlling the adjacent tissue, though obvious candidates were excluded. An alternative explanation would be that, as it is proposed in mammals, the two tissues are interdependent for specific metabolites, although it would be independent of TORC1, a classical sensor of metabolic activity. Finally, an attractive hypothesis would be that growth regulation between the soma and the germline depends on a mechanical steady state. Germline growth creates a tension on the follicle cell, leading to the proposal that this tension could trigger epithelial growth. If so, it would also mean that follicle cells provide a mechanical strain limiting germline growth. The mechanical control of growth in epithelial cells is usually devoted to the Hippo pathway, which is not involved in this instance. Thus, this work does not allow favoring one or the other of these nonexclusive mechanisms (Vachias, 2014).

Altogether, these results highlight several dimensions of coordination between cell growth, cell shape, and cell identity and all this between two distinct tissues. These different functional links offer a highly robust program in space and time. The relevance for such robustness has been very recently highlighted because it probably confers the reproducibility on embryonic development. Since usual pathways controlling growth are not involved in this two-way communication, this multidimensional coordination will be a useful framework for identifying molecular actors ensuring tissue homeostasis in the recurrent context of the development of two adjacent tissues (Vachias, 2014).

The ABC transporter Eato promotes cell clearance in the Drosophila melanogaster ovary

Two evolutionarily conserved signaling pathways that act redundantly to regulate engulfment of dead cells: the ced-1/-6/-7 and ced-2/-5/-12 pathways. Homology searches revealed a family of putative ced-7/ABCA1 homologs encoding ABC transporters in Drosophila. To determine which of these genes functions similarly to ced-7/ABCA1, mutants were analyzed for engulfment phenotypes in oogenesis, during which nurse cells in each egg chamber undergo programmed cell death and are removed by neighboring phagocytic follicle cells. Genetic analyses indicate that one of the ABC transporter genes, which was named Eato (CG31731), is required for nurse cell clearance in the ovary and acts in the same pathways as drpr, the ced-1 ortholog, and in parallel to Ced-12 in the follicle cells. Additionally, Eato acts in the follicle cells to promote accumulation of the transmembrane receptor Drpr, and promote membrane extensions around the nurse cells for their clearance. Since ABCA class transporters, such as CED-7 and Eato, are involved in lipid trafficking, it is proposed that Eato acts to transport membrane material to the growing phagocytic cup for cell corpse clearance. This work identifies Eato as the ced-7 ortholog in D. melanogaster, and demonstrates a role for Eato in Drpr accumulation and phagocytic membrane extensions during nurse cell clearance in the ovary (Santoso, 2018).

The temporally controlled expression of Drongo, the fruit fly homolog of AGFG1, is achieved in female germline cells via P-bodies and its localization requires functional Rab11

To achieve proper RNA transport and localization, RNA viruses exploit cellular vesicular trafficking pathways. AGFG1, a host protein essential for HIV-1 and Influenza A replication, has been shown to mediate release of intron-containing viral RNAs from the perinuclear region. It is still unknown what its precise role in this release is, or whether AGFG1 also participates in cytoplasmic transport. This study reports the expression patterns during oogenesis for Drongo, the fruit fly homolog of AGFG1. It was found that temporally controlled Drongo expression is achieved by translational repression of drongo mRNA within P-bodies. A link was found between the recycling endosome pathway and Drongo, and proper Drongo localization at the oocyte's cortex during mid-oogenesis was found to require functional Rab11 (Catrina, 2016).

Evidence for the mechanosensor function of filamin in tissue development

Cells integrate mechanical properties of their surroundings to form multicellular, three-dimensional tissues of appropriate size and spatial organisation. Actin cytoskeleton-linked proteins such as talin, vinculin and filamin function as mechanosensors in cells, but it has yet to be tested whether the mechanosensitivity is important for their function in intact tissues. This study tested how filamin mechanosensing contributes to oogenesis in Drosophila. Mutations that require more or less force to open the mechanosensor region demonstrate that filamin mechanosensitivity is important for the maturation of actin-rich ring canals that are essential for Drosophila egg development. The open mutant was more tightly bound to the ring canal structure while the closed mutant dissociated more frequently. Thus, these results show that an appropriate level of mechanical sensitivity is required for filamin's function and dynamics during Drosophila egg growth and support the structure-based model in which the opening and closing of the mechanosensor region regulates filamin binding to cellular components (Huelsmann, 2016).

