prospero is transcribed in all neuroblasts (NB) and ganglion mother cells (GMC). PROS protein is found in the NB, but is localized to the cortex and excluded from the nucleus. Asymmetric PROS localization follows centrosome migration to the basal pole of the NB during mitosis. This localization is blocked in string-mutant embryos arrested in the G-2 phase of the cell cycle (Spana, 1995).

See Chris Doe's Hyper-Neuroblast map site for information on the expression of prospero in neuroblasts.

For more information on Drosophila neuroblast lineages, see Linking neuroblasts to their corresponding lineage, a site carried by Flybrain, an online atlas and database of the Drosophila nervous system.

String is the Drosophila CDC-25 homolog. String encodes a phosphatase required to activate CDC2 kinase, which regulates entry into mitosis. It is unknown whether the defect in Prospero location in stg mutants is direct or not (Spana, 1995). pros is also expressed in glioblasts, the precursors of longitudinal and midline glia. Asymmetric localization of PROS protein to the cortex is also detected in precursors of the peripheral nervous system for both external sensory bristle organ lineage and abdominal lateral chordotonal sense organs (Doe, 1995). The same process also takes place in large intestinal cell precursors of the adult midgut endoderm (Doe, 1995).

prospero is expressed in specific neuroblasts of the fly ventral nervous system. In the Drosophila CNS, early neuroblast formation and fate are controlled by the pair-rule class of segmentation genes. The distantly related Schistocerca (grasshopper) embryo has a similar arrangement of neuroblasts, despite lack of known pair-rule gene function. Four molecular markers have been used to compare Drosophila and Schistocerca neuroblast identity: seven-up, prospero, engrailed, and fushi-tarazu/Dax. In both insect species some early-forming neuroblasts share key features of neuroblast identity (position, time of formation, and temporally accurate gene expression). Thus different patterning mechanisms can generate similar neuroblast fates. In contrast, several later-forming neuroblasts show species-specific differences in position and/or gene expression. These neuroblast identities seem to have diverged, suggesting that evolution of the insect central nervous system can occur through changes in embryonic neuroblast identity (Broadus, 1995b).

A specific P-element insertion into prospero expresses beta-gal in the early stomatogastric nervous system precursor cells that arise at approximately stage 10 from a region anterior to the SNS analge. beta-gal is also found in cells delaminating dorsally from the SNS vesicles, in a large number of neuroblasts in the CNS and PNS and in garland cells, cells that form a ring around the anterior-ventral side of the proventriculus. These cells are involved in intense endocytosis and exocytosis and have been proposed to function as nephrocytes (removing waste from the hemolymph by endocytosis), yet they express a number of neural specific markers including prospero, fasciclin II and syntaxin. This suggests that garland cells have a neural character and that their endocytic and exocytic activity is in fact synaptic activity involved in regulating the proventriculus. Later expression of prospero in the SNS is seen in the commissural glial cells. The frontal connectives in P-element insertion prospero mutants are often severly reduced or absent altogether, and there is defaciculation, particularly in the recurrent nerve, which connects the frontal ganglion of the CNS with the esophageal ganglia (Forjanic, 1997).

Biochemical analysis of prospero protein during asymmetric cell division: cortical prospero is highly phosphorylated relative to nuclear prospero

The Prospero transcript is alternatively spliced to encode two proteins: ProsL protein (1403 amino acids, predicted 165 kDa) and ProsS protein (1374 amino acids, predicted 160 kDa). The extra 29 amino acids in ProsL are at the beginning of the homeodomain (Chu-LaGraff, 1991) and thus, both ProsS and Pros should be recognized by N- and C-terminal monoclonal antibodies. Western blots were performed on hand-picked, staged embryos. Abundant Prospero protein isoforms at 220 and 210 kDa are observed as well as lower levels of protein migrating at 144 kDa. The 220-kDa isoform is present in stage 1-3 embryos and ovaries, suggesting that it is maternally inherited; isoforms of this size are detected throughout embryogenesis. The 210-kDa band first becomes abundant at stage 7, with levels steadily increasing at subsequent stages of development. The 144-kDa band appears at stage 9 and persists at low levels at later stages. The 220-, 210-, and 144-kDa isoforms are recognized by two independent monoclonal antibodies; the MR1 mouse monoclonal recognizes a C-terminal epitope and the P3D4 rat monoclonal recognizes an N terminal epitope. In addition, these bands are specifically absent in old (stage 17) embryos homozygous for a prospero null allele. It is concluded that prospero encodes multiple protein isoforms; the major two isoforms migrate slower than predicted, and the minor isoform migrates faster than predicted. Both 220-kDa and 210-kDa isoforms are phosphorylated; after phosphatase treatment, both lose their most acidic species (Srinivasan, 1998).

Prospero is translocated into the GMC nucleus, where it is necessary to establish GMC-specific gene expression. Cortical localization of Prospero protein is observed only during mitosis; cortical localization requires entry into mitosis and cortical delocalization requires exit from mitosis. The tight correlation and functional requirement between mitosis and cortical Prospero localization suggests that mitosis-specific posttranslational modifications may be involved in regulating Prospero subcellular localization. Monoclonal antibodies recognizing the N-terminal or C-terminal region of Prospero were used to explore its posttranslational regulation. Developmental 2D Western blots, cell fractionation assays, and analysis of a missense prospero mutation show that cortical Prospero protein is highly phosphorylated compared to nuclear Prospero protein. In precellular stage 1-5 embryos Prospero protein is cytoplasmic, with perhaps some associated with the embryo cortex. Only the acidic 220-kDa/pI5.0 phosphorylated Prospero isoform is present. In cellularized stage 7 embryos, Prospero is also cortical or cytoplasmic in the ventral ectoderm and procephalic region, but there are a few cells with nuclear staining in the procephalic region. Biochemical analysis reveals the acidic 220-kDa/pI5.0 isoform, as well as the first appearance of the more basic 210-kDa/pI5.6-6.0 isoforms; over time, these 210-kDa isoforms become more acidic (phosphorylated). During stages 9-13, basal cortical Prospero is found during mitosis in neuroblasts, sense organ precursors, and posterior midgut precursors; however, a much larger number of GMCs and glia have nuclear Prospero. The 210-kDa isoforms are shown to be the nuclear species. The 144-kDa isoform cannot be correlated with a particular subcellular distribution. Examination of embryos homozygous for a mutant prospero allele, in which Prospro remains cortical throughout interphase in GMCs and neurons, reveals an enrichment of the phosphorylated Prospero 220-kDa/pI5.0 isoform. These results are consistent with two functions of Prospero phosphorylation: (1) phosphorylation may be required for Prospero cortical localization; or (2) phosphorylation may be a consequence of Prospero cortical localization, in which case it may facilitate subsequent events, such as Prospero cortical release or nuclear localization (Srinivasan, 1998).

Transient nuclear Prospero induces neural progenitor quiescence

Stem cells can self-renew, differentiate, or enter quiescence. Understanding how stem cells switch between these states is highly relevant for stem cell-based therapeutics. Drosophila neural progenitors (neuroblasts) have been an excellent model for studying self-renewal and differentiation, but quiescence remains poorly understood. This study shows that when neuroblasts enter quiescence, the differentiation factor Prospero is transiently detected in the neuroblast nucleus, followed by the establishment of a unique molecular profile lacking most progenitor and differentiation markers. The pulse of low level nuclear Prospero precedes entry into neuroblast quiescence even when the timing of quiescence is advanced or delayed by changing temporal identity factors. Furthermore, loss of Prospero prevents entry into quiescence, whereas a pulse of low level nuclear Prospero can drive proliferating larval neuroblasts into quiescence. It is proposed that Prospero levels distinguish three progenitor fates: absent for self-renewal, low for quiescence, and high for differentiation (Lai, 2014).

Mutations in lottchen cause cell fate transformations in both neuroblast and glioblast lineages in the Drosophila embryonic central nervous system

lottchen (ltt) is a novel gene whose loss of function causes a change in the identity of at least one NB as well as cell fate transformations within the lateral glioblast lineage. lottchen is known to code for the protein Muscle segment homeobox. The Drosophila embryonic central nervous system (CNS) develops from a stereotyped pattern of neuronal progenitor cells called neuroblasts (NB). Each NB has a unique identity that is defined by the time and position of its formation and a characteristic combination of genes it expresses. Each NB generates a specific lineage of neurons and/or glia. In wildtype embryos, the parental NB of the motoneuron RP2 is NB4-2. ltt embryos are distinguished by an additional RP2-like neuron, which appears later in development. The two RP2 neurons are derived from two distinct GMC4-2a-like cells that do not share the same parental NB, indicating that a second NB has acquired the potential to produce a GMC and a neuron: this potential is normally restricted to the NB4-2 lineage. Moreover, the ltt mutations lead to a loss of correctly specified longitudinal glia; this coincides with severely defective longitudinal connectives. Therefore, lottchen plays a role in specifying the identity of both neuroblast and glioblast lineages in the Drosophila embryonic CNS. ltt may act to differentiate NB identity along the medial lateral axis (Buescher, 1997).

ltt mutations affect the longitudinal glioblast (LG) lineage. Six LGs are derived from the LGB which forms at stage 10 in the lateral-most row of NBs at the anterior margin of each segment. Repo expression can be detected in the LGB shortly before its first division. At early stage 11, the LGB divides along the apical/basal axis to generate two progeny of approximately equal size. The dorsal cell is positive for nuclear Prospero (Pros) while the ventral cell remains negative for nuclear Pros. Both daughter cells migrate medially and anteriorly. During stages 11/12 the LGB progeny undergo further divisions that result in six Pros/Repo double positive cells, which are arrayed in a characteristic rhomboid pattern. At stage 15, eight to ten Repo-positive glia form two rows on the dorsal surface of the neural connectives; six of these cells are also positive for nuclear Pros. The ltt mutation causes a loss of pros expression in the LGB lineage. In ltt mutants, the division of the LGB occurs during stage 12, no further formation and/or maintenance of the longitudinal connectives is observed and frequently the longitudinal connectives are lost. Loss of pros expression alone cannot account for the LG phenotype: it has been shown previously that in pros loss-of-function mutants the six LG are formed, although these mutant LG appear spatially disorganized and fail to undergo terminal differentiation. Nevertheless, in pros loss-of-function mutants, the LG do express repo and can be identified unambigiously. Since in ltt embryos the six LG are either not present or fail to express Repo, it is concluded that the ltt mutation must cause defects in the LGB lineage, in addition to the loss of pros expression. Interestingly, in wild type embryos, many glial precursor cells do express Pros and the ltt mutation does not abolish pros expression in these cells. In contrast to the LG, the post-mitotic progeny of these glial precursors are Pros-negative. This suggests that pros expression is regulated differently within the LGB lineage and other glial lineages and that the ltt gene product is required for pros expression in the LGB lineage but not in other glial lineages (Buescher, 1997 and references).

The lack of correctly specified LG in ltt mutants coincides with a reduction of 22C10 (see Futsch) expression in early MP2 neurons and severe defects of the longitudinal axon tracts. However, the causal relationship between these defects is difficult to assess. The presence of correctly specified pros-expressing LG may be an absolute requirement for axon pathfinding and loss of the ltt function within the LGB lineage may be sufficient to cause a lack of longitudinal connectives. Alternatively, the neurons whose axons contribute to the longitudinal connectives may be affected by the mutation and may not be able to recognize the positional cues required for axon pathfinding. These scenarios are not mutually exclusive (Buescher, 1997).

Thus ltt mutation causes a duplication of the RP2 neuron and a lack of correctly specified LG. These results suggest that ltt function is required to restrict the number of RP2 neurons to one per hemisegment and to ensure that six Pros-positive LG per hemisegment are formed. The strongest ltt allele causes a duplication of RP2 in approx. 70% of the hemisegments but the LG are affected in all hemisegments. This observation suggests that the ltt function may be indispensable for the formation of the LG but may be partially redundant with respect to RP2 formation. These results raise the interesting possibility that ltt may belong to a class of genes that acts to differentiate NB identities between medial and lateral columns of NBs (Buescher, 1997).

Lineage, cell polarity and inscuteable function in the peripheral nervous system of the Drosophila embryo

The stereotyped pattern of the Drosophila embryonic peripheral nervous system (PNS) makes it an ideal system to use to identify mutations affecting cell polarity during asymmetric cell division. However, the characterization of such mutations requires a detailed description of the polarity of the asymmetric divisions in the sensory organ lineages. The pattern of cell divisions generating the vp1-vp4a mono-innervated external sense (es) organs is described. Each sensory organ precursor (SOP) cell follows a series of four asymmetric cell divisions that generate the four es organs cells (the socket, shaft, sheath cells and the es neuron) together with one multidendritic (md) neuron. This lineage is distinct from any of the previously proposed es lineages. Strikingly, the stereotyped pattern of cell divisions in this lineage is identical to those described for the embryonic chordotonal organ lineage and for the adult thoracic bristle lineage. This analysis reveals that the vp2-vp4a SOP cells divide with a planar polarity to generate a dorsal pIIa cell and a ventral pIIb cell. The pIIb cell next divides with an apical-basal polarity to generate a basal daughter cell that differentiates as an md neuron. Inscuteable specifically accumulates at the apical pole of the dividing pIIb cell and regulates the polarity of the pIIb division. This study establishes for the first time the function of Inscuteable in the PNS, and provides the basis for studying the mechanisms controlling planar and apical-basal cell polarities in the embryonic sensory organ lineages (Orgogozo, 2001).

The external sensory organ cells are arranged in a segmental, highly stereotyped fashion, and each es organ cell can be reliably identified using anti-Cut antibodies in stage 16 embryos. In order to describe the pattern of cell divisions in the es organ lineage, the divisions of the Cut-positive es precursor cells were followed between stages 11 and 16. Analysis focused on the five mono-innervated es organs located in the ventral region, vp1-vp4a, because this region is particularly outstretched following germ-band elongation, thus facilitating the identification of each es organ cell. In stage 16 embryos, the vp1, vp2, vp3, vp4 and vp4a (vp1-vp4a) organs are arranged in a circular arc. Each organ is composed of four Cut-positive cells. The socket and shaft cells, which lie within the epithelium, are strongly labelled by anti-Cut antibodies, whereas the neuron and the sheath cell, which are subepidermal, are more weakly labeled. Elav and Pros proteins accumulate specifically in the neuron and in the shaft cell, respectively. The vp4a organ is found relatively close to the weakly Cut-positive anterior ventral md neuron called vdaa. The vdaa and vp4a cells are born from the same md-es lineage. In the center of the ventral region, the four vdaA-D and the ventral bipolar (vbp) md neurons, which are clustered together, are also weakly labeled by anti-Cut antibodies (Orgogozo, 2001).