Discs large 5, an essential gene in Drosophila, regulates egg chamber organization

Discs large 5 (Dlg5) is a member of the MAGUK family of proteins that typically serve as molecular scaffolds and mediate signaling complex formation and localization. In vertebrates, Dlg5 has been shown to be responsible for polarization of neural progenitors and to associate with Rab11-positive vesicles in epithelial cells. In Drosophila, however, the function of Dlg5 is not well-documented. This study identified dlg5 as an essential gene that shows embryonic lethality. dlg5 embryos display partial loss of primordial germ cells (PGCs) during gonad coalescence between stages 12 and 15 of embryogenesis. Loss of Dlg5 in germline and somatic stem cells in the ovary results in the depletion of both cell lineages. Reduced expression of Dlg5 in the follicle cells of the ovary leads to a number of distinct phenotypes, including defects in egg chamber budding, stalk cell overgrowth, and ectopic polar cell induction. Interestingly, loss of Dlg5 in follicle cells results in abnormal distribution of a critical component of cell adhesion, E-cadherin, shown to be essential for proper organization of egg chambers (Reilly, 2015).

dlg5 was shown to be essential for normal division or maintenance of FSCs and GSCs, and is required in later stage follicle cells. Reduction of Dlg5 levels disrupts egg chamber formation, indicating that dlg5 is involved in processes fundamental to egg chamber organization (Reilly, 2015).

When Dlg5 was depleted in follicle cells for 4 d, epithelial follicle cells showed relatively normal cell shape and polarity, but the polar and stalk cells showed strong abnormalities in number, localization, and overall organization. The induction of ectopic polar cells and stalk cell overgrowth observed in these ovaries is significant because it has been suggested that the differentiation of these two cell types are controlled by similar signaling events, which distinguishes them from the other follicle cells. Loss of Dlg5 may interfere with the pathways that specify stalk and polar cell differentiation and maturation and, consequently, egg chamber organization (Reilly, 2015).

The significantly enhanced phenotype observed upon depletion of Dlg5 in follicle cells for 10 d agrees with the dlg5D48 clonal phenotype and is consistent with the proposition that the function of dlg5 is completely lost in dlg5D48. Further, complete loss of dlg5 function results in embryonic lethality, possibly also as a result of abnormal organization and integration of specific embryonic cells (Reilly, 2015).

Analysis of primitive embryonic gonad formation in dlg5D48 embryos suggested that it likely functions during germ cell migration and gonad coalescence. These data are reminiscent of requirements of Dlg5 in the maintenance of the cohesion of migrating border cells in the ovary (Aranjuez, 2012). In this regard, it is interesting to note that proper gonad coalescence has been shown to depend on cell adhesion molecule E-cadherin. E-cadherin is consistently upregulated during late stages of gonad formation. Because loss of dlg5 results in altered distribution of E-cadherin in follicle cells, it is conceivable that aberrant E-cadherin levels and/or localization could also contribute to the embryonic gonad-specific phenotypes. In this context, it will be interesting to analyze further the precise requirement and cell-type specificity of Dlg5 function in controlling cell adhesion and migration (Reilly, 2015).

Many of the dlg5 phenotypes could result from aberrant E-cadherin distribution. For instance, oocyte mislocalization is often seen as a result of loss or aberrant homophilic adhesion mediated by E-cadherin between posterior follicle cells and the oocyte. Formation of interfollicular stalks is also dependent on dynamic E-cadherin accumulation: E-cadherin accumulates first at the apical boundary of prefollicular cells, followed by the establishment of lateral cell contacts to initiate and complete intercalation to form a single wide stalk. Additionally, E-cadherin has been demonstrated to be required for recruiting and anchoring stem cells to their niche prior to adulthood in the Drosophila ovary. By clonal analysis, this study showed that both germline and follicle dlg5 ovary stem cells are unable to give rise to normal daughter cells, indicating that the gene is essential in these cells. This may suggest several possibilities. There may be a cell-autonomous requirement for dlg5 in follicle cells; however, the loss of stem cells may also be an indication of the requirement for E-cadherin-mediated adhesion to the stem cell niche. Finally, the lack of cohesion in migrating germ cells in dlg5D48 embryos is consistent with a perturbation in cell-cell adhesion, as described above. The observed phenotypes, therefore, and the role of Dlg5 in E-cadherin distribution may be related; however, more work is needed to determine the nature of the participation of Dlg5 in E-cadherin distribution before any further conclusions may be drawn (Reilly, 2015).

Dlg5 is involved in vesicle trafficking in vertebrates and has been reported to colocalize with several Rab GTPases. The punctate distribution of Dlg5 in the Drosophila ovary is consistent with a similar association of the protein with endosomes. Therefore, it seems possible that Dlg5 is involved in endosomal trafficking. The abnormal distribution of E-cadherin, which is recycled through the endosome, observed in Dlg5 KD ovaries supports this hypothesis. Further, reduction of Dlg5 in the mammalian epithelial cell line LLc-PK1 resulted in lower levels of E-cadherin, but it is not clear how Dlg5 controls E-cadherin levels (Reilly, 2015).

Thus, Dlg5, like its human homolog and Dlg1, may be involved in endosomal recycling of E-cad. But based on this characterization of dlg5 and its functional requirement, there are fundamental differences between these two genes. Although dlg5 shows embryonic lethality and is essential in ovarian stem cells, dlg1 larvae can survive for >10 d and show overgrowth phenotypes in larvae and follicle cells. Persistent dlg1 germ line clones develop into eggs, whereas follicle cells lacking dlg1 sometimes show tumor-like invasion into the interior of the egg chamber, a phenotype this study not observe in Dlg5 KD. The task of figuring out what each of these Dlg proteins contributes to apical protein trafficking and cell survival should prove informative and stimulating (Reilly, 2015).

Targeted downregulation of s36 protein unearths its cardinal role in chorion biogenesis and architecture during Drosophila melanogaster oogenesis

Drosophila chorion represents a model biological system for the in vivo study of gene activity, epithelial development, extracellular-matrix assembly and morphogenetic-patterning control. It is produced during the late stages of oogenesis by epithelial follicle cells and develops into a highly organized multi-layered structure that exhibits regional specialization and radial complexity. Among the six major proteins involved in chorion's formation, the s36 and s38 ones are synthesized first and regulated in a cell type-specific and developmental stage-dependent manner. In this study, an RNAi-mediated silencing of s36 chorionic-gene expression specifically in the follicle-cell compartment of Drosophila ovary unearths the essential, and far from redundant, role of s36 protein in patterning establishment of chorion's regional specialization and radial complexity. Without perturbing the developmental courses of follicle- and nurse-cell clusters, the absence of s36 not only promotes chorion's fragility but also induces severe structural irregularities on chorion's surface and entirely impairs fly's fertility. Moreover, s36 chorionic protein regulates the number and morphogenetic integrity of dorsal appendages in follicles sporadically undergoing aged fly-dependent stress (Velentzas, 2016).

Increased intracellular pH is necessary for adult epithelial and embryonic stem cell differentiation

Despite extensive knowledge about the transcriptional regulation of stem cell differentiation, less is known about the role of dynamic cytosolic cues. This study reports that an increase in intracellular pH (pHi) is necessary for the efficient differentiation of Drosophila adult follicle stem cells (FSCs) and mouse embryonic stem cells (mESCs). It was shown that pHi increases with differentiation from FSCs to prefollicle cells (pFCs) and follicle cells. Loss of the Drosophila Na+-H+ exchanger DNhe2 lowers pHi in differentiating cells, impairs pFC differentiation, disrupts germarium morphology, and decreases fecundity. In contrast, increasing pHi promotes excess pFC cell differentiation toward a polar/stalk cell fate through suppressing Hedgehog pathway activity. Increased pHi also occurs with mESC differentiation and, when prevented, attenuates spontaneous differentiation of naive cells, as determined by expression of microRNA clusters and stage-specific markers. These findings reveal a previously unrecognized role of pHi dynamics for the differentiation of two distinct types of stem cell lineages, which opens new directions for understanding conserved regulatory mechanisms (Ulmschneider, 2016).