At stage 11, five isolated cells that accumulate Cut appear at stereotyped positions in the ventral region. Based on their position, these cells correspond to the pI cells of the five vp1-vp4a organs.The analysis of the positions of the two pI daughter nuclei at telophase indicates that pI divides within the plane of the epithelium. Numb localizes asymmetrically in pI at metaphase and is inherited by one of the two daughter cells at telophase. The pI daughter cell inheriting Numb is the pIIb cell and its sister is pIIa. In the case of the vp2-vp4a organs, the two daughter nuclei are positioned along the dorsal-ventral (d-v) axis, and Numb forms a ventral crescent at metaphase and segregates to the ventral daughter cell at telophase. It is concluded that, at the vp2-vp4a position, pI divides with a stereotyped d-v planar polarity. In contrast, the division of the vp1 pI cell is randomly oriented within the plane of the epithelium with Numb segregating in only one daughter cell (Orgogozo, 2001).

pIIb divides asymmetrically with an apical-basal polarity. At stage 12, the anterior-ventral cell of each pIIa-pIIb cell pair seen at the vp2-vp4a position enters mitosis. The position of the daughter nuclei relative to the surface of the embryo at telophase indicates that pIIb divides roughly perpendicular to the plane of the epithelium. Numb, Pon, Miranda and Pros, which are first detected in the dividing pIIb cell, localize to the basal pole of the pIIb cell at metaphase and segregate to the basal daughter cell. Noticeably, at telophase, the basal daughter cell appears to be significantly smaller than its apical sister. This indicates that pIIb generates two cells of different size. However, following pIIb division, no difference in nuclear size is detected using the Pros and Cut markers. It is concluded that, at the vp2-vp4a position, the pIIb division is polarized along the apical-basal axis of the epithelium (Orgogozo, 2001).

At the vp1 position, one of the two pI daughter cells expresses Pros and divides with an apical-basal polarity to generate a basal cell that inherits both Numb and Pros. Based on these observations, it is concluded that the second cell division observed at the vp1 position is the pIIb division, as shown for the vp2-vp4a positions. The small basal pIIb daughter cell that has specifically inherited Pros has been termed X, and pIIIb is its apical sister. Soon after the pIIb division, the only es cell to accumulate Pros is the X cell. In early stage 13 embryos, in which all pIIb cells have divided, two Pros-positive cells are observed: the basal highly Pros-positive X cell and the apical weakly Pros-positive pIIIb cell (Orgogozo, 2001).

pIIa divides next to generate the socket and shaft cells. At early stage 13, a Pros-negative pIIa cell entering mitosis can be observed, while clusters of four cells are seen at the corresponding position in adjacent hemisegments. These clusters contain the highly Pros-positive X cell, the weakly Pros-positive pIIIb cell and two Pros-negative cells. It is concluded that the two Pros-negative cells are the pIIa daughter cells. These cells are localized in the superficial epidermal layer and are strongly Cut positive. These two strongly Cut-positive cells are observed in the epidermis at the vp1-vp4a positions from stage 13 onward. At stage 16, these two cells express A1-2-29, a socket and shaft cell marker. These observations indicate that the division of pIIa generates the socket and shaft cells. At late stage 13, the weakly Pros-positive pIIIb cell enters mitosis. Pros is asymmetrically localized in dividing pIIIb and is inherited by only one daughter cell at telophase. The X and pIIIb cells both accumulate Elav, a neuronal marker. The X cell can be easily identified as it accumulates a higher level of Elav. In contrast to Pros, Elav segregates equally into the two pIIIb daughter cells at telophase. Following the pIIIb division, the vp1-vp4a clusters are composed of five cells: the socket and shaft cells, the two pIIIb daughter cells and the X cell (Orgogozo, 2001).

At stage 13, each X cell occupies a stereotyped position. The vp4a X cell is located dorsally between the vp4a and vp4 clusters, and each of the vp1-vp4 X cells is found nearest the center of the circular arc formed by the vp1-vp4a cells. The accumulation of Elav in the X cell indicates that X may become a neuron. To determine the fate of the X cell, the positions of the Pros- and Elav-positive X cells were compared in adjacent hemisegments of late stage 13 embryos. This analysis suggests that the vp1-vp4 X cells migrate towards the center of the vp1-vp4a circular arc, while the vp4a X cell migrates dorsally. Consistent with a migratory behaviour, the X cells display long cytoplasmic extensions at this stage. The level of Pros accumulation in the migrating Cut- and Elav-positive X cells appears to decrease over time, and becomes undetectable when these cells cluster in the center of the circular arc at stage 14. At this stage, these Cut-positive X cells can still be identified on the basis of their stereotyped position and of their high level of Elav accumulation. These cells occupy the positions of the vdaA-D/vbp cluster and of the vdaa neuron and, from stage 14 onwards, express the E7-2-36 md marker. These data indicate that the vp4a X cell migrates dorsally and becomes the vdaa md neuron, whereas the four vp1-vp4 X cells migrate towards the center of the circular arc to form four of the five vdaA-D/vbp neurons. The fifth vdaA-D/vbp neuron corresponds to the additional Cut-, Pros-and Elav-positive cell that migrates (together with the vp1 md neuron) toward the center of the circular arc (Orgogozo, 2001).

This fifth md neuron probably originates from a Cut-positive precursor cell detected anterior to vp1. This precursor cell divides asymmetrically at late stage 12 to generate a Pros- and Elav-positive cell that migrates dorsally (Orgogozo, 2001).

The es neuron and sheath cell are born from the pIIIb cell. From stage 14 onwards, one of the two pIIIb daughter cells accumulates a higher level of Elav, and is therefore identified as the es neuron. Its sister cell accumulates a high level of Pros and is thus identified as the sheath cell. No additional division is observed in the vp1-vp4a lineages after the pIIIb division (Orgogozo, 2001).

In summary, this analysis shows that the vp1-vp4 es SOPs produce four md neurons that most likely correspond to the four vdaA-D organs. The vp4a SOP follows an identical lineage and generates the vdaa md neuron. In this novel md-es lineage, the md neuron is generated by the division of the pIIb cell. This study of the vp1-vp4a lineages rules out all three previously proposed models for the md-es lineage. Also, the pattern of cell divisions is identical in the vp1- vp4a, chordotonal and adult bristle lineages. It is therefore proposed that the lineage described here for the vp1-vp4a lineages applies to all mono-innervated es organs in the embryo (Orgogozo, 2001).

This detailed analysis of the vp1-vp4a lineages allowed for an investigation of the mechanisms regulating cell polarity in these lineages. Previous studies have indicated that insc is expressed in pI, suggesting a role for insc in regulating cell polarity in these lineages. The expression pattern of insc was examined in the vp1-vp4a lineages. Insc protein is not detectable in dividing pI, pIIa and pIIIb cells, but specifically accumulates in an apical crescent in dividing pIIb cells. The lack of insc expression in pI is further confirmed by the analysis of an insc-lacZ enhancer-trap marker. The expression of insc-lacZ is not detectable in pI and pIIa. However, it is first detected in the pIIb cell as it divides and specifically accumulates in both pIIb daughter cells. insc regulates the apical-basal polarity of the pIIb division The role of insc in regulating cell polarity was examined in the vp1-vp4a lineages. In insc mutant embryos, the vp1-vp4a pI divisions occur within the plane of the epithelium. The vp2, vp4 and vp4a pI cells divide with a d-v orientation with Numb localiz ing asymmetrically to the ventral pole of pI. Furthermore, the cell that divides next is always found at an antero-ventral position in both wild-type and insc mutant embryos, suggesting that the pIIa and pIIb cells are correctly specified. It is concluded that the loss of insc activity does not affect the polarity of the pI division. This is entirely consistent with the observation that the Insc protein is not present in the pI cell (Orgogozo, 2001).

To analyse the role of insc in the dividing pIIb cell, the asymmetric distribution of Miranda, an adaptor protein for Pros, was examined. In wild-type embryos, Miranda accumulates to the basal pole of pIIb at metaphase. In contrast, Miranda localizes asymmetrically to the basal pole in only 32% of insc mutant pIIb cells at metaphase. In the other pIIb cells, Miranda is either partly (52%) or largely (16%) delocalized around the cell cortex. This shows that insc is required for the basal localization of Miranda (Orgogozo, 2001).

The distribution of Pros, which is the earliest marker for the fate of the md and pIIIb cells in the vp1- vp4a lineages, was examined. An equal level of Pros accumulation was found in 27% of the pIIb daughter cells in insc mutant embryos. This indicates that insc is required to regulate the unequal segregation of Pros during the pIIb division and/or to establish a fate difference between the two pIIb daughter cells (Orgogozo, 2001).

The role of insc in regulating cell fate decisions in the vp1-vp4a lineages was examined. Attention was focused on the vp4a organ because this lineage generates one md neuron that migrates very little, which greatly facilitates the identification of all the cells produced by the vp4a lineage. Cut, Elav and E7-2-36 were used as cell fate markers to identify by stage 16 the vp4a socket, shaft and sheath cells, and the es neuron and the vdaa md neuron. At all vp4a positions in insc mutant embryos, the socket and shaft cells are always present. In some segments, however, the vdaa md neuron is duplicated and the vp4a es neuron and sheath cell are missing. This suggests that the pIIIb cell has been transformed into a second md neuron. In some other segments, a single Elav-positive, E7-2-36-negative cell is seen at the position of the vp4a es neuron and sheath cell. This suggests that the pIIIb cell has failed to divide. In yet other segments, the two cells at the position of the es neuron and sheath cell express variable levels of Elav, indicating that the two pIIIb daughter cells are not correctly specified. It is concluded that insc regulates the fate of the pIIb daughter cells (Orgogozo, 2001).

This analysis was extended to the vp1-vp4 lineages. Socket and shaft cells are always detected, while the neuron and sheath cells form properly in only 66% (n=124) of the vp1-vp4 organs. In 22% of the cases, the two cells at the position of the sheath cell and es neuron express a similar level of either Elav, or Pros, or both Pros and Elav. In 6% of the es organs, only one Elav-expressing cell is detected. Finally, in the remaining 6%, the es neuron and sheath cell are both missing. This defect is always associated with the presence of an additional md neuron at the vdaA-D/vbp position (7 cases out of 7. This indicates that the pIIIb cell has been transformed into an md neuron (Orgogozo, 2001).

In conclusion, the data show that the loss of insc activity results in cell polarity defects in the pIIb cell, as revealed by the mislocalization of Miranda at metaphase. This phenotype correlates with the abnormal accumulation of Pros into the apical pIIb daughter cell and with the mis-specification of the pIIIb cell (Orgogozo, 2001).

This study provides the first detailed description of each asymmetric cell division in an md-es lineage. The division of the vp2-vp4a pI cell is planar and takes place with a d-v polarity, revealing for the first time the existence of a planar polarity orienting asymmetric cell divisions in the embryo. Similarly, in the pupa, the pI cell divides in the plane of the epithelium and along the a-p axis. The polarity of this division is controlled by the Fz signaling pathway. In both pupae and embryos, the pIIb cell divides with an apical-basal polarity, with Numb, Pros and Miranda segregating to the basal cell. Moreover, Insc forms an apical crescent in the pIIb cell in the pupal lineage. This suggests that Insc regulates also the apical-basal polarity of the pIIb cell in the adult bristle lineage. It is clear, however, that a detailed analysis of the function of insc in regulating cell polarity in the adult PNS would have been much more difficult and time-consuming because insc mutations are embryonic lethal. In conclusion, this study clearly illustrates that the regulation of both planar and apical-basal polarities can now be studied in the embryonic PNS. This detailed analysis therefore provides the basis for future studies addressing the function of various candidate genes known to affect the development of the embryonic PNS (Orgogozo, 2001).

Slit/Robo signaling regulates cell fate decisions in the intestinal stem cell lineage of Drosophila

In order to maintain tissue homeostasis, cell fate decisions within stem cell lineages have to respond to the needs of the tissue. This coordination of lineage choices with regenerative demand remains poorly characterized. This study identified a signal from enteroendocrine cells (EEs) that controls lineage specification in the Drosophila intestine. EEs secrete Slit, a ligand for the Robo2 receptor in intestinal stem cells (ISCs) that limits ISC commitment to the endocrine lineage, establishing negative feedback control of EE regeneration. Furthermore, this lineage decision was shown to be made within ISCs and requires induction of the transcription factor Prospero in ISCs. This work identifies a function for the conserved Slit/Robo pathway in the regulation of adult stem cells, establishing negative feedback control of ISC lineage specification as a critical strategy to preserve tissue homeostasis. The results further amend the current understanding of cell fate commitment within the Drosophila ISC lineage (Biteau, 2014).

The data support a model in which Slit/Robo2 controls cell fate decisions in the ISC lineage by regulating the specification of ISCs into Prospero-expressing EE precursors before or during mitosis. Interestingly, manipulating the activity of Robo2 in ISCs did not affect the phenotype generated by expression of NotchRNAi (in which the formation of EC-committed EBs is specifically inhibited). In addition, no evidence was found that loss of Robo2 affects Delta expression in ISCs. Finally, the activation of the Notch pathway is sufficient to promote differentiation independently of Robo2 signaling. This supports the idea that Robo2 acts upstream and independently of the activation of the Notch signaling pathway, regulating lineage commitment in ISCs, whereas Notch specifically controls differentiation of daughter cells into the EC fate. In this model, the absence of Notch signaling results in default commitment of ISC daughter cells into an EE fate, and lineage commitment thus becomes independent of Robo2/Slit signaling, because EC differentiation is impaired (Biteau, 2014).

It is interesting to note that the intensity of the Slit signal is integrated by ISCs to generate an all-or-nothing response: above a defined Slit threshold, Prospero is expressed by around 6% of mitotic ISCs, whereas below this level, 15%-20% of ISCs express Prospero, and no intermediate expression of Prospero can be detected. Further studies will be required to characterize the signaling cascade that controls Prospero expression downstream of the Robo2 receptor in ISCs (Biteau, 2014).

Robo4 has recently been identified as a regulator of hematopoietic stem cell homing in mice. In addition, proteins of the Slit and Robo families have been suggested to act as tumor suppressors and be directly involved in the tumorigenesis process. This study identifies a mechanism by which differentiated cells engage this pathway to directly regulate stem cell function and lineage commitment. A role for Slit/Robo signaling in the control of fate decisions in mammalian normal or cancer stem cell lineages has not yet been tested. However, based on the conservation of mechanisms that control Drosophila ISC self-renewal and differentiation, it can be anticipated that this feedback control of stem cell fate decisions through Slit/Robo signaling also controls adult tissue homeostasis in higher organisms (Biteau, 2014).

The Snail protein family regulates neuroblast expression of inscuteable and string, genes involved in asymmetry and cell division in Drosophila

Snail, a zinc-finger transcriptional repressor, is a pan-neural protein, based on its extensive expression in neuroblasts. Previous results have demonstrated that Snail and related proteins, Worniu and Escargot, have redundant and essential functions in the nervous system. The Snail family of proteins control central nervous system development by regulating genes involved in asymmetry and cell division of neuroblasts. In mutant embryos that have the three genes deleted, the expression of inscuteable is significantly lowered, while the expression of other genes that participate in asymmetric division, including miranda, staufen and prospero, appears normal. The deletion mutants also have much reduced expression of string, suggesting that a key component that drives neuroblast cell division is abnormal. Consistent with the gene expression defects, the mutant embryos lose the asymmetric localization of Prospero RNA in neuroblasts and lose the staining of Prospero protein that is normally present in ganglion mother cells. Simultaneous expression of inscuteable and string in the snail family deletion mutant efficiently restores Prospero expression in ganglion mother cells, demonstrating that the two genes are key targets of Snail in neuroblasts. Mutation of the dCtBP co-repressor interaction motifs in the Snail protein leads to reduction of the Snail function in central nervous system. These results suggest that the members of the Snail family of proteins control both asymmetry and cell division of neuroblasts by activating, probably indirectly, the expression of inscuteable and string (Ashraf, 2001).

In mutants containing deletions that uncover escargot, worniu and snail, many early neuroblast markers are normal, but ftz expression in GMCs is abnormal. The regulation of ftz depends on Prospero, a homeodomain protein that controls GMC fate. Prospero protein and mRNA are preferentially segregated to GMCs from the neuroblast through the process of asymmetric division. Genes that are involved in asymmetric segregation of Prospero include inscuteable, miranda and staufen. The expression of these possible Snail family target genes was examined in neuroblasts (Ashraf, 2001).

The segregation of Prospero protein into GMCs from neuroblasts is a critical event during asymmetric cell division. Since inscuteable plays a role in the segregation of prospero gene products into GMCs, whether there is Prospero protein in GMCs of mutant embryos was examined. Prospero protein staining can be easily detected in many wild type GMC nuclei. The staining is largely absent in the deletion that uncovers the snail family locus; only a few cells with the size of normal GMCs had clear nuclear staining. A band of cells along the midline also had Prospero staining, but these cells probably represent an expansion of the midline. It has been well documented that in all snail mutants there is derepression of the mid-line determinant single-minded in the blastoderm stage embryo (Ashraf, 2001).

To determine whether there are defects within GMCs in addition to the loss of Prospero, the expression of Hunchback, which is present transiently in early neuroblasts and later in many GMCs was examined. In the deletion mutant, the Hunchback protein in GMCs is also absent, while staining in cells surrounding the amnioserosa appeared normal. Transgenes of snail, worniu and escargot rescue the staining of Prospero and Hunchback, indicating that these GMC determinants are downstream of the Snail family. The results also suggest that the regulation of ftz by the Snail family is indirect, probably through an earlier event such as segregation of Prospero from neuroblast to GMC (Ashraf, 2001).

If the misregulation of inscuteable in the deletion mutant is the cause of the loss of Prospero and ftz expression in GMCs, the expression of inscuteable should correct the defects even in the absence of Snail family of proteins. A line carrying an inscuteable transgenic construct driven by the 2.8 kb snail promoter was crossed into the osp29 deletion genetic background. However, the rescue of Prospero expression in GMCs was variable and not nearly as strong as those embryos expressing the snail family transgenes. This suggests that inscuteable may not be the only important target gene of Snail. Another line of evidence supporting the idea of an additional target gene comes from the comparison of the phenotypes in osp29 and inscuteable mutant embryos. In inscuteable mutants, the Prospero crescent is formed but the mitotic spindle rotation is randomized. As a result, the Prospero protein frequently is present both in neuroblasts and GMCs. This phenotype is less severe than the almost total loss of Prospero GMC staining in osp29 deletion mutant. Therefore, it is surmised that in addition to the misregulation of inscuteable, there may be other defects that lead to the more severe phenotype in the deletion mutants (Ashraf, 2001).

A study of APC1 and APC2 examines asymmetric protein localization in larval neuroblasts

The tumor suppressor APC and its homologs, first identified for a role in colon cancer, negatively regulate Wnt signaling in both oncogenesis and normal development, and play Wnt-independent roles in cytoskeletal regulation. Both Drosophila and mammals have two APC family members. The functions of the Drosophila APCs is further explored using the larval brain as a model. Both proteins are expressed in the brain. APC2 has a highly dynamic, asymmetric localization through the larval neuroblast cell cycle relative to known mediators of embryonic neuroblast asymmetric divisions. Adherens junction proteins also are asymmetrically localized in neuroblasts. In addition they accumulate with APC2 and APC1 in nerves formed by axons of the progeny of each neuroblast-ganglion mother cell cluster. APC2 and APC1 localize to very different places when expressed in the larval brain: APC2 localizes to the cell cortex and APC1 to centrosomes and microtubules. Despite this, they play redundant roles in the brain; while each single mutant is normal, the zygotic double mutant has severely reduced numbers of larval neuroblasts. These experiments suggest that this does not result from misregulation of Wg signaling, and thus may involve the cytoskeletal or adhesive roles of APC proteins (Akong, 2002).

One striking feature of the asymmetric localization of APC2 is that it is present throughout the cell cycle and is particularly strong during interphase. During embryonic neuroblast divisions, most asymmetric markers are localized only during mitosis. However, less is known about their localization in larval neuroblasts. Several asymmetric markers in larval neuroblasts were examined, and their localization was compared with that of APC2. In embryonic neuroblasts, the transcription factor Prospero (Pros) and its mRNA are GMC determinants that are asymmetrically localized to the GMC daughter. Pros protein then becomes nuclear and helps direct cell fate. In larval neuroblasts, a similar localization is observed. Pros is not detectable in interphase neuroblasts, when the cortical APC2 crescent is strongest. A small amount of Pros transiently localizes to an asymmetric crescent during mitosis. Pros is present at low levels in GMC nuclei and at higher levels in the nuclei of ganglion cells (Akong, 2002).

Mira is basally localized in embryonic neuroblasts, and required there for localization of Pros protein and mRNA. In central brain neuroblasts, Mira is diffusely cytoplasmic during interphase, when the APC2 crescent is the strongest. As cells enter mitosis, Mira first becomes cortical and then begins to accumulate asymmetrically on the side of the neuroblast where the daughter will be born. By metaphase, Mira asymmetry is very pronounced. The center of the Mira crescent is always precisely aligned with one spindle pole. As a result, in cells with the spindle pointing toward the center of the APC2 crescent, the Mira and APC2 crescents substantially overlap, while in cells in which the spindle points to the edge of the APC2 crescent, the two crescents are offset. Mira is partitioned into the GMC during anaphase, while APC2 relocalizes to the cleavage furrow. Mira could still be detected in some GMCs, which are thought to be those that were recently born (Akong, 2002).

In contrast to Mira and Pros, Inscuteable (Insc) and Bazooka (Baz) localize to the apical sides of embryonic neuroblasts, where they play essential roles in asymmetric divisions. Insc is asymmetrically localized in larval neuroblasts. Insc localizes to the side of the neuroblast opposite that of APC2 through much, if not all, of the cell cycle. Interestingly, there is a weak Insc crescent during interphase, that becomes stronger through prophase and metaphase. During anaphase, Insc localizes to the neuroblast cortex but not the GMC daughter. Baz localization was similar to that of Insc, though no cortical localization during interphase was detected. During prophase and metaphase, Baz localizes to a crescent opposite APC2, and as the chromosomes begin to separate, Baz localizes to a tight cap opposite the future GMC. Together, these data confirm that larval and embryonic neuroblasts asymmetrically localize many of the same proteins, and that APC2 localizes on the GMC side (basal) of the neuroblast, overlapping Mira and opposite Baz and Insc, which localize apically (Akong, 2002).

Arm also localizes asymmetrically in neuroblasts. Extending this, an examination was made of the localization of Arm's adherens junction partners DE-cadherin and ß-catenin. When central brain neuroblasts undergo a sequential series of asymmetric divisions, the GMCs remain associated with their neuroblast mother, resulting in a cap of GMCs in association with each neuroblast. APC2 localizes strongly to the boundary between the neuroblast and each GMC, and more weakly to the borders between the GMCs. APC2 is present at lower levels in ganglion cells and differentiating neurons (Akong, 2002).

The adherens junction proteins DE-cadherin, Arm, and ß-catenin all show a striking and asymmetric localization pattern in central brain neuroblasts. All precisely colocalize both at the boundary between neuroblasts and GMCs and at the boundaries between GMCs. DE-cadherin, Arm, and ß-catenin are also all expressed in epithelial cells of the outer proliferation center. The localization of DE-cadherin and the catenins is consistent with the idea that cadherin-catenin-based adhesion could help ensure that GMCs remain associated with each other, via association with their neuroblast mother (Akong, 2002).

To further explore this, how successive GMCs are positioned relative to their older GMC sisters was examined using two different approaches. First Mira was used to mark the newborn GMCs and DE-cadherin was used to mark the neuroblast and all of her GMC daughters. Mira localizes to a crescent on the side of the neuroblast where the daughter will be born (basal side), and then is segregated into the daughter. Mira persists for some time in newborn GMCs, and it remains detectable in the other GMCs as well, thus allowing the position of newborn GMCs to be examined relative to their older sisters. In many cases, new GMCs are clearly born at the edge of the cluster of older GMCs. This is particularly striking in neuroblasts with many progeny. It is worth noting that the cluster of daughters is three-dimensional, comprising a 'cap' of daughters in three dimensions rather than a two-dimensional line of daughters. It is thus suspected that new daughters are born near the edge of this cap (Akong, 2002).

These data suggest that neuroblasts and their GMC progeny remain closely associated. The GMCs then divide to form ganglion cells and ultimately neurons. The data further suggest that these latter cells may also remain associated and send their axons together toward targets in the central brain. When sections were made more deeply into the brain, below each cluster of neuroblasts and GMCs, structures that appear to be axons were detected projecting from these groups of cells. These axons label with Arm, DE-cadherin, and APC1. Arm also localizes to the axons of the neuropil, while DE-cadherin and APC2 are present at low levels or are absent from this structure (Akong, 2002).

Evidence that stem cells reside in the adult Drosophila midgut epithelium: pros+ cells probably define a population of enteroendocrine cells in the midgut

Adult stem cells maintain organ systems throughout the course of life and facilitate repair after injury or disease. A fundamental property of stem and progenitor cell division is the capacity to retain a proliferative state or generate differentiated daughter cells; however, little is currently known about signals that regulate the balance between these processes. A proliferating cellular compartment has been characterized in the adult Drosophila midgut. Using genetic mosaic analysis it has been demonstrated that differentiated cells in the epithelium arise from a common lineage. Furthermore, reduction of Notch signalling leads to an increase in the number of midgut progenitor cells, whereas activation of the Notch pathway leads to a decrease in proliferation. Thus, the midgut progenitor's default state is proliferation, which is inhibited through the Notch signalling pathway. The ability to identify, manipulate and genetically trace cell lineages in the midgut should lead to the discovery of additional genes that regulate stem and progenitor cell biology in the gastrointestinal tract (Micchelli, 2006).

The adult Drosophila midgut can be identified on the basis of two anatomical landmarks along the anterior-posterior axis of the gastrointestinal tract: the cardia and pylorus. The inner surface of the midgut is lined with a layer of cells that project into the gut lumen. These cells exhibit apical-basal polarity; staining for F-actin reveals the presence of a distinct striated border on their lumenal surface. This observation is consistent with the suggestion that the midgut is lined by a cellular epithelium (Micchelli, 2006).

Wild-type midguts were stained with 4,6-diamidino-2-phenylindole (DAPI) to reveal the distribution of cell nuclei within the tissue. Nuclei of the midgut display a distinct distribution and fall into two main categories. The most prominent cells lining the midgut contain large oval nuclei that stain strongly with DAPI. These cells exhibit a region of the nucleus that does not stain with DAPI, giving the nucleus a hollow appearance. This unstained region may correspond to the large nucleolus characteristic of differentiated cells. A second population of cells containing small nuclei can be detected at a basal position within the tissue. The small nuclei are distant from the gut lumen and often lie in close apposition to the two layers of overlying visceral muscle that surround the gut. On the basis of nuclear size, position and morphology two general populations of midgut cells can, therefore, be distinguished (Micchelli, 2006).

Previous studies in Drosophila have led to conflicting views over the existence of cell proliferation in the adult gastrointestinal tract. Early reports suggested that somatic stem cells were present in the adult because of morphological similarity to certain larval cells and by analogy to different insect species. In contrast, 3H-thymidine labelling experiments detected DNA synthesis in the adult Drosophila midgut, but no mitotic figures were observed in a large sample analysed. On the basis of these observations, it was concluded that no somatic cell division occurs during the lifetime of Drosophila. To distinguish between these possibilities, a series of three independent assays was used to test whether cell proliferation can be detected in the adult midgut. In the first assay genetically marked wild-type cell lineages were used to identify dividing cells. The production of marked clones after mitotic recombination depends upon subsequent cell division and is, therefore, a direct means to assay proliferation. In these experiments, wild-type lineages were positively marked in adult flies using the MARCM system. Mitotic recombination was induced by heat shock and green fluorescent protein (GFP)-marked clones could be detected in the midgut. Similar results were obtained when adults were heat shocked up to 10 days after eclosion. This suggests that the ability to generate clones is not transient, and probably persists throughout the entire life of the animal (Micchelli, 2006).

Under the experimental conditions used, the MARCM system produced some background GFP signal that could be detected in control animals. To quantify the background signal, the number of GFP-labelled cells was compared in control and experimental animals. A greater than sixfold increase in the number of GFP-labelled cells was detected after heat shock. A second independent clone marking method was used that did not rely on either Gal4 or Gal80. In these experiments, clones were marked by the loss of a ubiquitously expressed GFP and similar results were observed. It is concluded that a population of actively dividing somatic cells is present in the adult Drosophila midgut (Micchelli, 2006).

To extend these findings, 5-bromodeoxyuridine (BrdU) incorporation studies were constructed. Both large and small BrdU-labelled midgut cells were detected. Large nuclei adjacent to each other can be differentially labelled, suggesting asynchrony in the timing or extent of DNA synthesis over the course of the labelling period. This is consistent with the notion that the large nuclei are endoreplicating. However, both endoreplication and the canonical cell cycle require new DNA synthesis. To distinguish endoreplicating from dividing cells in the midgut the tissue was stained with an antibody raised against phospho-histone H3. Careful examination revealed that very low levels of phospho-histone H3 staining could be detected in all cells. However, double staining with DAPI revealed that elevated levels of phospho-histone H3 indicative of mitosis could be detected only among the population of cells with small nuclei. Thus, cells in the midgut seem to have two distinct cell cycles; whereas both large and small nuclei undergo DNA synthesis, only the cells with small nuclei undergo cell division (Micchelli, 2006).