Neutral competition for Drosophila follicle and cyst stem cell niches requires vesicle trafficking genes

The process of selecting for cellular fitness through competition plays a critical role in both development and disease. The germarium, a structure at the tip of the ovariole of a Drosophila ovary, contains two follicle stem cells (FSCs) that undergo neutral competition for the stem cell niche. Using the FSCs as a model, a genetic screen through a collection of 126 mutants in essential genes on the X chromosome was performed to identify candidates that increase or decrease competition for the FSC niche. Approximately 55% and 6% of the mutations screened were obtained as putative FSC hypo- or hypercompetitors, respectively. A large majority of mutations were found in vesicle trafficking genes (11 out of the 13 in the collection of mutants) are candidate hypocompetition alleles, and the hypocompetition phenotype was confirmed for four of these alleles. Sec16 and another COP II vesicle trafficking component, Sar1, are required for follicle cell differentiation. Lastly, it was demonstrated that although some components of vesicle trafficking are also required for neutral competition in the cyst stem cells (CySCs) of the testis, there are important tissue-specific differences. These results demonstrate a critical role for vesicle trafficking in stem cell niche competition and differentiation, and a number of putative candidates for further exploration were identified (Cook, 2017).

The follicle stem cells in the Drosophila ovary are a highly tractable model of stem cell niche competition. The Drosophila ovary is comprised of long strands of developing follicles, called ovarioles, and a pair FSCs resides at the anterior tip of each ovariole in a structure called the germarium. These FSCs divide during adulthood to provide the follicle cells that surround germ cell cysts during follicle formation. FSCs are regularly lost and replaced during adulthood, and several studies have identified genes that increase the rate of FSC loss. In most cases, the mutations investigated in these studies disrupt the ability of the mutant FSC to adhere to the niche or transduce niche signals and thus are presumed to cause the mutant stem cell to be lost in a cell-autonomous manner. However, the suggestion that stem cells may compete with the daughters of neighboring stem cells for niche occupancy raises the possibility that a mutation in a competing mutant lineage could act in a noncell-autonomous manner to influence the likelihood that a neighboring wild-type lineage will be lost and replaced (Cook, 2017).

This study has generated a new resource for investigating the genetic basis of stem cell niche competition. The collection of confirmed and candidate competition mutations demonstrates the breadth of cell functions that influence niche competition, and will allow for further study into many different facets of this process. One hyper-competition mutation and seven additional candidate hyper-competition mutations were generated in genes involved in a variety of functions, including mitochondrial function (sicily, tumor suppression (Rbf), and protein ubiquitination (bendless). The diversity of this list of candidates suggests that effects on many different cellular processes can lead to a hyper-competition phenotype. Yet the vast majority of mutations that were studied do not cause hyper-competition, suggesting that the ultimate cause(s) of hyper-competition are more constrained, and that the mechanism of hyper-competition may be similar in these diverse mutants (Cook, 2017).

A previously unstudied aspect of niche competition revealed by this screen is the involvement of vesicle trafficking genes. The Sec16A, shiA, wusA, and CragA mutations that that were examined in this study represent a broad range of vesicle trafficking functions. Specifically, Sec16 is a central regulator of exocytosis, whereas wurst and shibire function primarily during endocytosis, and Crag is required in follicle cells for trafficking of basement membrane components to the basal surface. The shiA (K10X) and wusA (Q307X) alleles, which produced the most severe effects on DE-cad localization, contain nonsense mutations and are most likely to be amorphic alleles; while CragA (C1372S) and Sec16A (P1926S) contain missense mutations and are more likely to be hypomorphic alleles. Comparison of the relatively mild phenotypes caused by homozygosity for Sec16A to the more severe phenotypes caused by RNAi knockdown of Sec16A further support the conclusion that Sec16A is a hypomorphic allele. Interestingly, despite these differences, all four mutations caused strong hypo-competition phenotypes in the FSC lineage. This suggests that the process of niche competition is very sensitive to the function of these genes and is able to efficiently eliminate stem cell lineages with even only mild defects in vesicle trafficking (Cook, 2017).