In order to characterize further the small cell population, an expression screen was conducted to identify cell-specific molecular markers. Three markers expressed in small cells were identified: escargot (esg), a transcription factor that belongs to the conserved Snail/Slug family; prospero (pros), a conserved homodomain transcription factor, and Su(H)GBE-lacZ, a transcriptional reporter of the Notch signalling. Simultaneous detection of esg expression (esg-Gal4, UAS-GFP), anti-Pros, Su(H)GBE-lacZ expression and DAPI has demonstrated that small cells can be subdivided into the following classes on the basis of differential gene expression: esg-positive (esg+), pros-positive (pros+), esg-negative pros-negative (esg- pros-), esg-positive Su(H)GBE-lacZ-positive [esg+ Su(H)GBE-lacZ+] and esg-positive Su(H)GBE-lacZ-negative [esg+ Su(H)GBE-lacZ-]. esg+ and pros+ expression define distinct cell populations, whereas Su(H)GBE-lacZ expression subdivides the esg+ class into esg+ Su(H)GBE-lacZ+ and esg+ Su(H)GBE-lacZ- subpopulations. Quantification reveals that each cell type is present in the midgut in different proportions. The ability to distinguish different cell types using molecular markers enabled determination of the cell lineage relationships in this tissue. If the large and small nuclei are lineally distinct then marked clones should be restricted to one or the other cell type. However, if a common stem cell progenitor exists in the adult midgut, then marked lineages should contain both large and small nuclei within a clone. To distinguish between these possibilities positively marked MARCM clones were generated and nuclei were labeled using DAPI. Lineage analysis shows that marked clones generated in the adult contain both large and small nuclei. In addition, both esg expression and anti-Pros-labelled cells could be detected within the clones. These lineage-tracing experiments suggest that a stem cell progenitor exists and is sufficient to generate the distinct cell types of the adult midgut. This cell is referred to as the adult intestinal stem cell (ISC) (Micchelli, 2006).

esg expression in diploid cells has been shown to be necessary for the maintenance of diploidy. In addition, the distribution of esg messenger RNA has been used as a marker for male germline stem cells. Together, these observations raise the hypothesis that esg expression may also mark a population of progenitors in the midgut. It was therefore asked whether esg expression correlates with markers of cell proliferation. Simultaneous staining with anti-BrdU and DAPI reveals that esg-expressing cells are among the population of cells that are also positively labelled by BrdU. To ask whether esg-expressing cells also undergo cell division, the midgut was double stained to detect both esg expression and phospho-histone H3. High levels of phospho-histone H3 can be detected specifically in esg-expressing cells. These results demonstrate that esg expression marks a population of proliferating progenitor cells in the midgut (Micchelli, 2006).

However, the esg+ cell population can be divided on the basis of Su(H)GBE-lacZ expression. To distinguish functionally the two esg+ populations, the consequences of altering Notch signalling in the adult midgut were examined. The effect of globally reducing Notch signalling was tested using the conditional Notch temperature-sensitive (Nts) mutant. Nts flies were first crossed to an allelic series that included N55e11, N264.47, Nts1 and Nnd.1. The strongest loss of function combinations (Nts/N55e11 and Nts/N264.47) failed to generate viable adult flies even at the permissive temperature, often dying as pharate adults. Nts/Nts flies produced viable adults at the permissive temperature with midguts similar to wild type. Nts/Nts flies shifted to the non-permissive temperature led to a mild increase in the number of small cells. The weakest allelic combination, Nts/Nnd.1, also produced viable adults at the permissive temperature but showed no detectable phenotype when shifted to the non-permissive temperature (Micchelli, 2006).

The requirement of N only in esg+ progenitor cells was tested. To obtain both spatial and temporal control over transgene expression in esg-expressing cells, the temperature-sensitive Gal80 inhibitor, Gal80ts was combined with the esg-Gal4 transcriptional activator. To verify that the Gal80ts transgene functions in the midgut, the temporal and spatial induction of a UAS-GFP transgene was characterized. Adult esg-Gal4,UAS-GFP, tub-Gal80ts flies grown at the permissive temperature showed no detectable GFP expression in their midguts In contrast, when these flies were shifted to the non-permissive temperature they showed high levels of GFP expression that were detectable after 1 day and maximal by 2 days (Micchelli, 2006).

The requirement of Notch was then tested in esg+ cells using a UAS-NRNAi transgene, to reduce Notch signalling. In control experiments, UAS-NRNAi; esg-Gal4,UAS-GFP, tub-Gal80ts flies grown at the permissive temperature appear to have wild-type midguts and show no detectable GFP expression, suggesting that under these conditions UAS transgenes are efficiently suppressed. In contrast, UAS-NRNAi; esg-Gal4,UAS-GFP, tub-Gal80ts flies shifted to the non-permissive temperature show an increase in the number of small cells (19 out of 20 midguts). Notably, the presence of esg-Gal4, UAS-GFP in this experiment enabled a determination that the increased number of small cells were also esg+. When these guts were co-stained with anti-Pros antibody ectopic small cells were observed that also expressed pros, and these cells were often associated with lower levels of esg expression. Taken together these experiments suggest that Notch signalling in esg+ cells is necessary to restrict proliferation (Micchelli, 2006).

The effect of Notch activation was tested in esg+ cells using Nintra, a constitutively active form of Notch. In control experiments, esg-Gal4,UAS-GFP, tub-Gal80ts; UAS-Nintra flies grown at the permissive temperature appear to have wild-type midguts and show no detectable GFP expression. In contrast, esg-Gal4,UAS-GFP, tub-Gal80ts; UAS-Nintra flies shifted to the non-permissive temperature showed a decrease in phospho-histone H3 staining compared to controls that were not shifted. In addition, although some esg+ cells appear to be wild type, a region-specific decrease was observed in the levels of esg expression and a concomitant increase in nuclear size similar to that of midgut epithelial cells. These observations demonstrate that Notch activation is sufficient to limit proliferation of esg+ cells and suggests that Notch may also be sufficient to promote early steps of epithelial cell differentiation (Micchelli, 2006).

This characterization of the adult Drosophila midgut suggests that a population of adult stem cells resides within this tissue. This analysis of the Notch signalling pathway in esg+ cells suggests that esg+ Su(H)GBE-lacZ- cells mark a population of dividing progenitors and that Notch is necessary and sufficient to regulate proliferation. A model is proposed in which esg+ Su(H)GBE-lacZ- progenitors generate at least two different types of daughter cells depending on the level of Notch activation. Under conditions of reduced Notch function an expansion of both esg+ progenitor cells and pros+ cells is observed. These observations suggest that esg+ cells give rise to pros+ cells in a Notch-independent manner. Under conditions of Notch activation a decrease is observed in the proliferation and promotion of epithelial cell fate differentiation, while the number of pros+ cells remains unaffected (Micchelli, 2006).

Several lines of evidence suggest that pros+ cells correspond to gut enteroendocrine cells. Previous studies show that prox1, the vertebrate pros homologue, is associated with post-mitotic cells and early steps of differentiation in the central nervous system. Furthermore, in Drosophila, pros is thought to be a pan-neural selector gene that is both necessary and sufficient to terminate cell proliferation. Finally, although vertebrate enteroendocrine cells arise from endodermal origins they are known to express neural-specific markers. Therefore, pros+ cells probably define a population of enteroendocrine cells in the midgut (Micchelli, 2006).

Studies of stem cell compartments in Drosophila have led to the characterization of two types of progenitor cells in the germ line. The first is referred to as the germline stem cell and is sufficient to give rise to the respective cells of either the male or female germ line. The second type of progenitor cell described is called the cystoblast in female germ line and gonialblast in the male germ line. Although the cystoblast and gonialblast both have the capacity to generate the differentiated cells of their respective tissues, they are thought to be more restricted in their fate than the germline stem cells. On this basis, it is suggested that an analogous progenitor may also exist in the adult Drosophila midgut; this cell is referred to as the enteroblast (EB). The population of esg+ Su(H)GBE-lacZ- progenitor cells, which has been described, displays characteristics of both the ISC and the EB; therefore, additional experiments will be necessary to distinguish unambiguously these alternatives (Micchelli, 2006).

Drosophila Incenp is required for cytokinesis and asymmetric cell division during development of the nervous system

The chromosomal passenger protein complex has emerged as a key player in mitosis, with important roles in chromatin modifications, kinetochore-microtubule interactions, chromosome bi-orientation and stability of the bipolar spindle, mitotic checkpoint function, assembly of the central spindle and cytokinesis. The inner centromere protein (Incenp; a subunit of this complex) is thought to regulate the Aurora B kinase and target it to its substrates. To explore the roles of the passenger complex in a developing multicellular organism, a genetic screen was performed looking for new alleles and interactors of Drosophila Incenp. A new null allele of Incenp has been isolated that has allowed a study of the functions of the chromosomal passengers during development. Homozygous incenpEC3747 embryos show absence of phosphorylation of histone H3 in mitosis, failure of cytokinesis and polyploidy, and defects in peripheral nervous system development. These defects are consistent with depletion of Aurora B kinase activity. In addition, the segregation of the cell-fate determinant Prospero in asymmetric neuroblast division is abnormal, suggesting a role for the chromosomal passenger complex in the regulation of this process (Chang, 2006).

Asymmetric cell division is key to the development of the Drosophila nervous system. Each dividing neuroblast produces one large daughter cell that remains a multipotent neuroblast and continues to divide, and a smaller daughter cell that becomes a ganglion mother cell that divides once more asymmetrically to produce neurons or glia cells. This cell-fate decision hinges on the segregation of Prospero, a homeodomain transcription factor that is segregated largely, if not exclusively, into the ganglion mother cell. This is accomplished by sequestering Prospero into a basal cortical crescent in the dividing neuroblast from prophase onwards (Chang, 2006).

In wild-type embryos, the expected asymmetric distribution of Prospero at the basal cell surface of dividing cells was observed. However, in early prophase, Prospero transiently associates with the condensing chromatin on entry into mitosis. The distribution of Prospero was abnormal in neuroblasts lacking detectable Incenp. Abnormalities observed in neuroblasts of incenpEC3747 embryos included defects in the shape and orientation of the basal Prospero crescent. Mitotic neuroblasts were observed with Prospero distributed all around the cell cortex, and not restricted to a basal crescent. These results reveal that Drosophila Incenp and, therefore, presumably the chromosomal passenger complex, is required for the correct localization of Prospero during asymmetric cell division in the developing Drosophila nervous system (Chang, 2006).


The division of postembryonic neuroblasts (Nbs) has been studied in the outer proliferation center (OPC) and central brain anlagen of Drosophila. Attention has been focussed on three aspects of these processes: the pattern of cellular division; the topological orientation of these divisions, and the expression of asymmetric cell fate determinants. Although larval Nbs are of embryonic origin, the results indicate that their properties appear to be modified during development. Several conclusions are summarized: (1) in early larvae, Nbs divide symmetrically to give rise to two Nbs while in the late larval brain most Nbs divide asymmetrically to bud off an intermediate ganglion mother cell (GMC) that very rapidly divides into two ganglion cells (GC); (2) symmetric and asymmetric divisions of OPC Nbs show tangential and radial orientations, respectively; (3) this change in the pattern of division correlates with the expression of Inscuteable, which is apically localized only in asymmetric divisions; (4) the spindle of an asymmetrically dividing Nb is always oriented on an apical-basal axis; (5) Prospero does not colocalize with Miranda in the cortical crescent of mitotic Nbs; (6) Prospero is transiently expressed in one of the two sibling GCs generated by the division of GMCs (Ceron, 2001).

In simple geometric terms, one may describe the OPC as a germ neuroepithelium forming a ring-like structure that covers the most lateral side of the lobe. Nbs occupy the external layer, close to the outside surface, and their progeny ganglion cells lay inside it, forming a thicker layer. This layered structure, which can be observed in frontal sections of optic lobes, allows an easy identification of the different cell types. If sectioning is similarly applied to BrdU-labeled optic lobes, one may observe that different time pulses give rise to different patterns of cell labeling in the OPC. Thus, short pulses result in preferential labeling of medium-size nuclei located just below Nbs that in turn are very often unlabeled. In contrast, longer pulses yield extensive labeling of large Nb nuclei and abundant small GC. Different pulse periods do not result in differential labeling of central brain (CB) Nbs and their progeny. Thus, short pulses yield pairs of labeled cells that consist of one Nb and a single daughter cell, while longer pulses produce labeling of one Nb together with a couple of daughter cells (Ceron, 2001).

The incorporation of BrdU in the progeny of Nbs during short pulses and the frequent observation of two labeled nuclei apparently undergoing cytokinesis very close to a Nb suggest the existence of GMCs that have a cell cycle shorter than their parent Nbs. Direct evidence for the existence of mitosis in those daughter cells was obtained by applying several immunochemical tools. Medium-size mitotic cells are detected just below the layer of OPC Nbs. Also, in the CB, where individual Nbs and their progeny can be observed, medium-size mitotic cells are detected immediately close to each Nb. In this case, all daughter cells are located at the same side of the Nb but no more than one is in mitosis. Interestingly, even in interphase Nbs, the centrosome is always located at the pole opposite that of the budding cells and the mitotic spindle of daughter cells is most often oriented at an oblique angle relative to that of the parent Nb. Altogether, labeling experiments with BrdU and mitotic markers demonstrate the presence of GMC-like cells in postembryonic proliferative anlagen (Ceron, 2001).

OPC Nbs stop producing more Nbs and begin to generate the final neuronal progeny around the third-instar larval period. This change in proliferative behavior could be explained by a change from an initial symmetric pattern of division to a late asymmetric one. Since asymmetric divisions of embryonic Nbs follow an apical/basal orientation, it would be also interesting to find out whether symmetric and asymmetric divisions of postembryonic Nbs have different orientations. This is indeed the case. The divisions of mitotic Nbs in the OPC of early third-instar larvae are preferentially oriented on an axis tangential to the surface, whereas those observed in late third-instar larvae show almost exclusively a radial orientation. Larval ventral ganglion Nbs, which divide asymmetrically, contain unequal centrosomes during mitosis. The larger centrosome is segregated into the resulting Nb and the smaller is inherited by the GMC. Radially oriented divisions of OPC Nbs have asymmetric centrosomes with the larger one close to the optic lobe surface, whereas tangentially oriented divisions have symmetric centrosomes. The metaphase plate of asymmetrically dividing Nbs is located close to the smaller (basal) centrosome. In contrast to the epithelial sheet-like organization of the OPC anlagen, CB Nbs are distributed in the most medial part of the optic lobe and each one shows a different direction of asymmetric division. Nevertheless, all the progeny of each Nb appear to be released by the same side and the interphase centrosome is maintained at the opposite side of the progeny (Ceron. 2001).