The finding of this study suggest at least two possible reasons why impaired vesicle trafficking causes hypo-competition. First, DE-cad is known to be required for FSC maintenance, so the disruption of DE-cad localization to the membrane in vesicle trafficking mutants could at least partially account for the hypo-competition phenotype. Second, vesicle trafficking is required for functional EGFR signaling in MDCK cells, and this study found decreased pERK levels in the vesicle trafficking mutant clones. Since EGFR signaling is also essential for FSC self-renewal, the reduction in EGFR signaling may be another factor that contributes to the hypo-competition phenotype. However, EGFR signaling is normally downregulated as cells exit the FSC niche region, so it is unclear whether the decreased pERK levels in these mutant clones are a cause of the hyper-competition phenotype or a consequence of reduced association with the niche (for example, because DE-cad levels on the membrane are low). In addition, it is also unclear why the loss of detectable pERK in these mutant clones is not associated with a cell polarity defect, as has been observed in EGFR null clones. It may be that the vesicle trafficking alleles investigated in this study reduce the levels of EGFR signaling to a level that is below the limit of detection but sufficient to maintain cell polarity; or that the vesicle trafficking mutants impair the branch of EGFR signaling leading to ERK phosphorylation, but not the branch going through LKB1 and AMPK that is important for the maintenance of cell polarity in the FSC lineage. Alternately, it could be that the decrease in EGFR signaling is more gradual in the vesicle trafficking mutant clones than it is in EGFR mutant clones, so the vesicle trafficking mutant clones are able to grow normally for some time before EGFR signaling decreases to the point at which it is both undetectable by pERK staining and unable to promote FSC self-renewal or follicle cell polarity. Additional studies of these and other FSC niche competition mutants identified here will be important to clarify these issues (Cook, 2017).

In the testis, knockdown of Sec16 also affected CySC niche competition rather than self-renewal or differentiation, whereas knockdown of shi likely impaired CySC self-renewal or survival. Interestingly, knockdown of Crag had no effect on CySC retention in the niche but caused an unusual differentiation defect in which mutant cells failed to move out of the niche region. These differences indicate that there are tissue-specific aspects to the process of self-renewal and niche competition, and provide additional evidence that the hypo-competition phenotypes observed in the FSC niche are not due to a generic defect that would affect all cells equally. Overall, these studies demonstrate that mutations in multiple types of vesicle trafficking genes cause hypo-competition in both the ovary and testis. Vesicle trafficking is essential for a diverse array of cellular functions, and thus may function as a node, integrating outputs from these different cellular functions into a readout that influences the overall fitness of the cell for occupying the niche. Further investigation will make it possible to organize these and other mutations that cause niche competition phenotypes into common pathways and, ultimately, to understand whether and how stem cell niche competition promotes the maintenance of a healthy population of stem cells in each tissue throughout adulthood (Cook, 2017).

The retromer subunit Vps26 mediates Notch signaling during Drosophila oogenesis

During endocytosis, molecules are internalized by the cell through the invagination of the plasma membrane. Endocytosis is required for proper cell function and for normal development in Drosophila. One component of the endocytic pathway is the retromer complex, which recycles transmembrane proteins to other parts of the cell such as the plasma membrane and the trans-Golgi network. Previous studies have shown that mutations to the retromer complex result in developmental defects in Drosophila. In humans, retromer dysfunction has been implicated in Alzheimer's and Parkinson's disease, but little is known about the role of the retromer complex in Drosophila oogenesis. This project examined the role of the retromer protein Vps26 in oogenesis by characterizing the phenotype of vps26 germline clones. Immunofluorescence was used to visualize the expression of membrane proteins and vesicular trafficking markers in mutant egg chambers. vps26 germline clones exhibit a signaling defect between the germline cells and follicle cells indicated by an increase in LysoTracker staining of the border cells in the mutants. This signaling defect in vps26 mutants may be the result of impaired Notch signaling based on the misexpression of multiple proteins in the Notch signaling pathway in vps26 mutants (Starble, 2017).