To determine whether the regulation of asymmetric divisions and the segregation of cell fate determinants of postembryonic Nbs follow a pattern similar to that described for embryos, the expression and localization of Insc, Mir, Numb, and Pros were examined in whole mounts of third-instar larval brain. Mir is widely expressed in all larval proliferative anlagen. During division, it shows a polarized distribution in the cell cortex of both CB and OPC Nbs, and it is segregated to the GMC during cytokinesis. Recently born GMCs show a very high expression of Mir both in the CB and in the OPC. Mir seems to be rapidly down-regulated in CB GMCs, whereas it seems to remain in OPC GMCs at high level for a rather long period of time, as judged by the relative higher proportion of labeled GMCs versus Nbs that can be detected in the OPC. Nevertheless, Mir seems to be completely down-regulated before GMCs begin mitosis (Ceron, 2001).

The tissue pattern of Pros expression in the larval brain is different from that of Mir. The expression of Pros protein in the CB and ventral (thoracic) anlagen is quite high, while in the OPC and inner proliferative center (IPC) it is rather low. Due to the higher level of expression, the localization of Pros can be studied in more detail in the CB. Pros protein is clearly observed only in the nucleus of daughter cells located away from the parent Nbs and, therefore, identified as GCs. Surprisingly, Pros protein is not consistently detected in dividing Nbs and GMCs either in the CB or in the OPC. This is especially clear by the lack of colocalization with Mir in the cortical crescent of dividing Nbs and newborn GMCs. The almost exclusive expression of Pros in GCs is also supported by the colocalization with Elav, a nuclear protein that is expressed in postmitotic cells and is absent in GMCs (Ceron, 2001).

It has been reported that Pros located at cortical sites of embryonic Nbs is highly phosphorylated compared to nuclear Pros. Nevertheless, this cannot be the reason for lack of detection of cortical Pros in Nbs of the larval optic lobe since the antiserum seems to recognize both phosphorylated and unphosphorylated forms of Pros (Ceron, 2001 and references therein).

The lack of Mir-Pros colocalization in postembryonic Nbs and GMCs opens the question of Mir's role in these cells. One possibility is that Mir might be involved in the localization of PROS mRNA, as has been shown for embryonic Nbs. To test this hypothesis, the expression of PROS mRNA was studied by in situ hybridization. PROS mRNA is detected in isolated cells of the optic lobe in both CB and OPC regions. In the CB, these cells correspond to single small daughter cells located closer to the Nb than those GCs that express Pros protein. In the OPC, it is rather obvious that Pros-expressing cells are located below the layer of GMCs. Also in the CB, PROS mRNA is detected neither in dividing GMCs nor in Mir-expressing cells. Therefore, it must be concluded that Pros is expressed at detectable levels only in GCs (Ceron, 2001).

In contrast to what it is known in the embryo and to previous data of the larval brain, these BrdU-labeling experiments clearly indicate that most postembryonic GMCs, especially those of the OPC, have a very transient life with cell cycle much shorter than that of parent Nbs. Another interesting difference is the large number of GMCs expressing high levels of Mir in the OPC. Taking into account the very short cell cycle of these intermediate cells, it is suggested that, in contrast to the rapid down-regulation observed in embryonic GMCs, Mir protein remains for a longer time in GMCs of the OPC. The rapid down-regulation of Mir in embryonic GMCs has been related to the requirement for a rapid release of the cell determinant Pros that has to translocate to the nucleus. The fact that Pros protein is not consistently detected in postembryonic GMCs makes it difficult to interpret the functional significance of this long lasting expression of Mir (Ceron, 2001).

Since Pros seems to be expressed neither in Nbs nor in GMCs, the expression of Numb, another asymmetric cell determinant of embryonic Nbs, was studied. Numb localizes in the cortical crescent of dividing Nbs of the OPC and CB; it is segregated to the membrane of GMCs where it seems to remain at low level, and it does not appear to be polarized during GMC division. Afterward, it seems to be down-regulated since it is hardly detected in GCs (Ceron, 2001).

Identification of Drosophila type II neuroblast lineages containing transit amplifying ganglion mother cells

Mammalian neural stem cells generate transit amplifying progenitors that expand the neuronal population, but these type of progenitors have not been studied in Drosophila. The Drosophila larval brain contains 100 neural stem cells (neuroblasts) per brain lobe, which are thought to bud off smaller ganglion mother cells (GMCs) that each produce two post-mitotic neurons. This study used molecular markers and clonal analysis to identify a novel neuroblast cell lineage containing transit amplifying GMCs (TA-GMCs). TA-GMCs differ from canonical GMCs in several ways: each TA-GMC has nuclear Deadpan, cytoplasmic Prospero, forms Prospero crescents at mitosis, and generates up to 10 neurons; canonical GMCs lack Deadpan, have nuclear Prospero, lack Prospero crescents at mitosis, and generate two neurons. It is concluded that there are at least two types of neuroblast lineages: a Type I lineage where GMCs generate two neurons, and a type II lineage where TA-GMCs have longer lineages. Type II lineages allow more neurons to be produced faster than Type I lineages, which may be advantageous in a rapidly developing organism like Drosophila (Boone, 2008).

During a clonal analysis of a larval neuroblast self-renewal mutant it was realized that wild type brains have two distinct types of neuroblast lineages. Mosaic analysis with repressible cell marker (MARCM) was used to generate GFP-marked single cell clones in the larval brain. Depending on the cell in which chromosomal recombination occurs, it is possible to label a single neuroblast and all its progeny, a single GMC and all its progeny, or a single neuron derived from a terminal mitosis. A low density of clones was induced randomly throughout the brain at either mid-first or mid-second larval instar and all clones were assayed 48 h after induction. Two distinct neuroblast lineages were found: a 'Type I' lineage that matches previously reported neuroblast lineages, and a novel 'Type II' lineage that is larger and more complex (Boone, 2008).

Type I neuroblast clones always contained one large neuroblast near the surface of the brain that had nuclear Dpn and cytoplasmic Pros. These clones always contained a column of smaller cells that lacked Dpn and had nuclear Pros, with the occasional presence of a single Dpn+ small cell contacting the neuroblast, which is likely to be a newborn GMC. The cells furthest from the neuroblast were Dpn Pros mature neurons that extend GFP1 axons into the central brain. Type I neuroblast lineages are the sole occupants of the dorsoanterior lateral (DAL) brain region, but can also be found in all other brain regions. To minimize regional variation in neuroblast lineages. Analysis of Type I neuroblasts was restricted to the DAL region (Boone, 2008).

Type I GMC clones were assayed only in the DAL region, where no Type II neuroblasts were observed. All clones lacking a large Dpn+ neuroblast were considered to be GMC clones, and these GMC clones generated at most just two cells. Thus, Type I lineages are identical to those reported for Drosophila embryonic neuroblasts, larval mushroom body neuroblasts, and grasshopper neuroblasts (Boone, 2008).

Type II neuroblast clones always contained one large Dpn+ neuroblast near the surface of the brain, but also contained a distinctive group of small Dpn+ cells that lack nuclear Pros. There are also usually 1-2 small cells in direct contact with the neuroblast that lack both Dpn and nuclear Pros. These two types of small cells are never observed in Type I clones and are a defining feature of Type II clones. Type II neuroblast clones are found in several brain regions, including a cluster within the DPM region. One Type II neuroblast appears to be the previously identified DPMpm1 neuroblast based on its distinctive axon projection that bifurcates over the medial lobe of the mushroom body before crossing the midline (Boone, 2008).

Type II GMC clones were identified by the lack of a large Dpn+ neuroblast. All brain regions that contained Type II neuroblast lineages produced GMC clones of greater than two cells; all brain regions that lacked Type II neuroblast lineages never generated >2 cell GMC clones. Type II GMC clones often contained Dpn+ Proscyto small cells that are unique to Type II neuroblast lineages, confirming that these clones are sublineages of a Type II neuroblast lineage. It is concluded that Type II neuroblasts generate GMCs that produce more than two neurons. Because Type II GMC clones could generate several fold more neurons than a Type I GMC, they were called 'transit amplifying GMCs' or TA-GMCs (Boone, 2008).

TA-GMC clones also contained small cells with nuclear Pros; it is suggested that these cells are equivalent to Type I GMCs based on their cell division profile, and because two cell clones were observed in regions of the brain that contained Type II neuroblast lineages. However, the possibility that some of these nuclear Pros cells are post-mitotic immature neurons cannot be ruled out (Boone, 2008).

If Type II lineages generate TA-GMCs that make an average of twice as many neurons as a Type I lineage, it would be expected that Type II lineages generate approximately twice as many cells over the same timespan compared with Type I lineages. Indeed, it was found that when Type I or Type II clones are grown for the same length of time (between clone induction and analysis), Type II clones generate approximately twice as many neurons. Type I clones in the DAL generate 40.4 +/ 3.1 cells, whereas Type II lineages in the DPM generate 71.2 +/- 6.3 cells . In all cases the final Type I and Type II neuroblast clones contained a single large Dpn+ neuroblast, ensuring that only single neuroblast clones were counted. It is concluded that Type II TA-GMCs generate more neurons than Type I GMCs, and that Type II lineages generate more neurons than Type I lineages (Boone, 2008).

This study characterized the cell division patterns within Type I and Type II lineages to help understand the relationship between different cell types in a lineage. It was first asked what cell type is directly produced by Type I and Type II neuroblasts? Type I neuroblasts in the DAL region always segregate Pros protein into the newborn GMC resulting in easily detectable levels of Pros in neuroblast progeny. Thus, Type I neuroblasts in the DAL generate nuclear Pros+ GMCs, as previously reported. In contrast, Type II neuroblasts of the DPM region often fail to segregate Pros protein, despite proper localization of other apical/ basal proteins, which would account for reduced Pros levels in newborn progeny. The variation in Pros localization among DPM neuroblasts could be due to the presence of some Type I neuroblasts in the region, or actual variation among Type II neuroblasts. It is concluded that Type II neuroblasts divide asymmetrically, but can fail to segregate Pros protein into their newborn progeny (Boone, 2008).

Next, the relationship between the Type II small cells that have high Dpn, low Pros (Dpn+ Proscyto) and those that contain high Pros, but no Dpn (Dpn- Prosnucl), was investiged. It was found that mitotic Dpn+ small cells always form Mira/Pros cortical crescents, with Pins protein localized to the opposite cortical domain, and the spindle aligned along this cortical polarity axis. This type of division is unique to Type II lineages, as all Type I GMCs always showed diffuse cytoplasmic Pros during mitosis. It is concluded that Type II Dpn+ small cells undergo molecularly asymmetric cell divisions to generate a Pros+ sibling and a Pros- sibling. It is proposed that the sibling with little or no Pros remains a Dpn+ TA-GMC, whereas the Pros+ sibling generates one or two post-mitotic neurons, similar to Pros+ GMCs in Type I lineages (Boone, 2008).

To characterize the cell cycle kinetics of Type I GMCs and Type II TA-GMCs, BrdU labeling experiments were performed. Larvae were exposed to a 4.5 h BrdU pulse and then immediately fixed and assayed for BrdU incorporation. As expected, both Type I and Type II neuroblasts always incorporated BrdU. Type I neuroblasts showed only a few closely-associated GMCs labeled, whereas Type II neuroblasts had a much larger number of labeled progeny. It is unlikely that the Type II neuroblasts generate all of these progeny during the 4.5 h labeling window, because the shortest neuroblast cell cycle time observed in any brain region was ~50 min, and thus it is concluded that Type II neuroblast progeny undergo more rounds of cell division that Type I GMCs (Boone, 2008).

To determine if the proliferative Type II neuroblast progeny are competent to differentiate into neurons, a BrdU pulse/chase experiment was performed. Larvae were fed BrdU for 4.5 h as described above, but then allowed to develop for 18 h without BrdU. Type I neuroblasts lacked BrdU incorporation, as expected due to label dilution during the chase interval, but BrdU was maintained in the Elav1 post-mitotic neurons born during the pulse window. Type II neuroblasts and most of their progeny also diluted out BrdU, confirming their status as proliferative cells, and some Elav1 post-mitotic neurons were born during the pulse interval and maintained BrdU labeling. It is concluded that Type II neuroblast progeny are proliferative but can still give rise to differentiated neurons (Boone, 2008).

There are currently no molecular markers that can be used to unambiguously identify Type II neuroblasts. The inability to form Pros crescents may be shared by all Type II neuroblasts, but even so, it would only be a useful marker for mitotic neuroblasts. In the DPM brain region (enriched for Type II lineages) it was found about 50% of the mitotic neuroblasts have little or no Pros crescent, and based on the distinctive lack of Pros in some Type II neuroblast progeny, it is concluded that these are Type II neuroblasts. (The 50% of the DPM neuroblasts that form Pros crescents may be Type I neuroblasts within the region, a special subset of Type II neuroblasts, or there may be stochastic variability in Pros crescent-forming ability among Type II neuroblasts.) In any case, these findings may explain why some labs report seeing Pros crescents whereas others report that neuroblasts do not form Pros crescents; both are correct because there are two types of larval neuroblast lineages (Boone, 2008).

It is unknown whether neuroblasts can switch back and forth between Type I and Type II modes of cell lineage. If the level of Pros in the neuroblast is the key factor distinguishing these modes of division, then experimentally raising Pros levels in Type II lineages may switch them to Type I lineages; conversely, reducing Pros levels in Type I lineages may switch them to Type II lineages. As more brain neuroblasts become uniquely identifiable it will be interesting to address this question. It will also be interesting to search for Type II neuroblast lineages in other insects or crustaceans where Type I neuroblast lineages have been documented (Boone, 2008).

What terminates the TA-GMC lineage? The TA-GMC may fall below a size threshold for continued proliferation. Alternatively, TA-GMCs may lose contact with a niche-derived signal that maintains their proliferation; Hedgehog, Fibroblast growth factor, and Activin are all required for larval brain neuroblast proliferation, but none have been tested for a role in TA-GMC proliferation. Lastly, there may be lineage-specific factors segregated into the TA-GMCs that limit their mitotic potential. TA-GMCs may die at the end of their lineage, as do some neuroblasts, or they may differentiate. It has been shown that loss of Pros and Brat together can generate a more severe neuroblast tumor phenotype than either alone. This suggests that the Type II lineages may be especially sensitive to further loss of differentiation promoting factors due to their low levels of endogenous Pros. Indeed, a dramatic neuroblast tumor phenotype has been observed in type II lineages in lethal giant discs mutants. This raises the question of how Type II lineages benefit the fly. They have the ability to generate more neurons in a faster period of time, due to the presence of TA-GMCs, and may be an evolutionary adaptation to the rapid life cycle of Drosophila. Slower developing insects may not require such rapid modes of neurogenesis (Boone, 2008).

Temporal transcription factors and their targets schedule the end of neural proliferation in Drosophila

The timing mechanisms responsible for terminating cell proliferation toward the end of development remain unclear. In the Drosophila CNS, individual progenitors called neuroblasts are known to express a series of transcription factors endowing daughter neurons with different temporal identities. This study shows that Castor and Seven-Up, members of this temporal series, regulate key events in many different neuroblast lineages during late neurogenesis. First, they schedule a switch in the cell size and identity of neurons involving the targets Chinmo and Broad Complex. Second, they regulate the time at which neuroblasts undergo Prospero-dependent cell-cycle exit or Reaper/Hid/Grim-dependent apoptosis. Both types of progenitor termination require the combined action of a late phase of the temporal series and indirect feedforward via Castor targets such as Grainyhead and Dichaete. These studies identify the timing mechanism ending CNS proliferation and reveal how aging progenitors transduce bursts of transcription factors into long-lasting changes in cell proliferation and cell identity (Maurange, 2008).

Initially investigated was whether distinct temporal subsets of neurons are generated throughout the larval CNS. Chinmo and Broad Complex (Br-C), two BTB-zinc finger proteins known to be expressed in the postembryonic CNS, are distributed in complementary patterns in the central brain, thoracic, and abdominal neuromeres at the larval/prepupal transition stage at 96 hr (timings are relative to larval hatching at 0 hr). Chinmo is expressed by early-born neurons located in a deep layer, whereas Br-C marks later-born neurons in a largely nonoverlapping and more superficial layer. The deep Chinmo+ layer comprises most/all neurons born in the embryo plus an early subset of those generated postembryonically. Thoracic postembryonic neuroblasts undergo the Chinmo --> Br-C switch at ~60 hr such that they have each generated an average of 15 Chinmo+ cells expressing little or no Br-C and 39 Chinmo- Br-C+ cells by 96 hr. The Chinmo+ and Br-C+ neuronal identities can be recognized as distinct cell populations on the basis of an ~2-fold difference in cell-body volume. This equates to an average cell-body diameter for Chinmo+ neurons of 4.5 μm, compared to only 3.6 μm for Br-C+ neurons. Plotting cell diameter versus deep-to-superficial position within postembryonic neuroblast clones reveals an abrupt decrease in neuronal size at the Chinmo --> Br-C transition. Together, these results provide evidence that most, if not all, postembryonic neuroblasts sequentially generate at least two different populations of neurons. First they generate large Chinmo+ neurons and then they switch to producing smaller Br-C+ neurons (Maurange, 2008).

To begin dissecting the neuronal switching mechanism, the functions of Chinmo and Br-C were investigated, but neither factor was found to be required for the transition in cell identity and cell size. Next it was asked whether a temporal transcription factor series related to the embryonic Hb --> Kr --> Pdm --> Cas sequence might be involved. Cas is known to be expressed in the larval CNS, and this study shows that many different thoracic neuroblasts progress through a transient Cas+ phase during the 30-50 hr time window. Thoracic neuroblasts transiently express another member of the embryonic temporal series, Svp, during a somewhat later time window, from ~40 to ~60 hr. These results indicate that postembryonic Cas and Svp bursts are observed in many, but probably not all, thoracic progenitors and that their timing varies from neuroblast to neuroblast (Maurange, 2008).

To determine Svp function, thoracic neuroblast clones were generated homozygous for svpe22, an amorphic allele. In ~53% of svpe22 neuroblast clones induced in the early larva (at 12-36 hr), the Br-C+ neuronal identity is completely absent, all neurons express Chinmo, and there is no sharp decrease in neuronal size. The proportion of lineages failing to generate Br-C+ neurons rises to ~70% when clones are induced in the embryo and falls to only ~7% with late-larval (65-75 hr) induction. This is consistent with a previous finding that Svp bursts are asynchronous from neuroblast to neuroblast. The expression and clonal analyses together demonstrate that a progenitor-specific burst of Svp is required in many lineages for the switch from large Chinmo+ to small Br-C+ neurons (Maurange, 2008).

Thoracic neuroblast lineages homozygous for a strong cas allele, cas24, show no obvious defects in the Chinmo --> Br-C transition when induced at 12-36 hr. However, since Cas is expressed in many postembryonic neuroblasts before their first larval division, it can only be removed by inducing clones during embryonic neurogenesis. Such cas24 clones generate supernumerary Chinmo+ neurons and completely lack Br-C+ neurons at 96 hr, although this switching phenotype is restricted to only ~16% of thoracic neuroblasts. Constitutively expressing Cas blocks the Chinmo --> Br-C switch in a similar manner, with a frequency dependent upon whether thoracic UAS-cas clones are induced during embryogenesis (~47%), at early-larval (~10%) or at late-larval (0%) stages. This indicates that the response to Cas misexpression decreases as neuroblasts age. Together, the expression and loss- and gain-of-function analyses demonstrate that Chinmo and Br-C are negative and positive targets, respectively, of Cas and Svp. They also strongly suggest that progression through transient Cas+ and Svp+ states permits many postembryonic neuroblasts to switch from generating large to small neurons (Maurange, 2008).

To investigate whether Cas and Svp regulate neural proliferation as well as neuronal fates, the effector mechanism ending neurogenesis in the central brain and thorax was identified. In these regions, most neuroblasts cease dividing in the pupa at ~120 hr. Correspondingly, neurogenesis in all regions of the wild-type CNS ceases before the adult fly ecloses such that no adult neuroblasts are detected. In contrast to the central abdomen, blocking cell death by removing Reaper, Hid, and/or Grim (RHG) activity in the central brain and thorax does not prevent or delay pupal neuroblast disappearance. However, time-lapse movies of individual thoracic neuroblasts at ~120 hr reveal an atypical mitosis that is much slower than at ~96 hr, producing two daughters of almost equal size. This is largely accounted for by a reduction in the average diameter of neuroblasts from 10.4 μm at 96 hr to 7 μm at 120 hr, as GMC size does not vary significantly during this time window. The end of this atypical progenitor mitosis temporally correlates with reduced numbers of Mira+ cells and disappearance of the M phase marker phosphorylated-Histone H3 (PH3), indicating that it marks the terminal division of the neuroblast (Maurange, 2008).

Next whether late changes in basal complex components might underlie loss of neuroblast self-renewal was addressed. At 120 hr, it was found that Mira becomes delocalized from the cortex to the cytoplasm and nucleus of many interphase neuroblasts. In metaphase neuroblasts, Mira fails to localize to the basal side of the cortex, although it does selectively partition into one daughter during telophase. This late basal restoration resembles the 'telophase rescue' associated with several apical complex mutations. Pros, the Mira-binding transcription factor and GMC-determinant, is not detectable in the neuroblast nucleus at 96 hr, but at 120 hr a burst of Pros was observe in the nucleus of many Mira+ cells of intermediate size indicative of neuroblasts in the interphase preceding the terminal mitosis. Clones lacking Pros activity contain multiple Mira+ neuroblast-like cells. It was found that they do not respect the ~120 hr proliferation endpoint and even retain numerous dividing Mira+ progenitors into adulthood. In addition, GAL80ts induction was used to induce transiently the expression of a YFP-Pros fusion protein well before the normal ~120 hr endpoint. Under these conditions, YFP-Pros can be observed in the nucleus of neuroblasts, most Mira+ progenitors disappear prematurely, and neural proliferation ceases much earlier than normal. Together, these results provide evidence that most neuroblasts terminate activity in the pupa via a nuclear burst of Pros that induces cell-cycle exit. These progenitors are referred to as type I neuroblasts to distinguish them from the much smaller population of type II neuroblasts that terminate via RHG-dependent apoptosis (Maurange, 2008).

Whether the postembryonic pulses of Cas and Svp in type I neuroblasts are implicated in scheduling their subsequent cell-cycle exit was tested. Remarkably, it was observed that many svpe22 clones induced at early-larval stages retain a single Mira+ neuroblast at 7 days into adulthood. The persistent adult neuroblasts in a proportion of these clones also express the M phase marker, PH3, indicating that they remain engaged in the cell cycle and, accordingly, they generate approximately twice the normal number of cells by 3 days into adulthood. Furthermore, superficial last-born neurons in these over proliferating svpe22 adult clones are Chinmo+ Br-C- indicating a blocked Chinmo --> Br-C transition. Similar phenotypes are obtained in some UAS-cas and cas24 clones. This analysis demonstrates that stalling the temporal series not only inhibits the late-larval switch to Br-C+ neuronal identity but also prevents the pupal cell-cycle exit of type I neuroblasts (Maurange, 2008).

To test the regulatory relationship between Pros and the temporal series, svpe22 clones were examined at pupal stages. It was found that mutant interphase neuroblasts fail to switch on nuclear Pros at 120 hr, although svpe22 GMCs express nuclear Pros as normal. This likely accounts for why adult clones lacking Svp retain only a single neuroblast, whereas those lacking Pros contain multiple neuroblast-like progenitors. Importantly, these results demonstrate that nuclear Pros acts downstream of the temporal series in type I neuroblasts. Together, the genetic and expression analyses of Svp and Pros show that the temporal series triggers a burst of nuclear Pros in type I neuroblasts, thus inducing their cell-cycle exit (Maurange, 2008).

To determine how the temporal series is linked to the cessation of progenitor divisions, two transcription factors expressed in neuroblasts in a temporally restricted manner were examined. Dichaete (D), a member of the SoxB family, is dynamically expressed in the early embryo and is required for neuroblast formation. Consistent with the previous studies, it was observed that most or all embryonic neuroblasts progress through a transient D+ phase, but those in the lateral column of the ventral nerve cord initiate expression after their medial and intermediate counterparts. Dichaete subsequently becomes repressed in ~85% of neuroblasts during late-embryonic and postembryonic stages. Grainyhead (Grh) is first activated in neuroblasts in the late embryo and is required for regulating their mitotic activity during larval stages. Blocking early temporal series progression in the embryo, either by persistent Hb or loss of Cas activity, prevents most neuroblasts from downregulating D and also from activating Grh at late-embryonic and postembryonic stages. As forcing premature Cas expression leads to precocious D repression and Grh activation, both factors are likely to be regulated by Cas rather than by a later member of the temporal series. These results demonstrate that transient embryonic Cas activity permanently switches the expression of Grh on and D off. They also identify Grh and D as positive and negative targets, respectively, of the temporal series in many neuroblasts (Maurange, 2008).

Loss of Grh activity in thoracic neuroblasts (here defined as type I neuroblasts) leads to their reduced cell-cycle speed and disappearance during larval stages. At 96 hr, it was observed that 65% of grh370 type I neuroblasts are smaller than normal (~6.4 μm in diameter), delocalize Mira from the cortex to the cytoplasm and nucleus, and strongly express Pros in the nucleus. These events, reminiscent of the 120 hr terminal cell cycle of wild-type progenitors, show that Grh is required to prevent the premature cell-cycle exit of type I neuroblasts. Given this finding, and that Cas is required to activate Grh, the question arises as to how some neuroblasts lacking embryonic Cas activity are able to continue dividing into adulthood. However, cas24 neuroblasts persisting in adults all retain Grh, suggesting that they may derive from clones that lacked only the last of the two Cas pulses observed in some embryonic neuroblasts, perhaps retaining enough Cas activity to support Grh activation but not later progression of the temporal series (Maurange, 2008).

To determine if late temporal series inputs, after embryonic Cas, are also required to maintain the long-lasting postembryonic expression of Grh, svpe22 clones were induced in early larvae. Although type I neuroblasts in these mutant clones have a stalled temporal series, they retain postembryonic Grh expression through to adult stages. Thus, two sequential inputs from the temporal series are required for type I neuroblasts to undergo timely Pros-dependent cell-cycle exit. First, embryonic Cas activity switches on sustained Grh expression, inhibiting premature nuclear Pros and permitting continued mitotic activity. Second, a late postembryonic input, requiring Svp, counteracts this activity of Grh by triggering a pupal burst of nuclear Pros (Maurange, 2008).

Despite undergoing premature cell-cycle exit, it was noticed that grh mutant neuroblasts can still generate both Chinmo+ and Br-C+ neurons. Thus, Grh is not required for the neuronal Chinmo --> Br-C switch. Conversely, neither Chinmo nor Br-C appears to be required postembryonically for neuroblast cell-cycle exit. In summary, the properties of both neuroblasts and neurons are regulated by downstream targets of the temporal series (Maurange, 2008).

Next whether the temporal series and its targets also function in type II neuroblasts, which terminate via RHG-dependent apoptosis rather than Pros-dependent cell-cycle exit, was tested. Focus was placed on one identified type II neuroblast in the central abdomen, called dl, which undergoes apoptosis at 70-75 hr and produces only a small postembryonic lineage of ~10 neurons. It was observed that the dl neuroblast expresses bursts of Cas (~45 to ~60 hr) and Svp (~62 to ~65 hr) and sequentially generates Chinmo+ (~7 deep) and Br-C+ (~3 superficial) neurons. Loss of Cas or Svp activity, or prevention of temporal series progression in several other ways all lead to a blocked Chinmo --> Br-C transition, a failure to die at 70-75 hr, and the subsequent generation of many supernumerary progeny. These results show that the temporal series performs similar functions in type I and type II neuroblast lineages, regulating both the Chinmo --> Br-C neuronal switch and the cessation of progenitor activity. Next, the mechanism linking the temporal series to RHG-dependent death of type II neuroblasts was tested. As for type I neuroblasts, dl progenitors in cas24 clones, induced in the embryo, fail to activate Grh and repress D. Moreover, if grh activity is reduced, or if D is continuously misexpressed, dl progenitors persist long after 75 hr. Therefore, both of these early Cas-dependent events are essential for subsequent type-II neuroblast apoptosis. However, in contrast to members of the temporal series themselves, persistent misexpression of their target D does not block the Chinmo --> Br-C switch in the dl lineage. Thus, the D- Grh+ state of type I and type II neuroblasts, installed via an embryonic Cas pulse, appears to be necessary for progenitor termination but not for Chinmo --> Br-C switching. Nevertheless, dl neuroblasts stalled at a postembryonic stage in svpe22 and UAS-cas clones retain the D- Grh+ code yet still fail to undergo apoptosis. Therefore, as with type I cell-cycle exit, timely type II apoptosis requires both embryonic Cas-dependent and postembryonic Svp-dependent inputs from the temporal series (Maurange, 2008).

Finally, the regulatory relationship between the temporal series and AbdA, a Hox protein transiently expressed in postembryonic type II (but not type I) neuroblasts and required for their apoptosis was dissected. dl neuroblasts lacking postembryonic Svp activity or persistently expressing Cas still retain AbdA expression yet do not die. This suggests that AbdA is unable to kill neuroblasts unless they progress, in a Svp-dependent manner, to a late Cas- temporal state. To test this prediction directly, use was made of the previous finding that ectopic AbdA is sufficient to induce neuroblast apoptosis, albeit only within a late time window. Constitutive AbdA-induced apoptosis is efficiently suppressed by persistent Cas, not only in type II but also in type I neuroblasts. This result demonstrates that, in order to terminate, type II neuroblasts must progress to a late Cas- state, thus acquiring a D- Grh+ Cas- AbdA+ code. It also suggests that AbdA is sufficient to intercept progression of the temporal series in type I neuroblasts, inducing an early type II-like termination (Maurange, 2008).