Collective growth in a small cell network

Theoretical studies suggest that many of the emergent properties associated with multicellular systems arise already in small networks. However, the number of experimental models that can be used to explore collective dynamics in well-defined cell networks is still very limited. This study focused on collective cell behavior in the female germline cyst in Drosophila melanogaster, a stereotypically wired network of 16 cells that grows by approximately 4 orders of magnitude with unequal distribution of volume among its constituents. Multicellular growth was quantified with single-cell resolution, and it was shown that proximity to the oocyte, as defined on the network, is the principal factor that determines cell size; consequently, cells grow in groups. To rationalize this emergent pattern of cell sizes, a tractable mathematical model is proposed that depends on intercellular transport on a cell lineage tree. In addition to correctly predicting the divergent pattern of cell sizes, this model reveals allometric growth of cells within the network, an emergent property of this system and a feature commonly associated with differential growth on an organismal scale (Imran, 2017).

Drosophila glob1 is required for the maintenance of cytoskeletal integrity during oogenesis

Hemoglobins (Hbs) are evolutionarily conserved heme-containing metallo-proteins of the "Globin" protein family which harbour the characteristic "globin fold". Hemoglobins have been functionally diversified during evolution and its usual property of oxygen transport is rather a recent adaptation. Drosophila genome possesses three globin genes (glob1, glob2 and glob3). and earlier work has reported that adequate expression of glob1 is required for the various aspects of development and also to regulate the cellular level of reactive oxygen species (ROS). The present study illustrates the explicit role of Drosophila globin1 in progression of oogenesis. A dynamic expression pattern is reported of glob1 in somatic and germ cell derivatives of developing egg chambers during various stages of oogenesis which largely confines around the F-actin rich cellular components. Reduced expression of glob1 leads to various types of abnormalities during oogenesis which were primarily mediated by the inappropriately formed F-actin based cytoskeleton. Subsequent analysis in the somatic and germ line clones shows cell autonomous role of glob1 in the maintenance of the integrity of F-actin based cytoskeleton components in the somatic and germ cell derivatives. This study establishes a novel role of glob1 in maintenance of F-actin based cytoskeleton during progression of oogenesis in Drosophila (Yadav, 2016).

Outer nuclear membrane protein Kuduk modulates the LINC complex and nuclear envelope architecture

Linker of nucleoskeleton and cytoskeleton (LINC) complexes spanning the nuclear envelope (NE) contribute to nucleocytoskeletal force transduction. A few NE proteins have been found to regulate the LINC complex. This study identified one, Kuduk (Kud), which can reside at the outer nuclear membrane and is required for the development of Drosophila melanogaster ovarian follicles and NE morphology of myonuclei. Kud associates with LINC complex components in an evolutionarily conserved manner. Loss of Kud increases the level but impairs functioning of the LINC complex. Overexpression of Kud suppresses NE targeting of cytoskeleton-free LINC complexes. Thus, Kud acts as a quality control mechanism for LINC-mediated nucleocytoskeletal connections. Genetic data indicate that Kud also functions independently of the LINC complex. Overexpression of the human orthologue TMEM258 in Drosophila proved functional conservation. These findings expand understanding of the regulation of LINC complexes and NE architecture (Ding, 2017).

Variations in basement membrane mechanics are linked to epithelial morphogenesis

The regulation of morphogenesis by the basement membrane (BM) may rely on changes in its mechanical properties. To test this, an atomic force microscopy-based method was developed to measure BM mechanical stiffness during two key processes in Drosophila ovarian follicle development. First, follicle elongation depends on epithelial cells that collectively migrate, secreting BM fibrils perpendicularly to the anteroposterior axis. These data show that BM stiffness increases during this migration and that fibril incorporation enhances BM stiffness. In addition, stiffness heterogeneity, due to oriented fibrils, is important for egg elongation. Second, epithelial cells change their shape from cuboidal to either squamous or columnar. This study proves that BM softens around the squamous cells and that this softening depends on the TGFbeta pathway (the ligands Gbb and Dpp signalling to follicle cells). It was also demonstrated that interactions between BM constituents are necessary for cell flattening. Altogether, these results show that BM mechanical properties are modified during development and that, in turn, such mechanical modifications influence both cell and tissue shapes (Chlasta, 2017).


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date revised:  10 August 2017

genes involved in oogenesis

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