This study has found that the Drosophila CNS contains two distinct types of self-renewing progenitors: type I neuroblasts terminate divisions by cell-cycle withdrawal and type II neuroblasts via apoptosis. Despite these different exit strategies, both progenitor types use a similar molecular timer, the temporal series, to shut down proliferation and thus prevent CNS overgrowth. These findings demonstrate that the temporal series does considerably more than just modifying neurons; it also has multiple inputs into neural proliferation. The identification and analysis of several pan-lineage targets of the temporal series also begins to shed light on the mechanism by which developmental age modifies the properties of neuroblasts and neurons. Two targets, Chinmo and Br-C, are part of a downstream pathway temporally regulating the size and identity of neurons. Two other temporal series targets, Grh and D, function in neuroblasts to regulate Prospero/RHG activity, thereby setting the time at which proliferation ends. The temporal series regulates both cell proliferation and cell identity; a feedforward mechanism is proposed for generating combinatorial transcription factor codes during progenitor aging (Maurange, 2008).

This study has found that the temporal series regulates a widespread postembryonic switch in neuronal identity. Most, if not all, type I and type II neuroblasts first generate a deep layer of large Chinmo+ neurons and then switch to producing a superficial layer of small Br-C+ neurons. Two lines of evidence argue that this postembryonic neuronal switch is likely to be regulated by a continuation of the same temporal series controlling early/late neuronal identities in the embryo. First, the postembryonic Chinmo --> Br-C neuronal switch is promoted by the transient redeployment of two known components of the embryonic temporal series, Cas and Svp. Second, this switch remains inhibitable by misexpression of the other embryonic temporal factors such as Hb. Since both Cas and Svp are expressed somewhat earlier than the neuronal-size transition, it is likely that they promote bursts of later, as yet unknown, members of the temporal series that more directly regulate Chinmo and Br-C. Although neuronal functions for both BTB zinc-finger targets have yet to be characterized, a progressive early-to-late decrease in postmitotic Chinmo levels is known to regulate the temporal identities of mushroom-body neurons. The current results now suggest that this postmitotic gradient mechanism may be linked to, rather than independent from, the temporal series (Maurange, 2008).

Type I neuroblasts in clones lacking postembryonic Cas/Svp activity or retaining an early temporal factor, fail to express nuclear Pros during pupal stages and thus continue dividing long into adulthood. These overproliferating adult clones each contain only a single neuroblast, sharply contrasting with adult clones lacking Brat or Pros, in which there are multiple neuroblast-like progenitors. Hence, manipulations of the temporal series and its progenitor targets offer the prospect of immortalizing neural precursors in a controlled manner, without disrupting their self-renewing asymmetric divisions (Maurange, 2008).

This study demonstrates that type I and type II neuroblasts must progress through at least two critical phases of the temporal series in order to acquire the D- Grh+ Cas- combinatorial transcription factor code that precedes Pros/RHG activation. The early phase corresponds to embryonic Cas activity switching neuroblasts from D+ Grh- to D- Grh+ status. The equally essential, but less well-defined, late postembryonic phase of the temporal series requires transition to a Cas- state and a late Svp burst. For type I neuroblasts, Grh and a late Cas- temporal identity are both required for timely expression of nuclear Pros and subsequent cell-cycle withdrawal. For type II neuroblasts, these two inputs are also necessary for RHG-dependent apoptosis, with the additional requirement that D must remain repressed. Although the temporal series and its targets are similarly expressed in type I and type II neuroblasts, only the latter progenitors undergo a larval burst of AbdA. This AbdA expression is likely to be the final event required to convert the D- Grh+ Cas- state, installed by the temporal series, into the D- Grh+ Cas- AbdA+ combinatorial code for RHG-dependent apoptosis. This code prevents type II neuroblasts in the abdomen from reaching the end of the temporal series and accounts for why they generate fewer progeny and terminate earlier than their type I counterparts in the central brain and thorax (Maurange, 2008).

The data in this study support an indirect feedforward model for neuroblast aging. Key to this model is the finding that, although members of the temporal series are only expressed very transiently, some of their targets can be activated or repressed in a sustained manner, as observed for Chinmo/Br-C in neurons and also for Grh/D in neuroblasts. In principle, this indirect feedforward allows aging progenitors to acquire step-wise the combinatorial transcription factor codes modulating cell-cycle speed, growth-factor dependence, competence states, and neural potential. Like Drosophila neuroblasts, isolated mammalian cortical progenitors can sequentially generate neuronal fates in the correct in vivo order. These studies suggest that it will be important to investigate whether the transcription factors controlling this process also regulate cortical proliferation and whether their targets include BTB-zinc finger, Grh, SoxB, Prox, or proapoptotic proteins. Some insect/mammalian parallels seem likely, since it is known that Sox2 downregulation and Prox1 upregulation can both promote the cell-cycle exit of certain types of vertebrate neural progenitors (Dyer, 2003, Graham, 2003). Thus, although insect and mammalian neural progenitors do not appear to use the same sequence of temporal transcription factors, at least some of the more downstream components identified in this study might be functionally conserved (Maurange, 2008).

Progenitor properties of symmetrically dividing Drosophila neuroblasts during embryonic and larval development

Asymmetric cell division generates two daughter cells of differential gene expression and/or cell shape. Drosophila neuroblasts undergo typical asymmetric divisions with regard to both features; this is achieved by asymmetric segregation of cell fate determinants (such as Prospero) and also by asymmetric spindle formation. The loss of genes involved in these individual asymmetric processes has revealed the roles of each asymmetric feature in neurogenesis, yet little is known about the fate of the neuroblast progeny when asymmetric processes are blocked and the cells divide symmetrically. Such neuroblasts were genetically created, and it was found that in embryos they were initially mitotic and then gradually differentiated into neurons, frequently forming a clone of cells homogeneous in temporal identity. By contrast, larval neuroblasts with the same genotype continued to proliferate without differentiation. These results indicate that asymmetric divisions govern lineage length and progeny fate, consequently generating neural diversity, while the progeny fate of symmetrically dividing neuroblasts depends on developmental stages, presumably reflecting differential activities of Prospero in the nucleus (Kitajima, 2010).

This study investigated how the asymmetric mode of neuroblast division contributes to the specification and diversification of neuronal cell fate by generating neuroblasts that divide symmetrically. Combinations of dlg and Gβ13F mutants and of baz and Gβ13F mutants successfully generated neuroblasts that divide symmetrically with respect to both partition of determinants and daughter cell size during embryonic stages, allowing all progeny to differentiate into neurons that are often clonally homogeneous in temporal identity. At larval stages, dlg-Gβ13F neuroblasts generated overgrowing cell populations. Based on these results, the roles of asymmetric features of neuroblast division in the choice of self-renewal vs. differentiation and in cellular diversification are discussed (Kitajima, 2010).

Based on the observations, at embryonic stages, dlg-Gβ13F neuroblast divisions occur essentially without asymmetry in either daughter cell size or in the partition of the determinants from the first division. It is possible, however, that two daughter cells occasionally inherit different amounts of the determinants, leading to the generation of cell clusters expressing differential temporal identity genes in a single neuroblast progeny such as NB7-3. Such fluctuations in the partition of the determinants may occur stochastically because the apical/basal components are not tightly associated with cortex in dlg-Gβ13F neuroblasts (Kitajima, 2010).

All progenies of dlg-Gβ13F neuroblasts eventually differentiate in the embryonic stages. Which feature is then critical for the differentiation of all progeny; the asymmetric partition of determinants or of cell volume? On the one hand, the basal determinants are known to function in daughter cells' commitment to differentiation. On the other hand, all available results suggest that reduction in neuroblast cell size contributes to attenuation of cell cycle progression but not to the induction of differentiation. In the wild type, neuroblasts gradually reduce their size by budding off GMCs, and eventually enter the dormant state (Miranda+, Pros−, Elav−). In the Gβ13F single mutant, neuroblasts more rapidly lose volume by generating equal-sized daughters with the basal determinants normally segregating to one daughter, and remain in the same Miranda+, Pros−, Elav− state with the characteristic cell morphology of quiescent neuroblasts. This suggests that in the Gβ13F single mutant, neuroblasts also eventually enter the dormant state after the generation of a fewer number of GMCs. Thus, cell size reduction alone is not likely to cause neuronal differentiation of progenitors, but instead appears to cause them to remain in the undifferentiated state unless the basal determinants are present. This was confirmed in Gβ13F mutant neuroblasts at the larval stage (Kitajima, 2010).

What amount of the basal determinants is necessary to induce GMC fate? In Gβ13F mutants where neuroblast divisions give rise to daughters of equal size, a large daughter at the first division inherits most of the basal determinants and becomes differentiated into a GMC, indicating that a full amount of basal determinants can cause a daughter cell half the size of a newly born neuroblast to commit to the GMC fate. By contrast, neuroblasts undergoing symmetric divisions (dlg-Gβ13F mutants) appear to subsequently undergo at least two cell cycles and do not immediately commit to a GMC-like fate. This difference between embryonic Gβ13F mutant and dlg-Gβ13F mutant first daughters may mean that a half amount of the basal determinants is not sufficient to commit a daughter cell to the GMC fate. Alternatively, it has been argued that neuroblasts may express self-renewal factors that promote self-renewal and thereby proliferation and that asymmetrically segregate into the neuroblast. When dlg-Gβ13F neuroblasts undergo their first division symmetrically, those postulated factors and the basal determinants will be partitioned into both daughters and will counteract each other. This may cause a delayed commitment to the GMC fate, compared with the first GMC of Gβ13F mutant neuroblasts, which do not receive the self-renewal factors (Kitajima, 2010).

In the Drosophila CNS, the expression of the temporal identity genes changes sequentially in mother neuroblasts but is persistent in the sibling GMC progeny. Hence, the expression of such genes should also depend on the asymmetric mode of division. A significant difference was found in the expression of the temporal identity genes between normal neuroblast lineages and symmetrically dividing dlg-Gβ13F neuroblasts. In the latter, the neuroblast progeny frequently forms a clone of cells homogeneous for the expression of temporal identity genes, providing evidence for the importance of asymmetric division for the generation of neuronal diversity (Kitajima, 2010).

It has been shown that the first transition of temporal identity genes in embryonic neurogenesis, from Hb to Kr, requires cytokinesis, whereas the transition from Kr to Cas occurs without cell cycle progression. Symmetrically dividing neuroblasts pass through the initial transition from Hb to Kr in all lineages examined in this study (NB1-1, NB4-2, NB3-3 and NB7-3). A large proportion of neuroblast lineages appears to continue expressing the temporal genes in succession, but terminates earlier than normal, as revealed by their lack of Grh expression. Two observations suggest that the transition of temporal identity genes occurs sequentially (in the order of Hb to Kr to Pdm to Cas) in the majority of dlg-Gβ13F neuroblasts undergoing clonal expansion during early stages of neurogenesis; first, the size of Cas clusters is mainly 4 or 8 cells when Cas expression appears at 6–8 h AEL, suggesting that Cas expression starts in the clones that have already divided two or three times (some neuroblasts like NB3-3 start with Kr). Second, the cluster size of the clones become larger in the order of Hb, Kr, and Cas clusters at 8–10 h AEL (Kitajima, 2010).

The terminal temporal identity and the size of a particular neuroblast lineage are, however, not constant in dlg-Gβ13F mutants. Furthermore, a neuroblast progeny occasionally splits into two clusters with different temporal identities, as observed in the lineages of NB7-3. These observations suggest that stochastic processes are involved in the expression of temporal identity genes and cell cycle progression in the dlg-Gβ13F mutant neuroblast lineages (Kitajima, 2010).

Analysis of the relationship between clone size and clone homogeneity of NB7-3 reveals two characteristic features regarding dlg-Gβ13F neuroblast progenies. First, larger clones tend to be heterogeneous, containing both Kr-positive and Kr-negative cells (presumably Pdm-positive in their next identity), when compared to small-sized clones. Second, in heterogeneous clones, neurons with the same temporal identity form a cluster (Kr-positive and Kr-negative) and do not intermingle with each other, suggesting that cells with a different identity are also clonal instead being formed randomly during the expansion into a large heterogeneous clone. These observations regarding a single neuroblast lineage raise the possibility that a slight heterogeneity or difference created between sibling cells in early divisions become more pronounced in temporal identity in later stages as cells go through cell cycles. This and the remaining presence of a few Hb/Kr-double positive clones at late stages indicate that, in dlg-Gβ13F neuroblasts, cell cycle progression is not always linked to temporal identity progression as expected from looking at corresponding wild type lineages, although the progression of temporal identity is seen in this mutant (Kitajima, 2010).

Termination of temporal identity progression may depend on the amount of the basal determinants, including Prospero, given that the transition of temporal identity genes do not occur in wild type GMCs. Indeed, in dlg-Gβ13F neuroblasts, as cells divide, the size of the cell size is rapidly reduced to approach a GMC-like state. It is thus speculated that the progeny of symmetrically dividing neuroblasts eventually assume a GMC-like state, thereby terminating temporal identity gene progression prematurely (Kitajima, 2010).

A remarkable finding in this study is the opposite nature of the progeny of dlg-Gβ13F mutant neuroblasts in embryos and in larvae. When created at larval stages, dlg-Gβ13F mutant neuroblasts generate continuously proliferating progeny after reducing their cell size, in contrast to the embryonic situation. This difference would appear to reflect differences in the proliferation control of neuroblasts in the embryonic and larval stages. The function of the pros gene, which negatively regulates genes promoting cell cycle progression, appears to be pivotal because Pros functions as a tumor suppressor in larval brains but not in embryos. This difference in the effect of the loss of Pros has been attributed to the redundancy of Pros with Brat in embryos, while they are both necessary for normal larval lineages. pros mutant larval clones are thought to form tumors by the transformation of GMCs into proliferative cells, although proliferative dlg-Gβ13F mutant cells are likely derived from the transformation of neuroblasts into proliferative cells that undergo symmetric divisions (Kitajima, 2010).

Given that embryonic dlg-Gβ13F mutant neuroblasts appear to become GMC-like cells that inherit sufficient amounts of the basal determinants to differentiate, a simple explanation for the continuous proliferation of larval dlg-Gβ13F mutant cells is that the effective dosage of Pros (or other basal determinants) in those cells is insufficient to induce differentiation, unlike in their embryonic counterparts. Indeed, it was shown that elevation of Pros expression can induce proliferating cells in the dlg-Gβ13F mutant clones to exit the cell cycle and differentiate. It is of interest that, in interphase dlg-Gβ13F mutant cells of both embryonic and larval stages, Miranda is mainly cytoplasmic and Pros is largely nuclear, while during mitosis these proteins appear to form a cortical complex. There may be a larval mechanism by which neuroblasts reduce the nuclear entry of Pros in both wild-type and dlg-Gβ13F mutant cells. The ability of neuroblasts to prevent the nuclear import of Pros when it is overexpressed under the heatshock promoter was tested, and it was found that larval neuroblasts do not accumulate Pros protein in the nucleus at all under conditions in which embryonic neuroblasts show Pros nuclear accumulation. These results suggest that, compared with embryonic neuroblasts, larval neuroblasts have a strong ability to prevent nuclear accumulation of Pros (Kitajima, 2010).

A recent study has shown that cell cycle exit at the end of larval thoracic neurogenesis is programmed to reduce cell volume by symmetric divisions and nuclear localization of Pros; this is regarded as the mechanism terminating neuroblast division and allowing differentiation. As shown in larval Gβ13F mutant neuroblasts, the reduction of cell volume only limits the proliferative state or rate by idling or slowing the cell cycle progression, but does not induce differentiation. Furthermore, symmetric neuroblast divisions in the dlg-Gβ13F mutant resulted in reduction of cell volume and nuclear accumulation of Pros (although at a low level), but caused continuous proliferation of daughter cells. It may be that unlike the dlg-Gβ13F mutant, the level of nuclear Pros becomes high enough to terminate the cell cycle when wild-type neuroblasts stop division in the larval thorax. Alternatively, the progression of temporal identity in neuroblasts may induce additional mechanisms that cause neuroblasts to exit from the cell cycle into the differentiated state, as in the case for embryonic neuroblasts (Kitajima, 2010).

Drosophila type II neuroblast lineages keep Prospero levels low to generate large clones that contribute to the adult brain central complex

Tissue homeostasis depends on the ability of stem cells to properly regulate self-renewal versus differentiation. Drosophila neural stem cells (neuroblasts) are a model system to study self-renewal and differentiation. Recent work has identified two types of larval neuroblasts that have different self-renewal/differentiation properties. Type I neuroblasts bud off a series of small basal daughter cells (ganglion mother cells) that each generate two neurons. Type II neuroblasts bud off small basal daughter cells called intermediate progenitors (INPs), with each INP generating 6 to 12 neurons. Type I neuroblasts and INPs have nuclear Asense and cytoplasmic Prospero, whereas type II neuroblasts lack both these transcription factors. This study tested whether Prospero distinguishes type I/II neuroblast identity or proliferation profile, using several newly characterized Gal4 lines. Prospero was misexpressed using the 19H09-Gal4 line (expressed in type II neuroblasts but no adjacent type I neuroblasts) or 9D11-Gal4 line (expressed in INPs but not type II neuroblasts). It was found that differential prospero expression does not distinguish type I and type II neuroblast identities, but Prospero regulates proliferation in both type I and type II neuroblast lineages. In addition, 9D11 lineage tracing was used to show that type II lineages generate both small-field and large-field neurons within the adult central complex, a brain region required for locomotion, flight, and visual pattern memory (Bayraktar, 2010).

The recent identification of the type II lineages containing transit amplifying intermediate progenitors provides an important new model for investigating progenitor self-renewal and differentiation. However, little is known about their development, cell biology, gene expression, and functional importance in the Drosophila central nervous system. This is primarily due to a lack of genetic tools and markers that are specifically expressed in type II NBs and/or INPs. This study characterized the 19H09-Gal4 line expressed in type II NBs, and the 9D11-Gal4 line expressed in INPs but not their parental type II NBs. Using 19H09 it was shown that Ase is upregulated before Dpn during INP maturation. Using both lines, Prospero misexpression was shown to regulate proliferation but not identity within type II lineages. And using 9D11 the majority of type II-derived neurons was permanently labelled to show they are major contributors to the adult central complex brain region (Bayraktar, 2010).

The 19H09-Gal4 and 9D11-Gal4 lines can also be used to monitor the development of type II NBs and INPs in different mutant backgrounds to help clarify the origin of a mutant phenotype. For example, early studies on tumor suppressor genes showed increases in global brain NB numbers; for some of these mutants (for example, brat, numb) this study now shows that the phenotype arises specifically within the type II lineage. The 19H09-Gal4 and 9D11-Gal4 lines can also be used to drive UAS-RNAi, UAS-GFP constructs to test the role of any gene within these lineages. In addition, because these lines are made from defined enhancer fragments driving Gal4 placed into a specific attP site in the genome, it is easy to generate different transgenes with precisely the same expression pattern. Some future uses would be: using 19H09-FLPase to generate mutant clones or MARCM genetic screens in type II lineages; using 9D11 to drive expression of uracil phosphoribosyltranferase to isolate RNA from INP sublineages; or using 9D11-grim to ablate specifically type II neurons to determine their role in larval or adult behavior (Bayraktar, 2010).

The 19H09 and 9D11 lines were used to show that misexpression of Prospero can suppress proliferation within type II NBs and INPs without altering NB identity. As 19H09 is expressed only during the late larval stages, Prospero misexpression with 19H09 clearly distinguishes the effects of Prospero on NB proliferation from its effects on NB fate specification, which occurs in the embryonic stages. Misexpression with both 19H09 and 9D11 lead to a reduction in the number of INPs and neurons made by each type II NB. This reduction is unlikely to be due to an effect on the parental type II NBs, such as slowed down cell cycle or compromised NB survival, for the following reasons: first, low levels of ectopic Prospero are cytoplasmic in type II NBs, where Prospero has no known function; second, ectopic Prospero does not transform type II lineages to a type I identity based on the failure to upregulate ase expression; and third, misexpression of Prospero with both 9D11 and 19H09 give similar phenotypes, yet 9D11 is not expressed in type II NBs. It is suggested that the reduction of clone size is due to an effect in the INP cell type. Possible mechanisms include INP apoptosis, INP cell cycle lengthening, premature cell cycle exit, or transforming INPs into central brain type II GMCs, which generate lineages with bifurcated axon fascicles. While it was not possible to distinguish between these possibilities, the mechanism of a transformation of INP to central brain type I GMC identity can now be tentatively excluded because the neurons still retained their ability to form bifurcated axon fascicles, which are not a feature of central brain type I GMCs (Bayraktar, 2010).

Type II NBs lack both Ase and Prospero, whereas type I NBs contain both proteins. Yet only misexpression of Ase can transform type II into type I NBs, suggesting that Ase is sufficient to upregulate prospero expression in NBs. However, loss of Ase does not transform type I NBs into type II NBs, so there must be additional factors promoting the expression of Prospero in type I NBs. The analysis of gene expression differences between type I and II NBs would be one way of uncovering genes that control the difference between them (Bayraktar, 2010).

Lineage-tracing of INP-derived neurons shows that type II lineages make major contributions to all aspects of the central complex of the adult brain, as well as the bulbs (BUs; also known as lateral triangles) and lateral accessory lobes (LAL) accessory structures, including both small-field and large-field neurons. Central complex neurons derived from type II lineages likely include several small-field types, such as ventral fiber system (VFS), pontine, fb-eb, fb-no, and pb-eb-no neurons, and, to a lesser extent, large-field types, such as F neurons, including Fm, Fl and ExFl subtypes and some extrinsic R neurons. A recent study found that type II NB clones in the pupal brain projected to the PB (the largest structure in the central complex, divided into several vertical staves and horizontal stratifications), FB (the fan-shaped body) and NO (paired noduli) regions, with some projections forming restricted arbors at the PB (protocerebral bridge) and innervating domains of the FB and NO, while others made widespread arborizations outside the central complex. The data showing labeling of the majority of type II neuronal progeny are consistent with those of a previous study: while this study did not have the resolution to link cell bodies with axon projections, it is possible to provide a more comprehensive view showing that type II lineages contribute to all central complex neuropils and accessory areas in the adult brain. Future studies that selectively ablate different spatial or temporal cohorts of type II neurons will be necessary to determine if all type II-derived neurons share a common function (Bayraktar, 2010).

Although a large subset of central complex neurons derive from type II lineages, there are clearly some central complex neurons that originate from type I NBs or embryonic type II lineages. For example, no projections were seen that match those of the well-characterized large-field R neurons (R1 to R4). It is not clear which small-field types are not derived from type II lineages as they are difficult to distinguish. However, it is clear that the type II lineages do not make up the entire central complex so there must be contributions from type I lineages as well (Bayraktar, 2010).

Outside the central complex, labeling was observed of the region-specific staining of both the mushroom body and ALs; staining in the ALs was restricted to a subset of glomeruli. These could be novel connections from the central complex to the mushroom body and ALs formed by large-field or poorly understood extrinsic small-field neurons, or the projections of non-central complex neurons labeled by 9D11. Previous studies have revealed no direct connection between central complex and mushroom bodies or between LALs and ALs, and very few connections from LALs to mushroom bodies. The type II projection patterns from larval and pupal brains suggest that the lineages are not dedicated to a single neuropile center, which is consistent with type II lineages giving rise to non-central complex neurons as well. Labeling of large regions in the protocerebrum was also observed outside the central complex. However, it was not possible to distinguish whether they were connected to the central complex or its accessory areas. Another caveat to this analysis is that 9D11 is also expressed in the larval optic lobes, and indeed labeling was observed in the adult optic lobes. It was not possible to distinguish the projections from these cells from those of the central brain cell bodies due to dense staining. Analysis of 1,200 Golgi-impregnated brains revealed direct connections between optic lobes and the BU neuropil, but not to the other central complex neuropils that were found to be labeled. This suggests that most if not all central complex labeling is due to type II-derived neurons (Bayraktar, 2010).

In addition to using 9D11 to lineage trace the contribution of larval-derived type II neurons to the adult brain, maintained expression of 9D11 was also detected in a small subset of adult neurons, which are likely to be P3 or P4 small-field pontine neurons, which are also detected by the Gal4 line NP2320. Thus, the 9D11 line, and others with similarly specific adult expression patterns, should be useful for future studies using TU-tagging to transcriptionally profile neuronal subsets, GRASP to identify pre/post-synaptic partner, or for expression of optogenetic modulators of neuronal activity to determine the role of specific neurons in behavior (Bayraktar, 2010).

This characterization of type II lineages suggests that as a group the type II NBs produce a wide variety of neuronal subtypes. This neural diversity can be achieved spatially if each type II NB generates just one or two types of neurons; this model is supported by clonal data showing that each type II NB produces neurons with distinct axon projection patterns. In addition, temporal identity could generate further neuronal diversity as seen in type I NB lineages. This model is supported by clonal analysis of a small central complex sublineage in the adult brain, which has revealed temporally distinct neuronal fates. Finally, hemilineages could provide a final doubling of neuronal diversity, in which each sibling neuron derived from a single GMC takes either an 'A' or a 'B' cell fate. The fact that bifurcating axon projections are seen even in the highly sparse type II lineages following Prospero overexpression is consistent with GMCs producing A/B neurons that have different fasciculation patterns. In the future, it will be important to determine the birth-order and identities of neurons in each type II lineage and the mechanisms that regulate spatial and temporal neural fate specification in these lineages (Bayraktar, 2010).

Functional genomics identifies neural stem cell sub-type expression profiles and genes regulating neuroblast homeostasis

The Drosophila larval central brain contains about 10,000 differentiated neurons and 200 scattered neural progenitors (neuroblasts), which can be further subdivided into ~95 type I neuroblasts and eight type II neuroblasts per brain lobe. Only type II neuroblasts generate self-renewing intermediate neural progenitors (INPs), and consequently each contributes more neurons to the brain, including much of the central complex. Six different mutant genotypes were characterized that lead to expansion of neuroblast numbers; some preferentially expand type II or type I neuroblasts. Transcriptional profiling of larval brains from these mutant genotypes versus wild-type allowed identification of small clusters of transcripts enriched in type II or type I neuroblast,s, and these clusters were validated by gene expression analysis. Unexpectedly, only a few genes were found to be differentially expressed between type I/II neuroblasts, suggesting that these genes play a large role in establishing the different cell types. A large group of genes predicted to be expressed in all neuroblasts but not in neurons were identified. A neuroblast-specific, RNAi-based functional screen was performed and 84 genes were identified that are required to maintain proper neuroblast numbers; all have conserved mammalian orthologs. These genes are excellent candidates for regulating neural progenitor self-renewal in Drosophila and mammals (Carney, 2012).

To identify genes expressed differentially between type I and type II neuroblasts, genes were sought clustered with pros and ase, the only two genes known to be differentially expressed in type II neuroblasts. It was found that pros and ase reside together in a small sub-cluster of only 11 genes within group B. This sub-cluster as a whole exhibits reduced expression in brat, lgl, and lgl lgd mutants and enrichment in aur, aPKCCAAX, and lgl pins; remarkably, no other sub-cluster exhibits such a pattern. This suggests that the other nine genes in the cluster may also be specifically expressed in type I neuroblasts, like pros and ase, and that these are potentially the only genes that exhibit this unique pattern (Carney, 2012).

To test whether other genes in the small pros/ase cluster are also expressed in type I neuroblasts but not type II neuroblasts, an antibody was obtained to a candidate from this cluster, Retinal homeobox (Rx), a homeodomain-containing transcription factor. It was found that Rx is completely absent from type II neuroblasts, similar to Pros and Ase; Rx is detected in several type I neuroblasts as well as in a subset of differentiated type II progeny. Consistent with this expression pattern, it was found that brat mutants, which overproduce type II neuroblasts, show a loss of Rx staining. In contrast, lgl pins mutants, which have ectopic type I neuroblasts, show territories of strong Rx expression which is confined to Pros+ (likely type I-originating) cells. The fact that only a small patch of lgl pins mutant brain tissue is Rx+ is probably because Rx is normally expressed in a subset of type I neuroblasts. It is concluded that Rx, like Pros and Ase, is expressed in type I but not type II neuroblasts. Thus, most or all of the 11 genes in the pros/ase sub-cluster may be expressed in type I but not type II neuroblasts (Carney, 2012).

Genes expressed in type II neuroblasts but not type I neuroblasts were sought, as there are currently no known markers specifically expressed in type II neuroblasts. It was reasoned that transcripts expressed in type II neuroblasts should be enriched in genotypes that overproduce type II neuroblasts: brat, lgl and lgl lgd. One small cluster was found enriched in two of the three mutants (brat and lgl lgd). This cluster contains just 10 genes, seven encoding transcription factors. To verify the expression pattern of this gene cluster, the expression of one gene product, Optix, was examined. Optix is a conserved homeodomain-containing transcription factor required for eye development. Consistent with the microarray data, it was found that most of the Optix expression in the brain is indeed restricted to type II lineages; four of the six dorso-medial type II neuroblasts (DM1, 2, 3, and 6) express Optix, as do most of the INPs, GMCs, and neurons in these lineages. In addtion, recent work has shown that another gene in this cluster, pointedP1, is also preferentially expressed in type II neuroblasts (Sijun Zhu and Y.N. Jan, personal communication to Carney, 2012). The other two dorso-medial type II lineages (DM4 and 5) exhibit some expression of Optix in a subset of neuronal progeny, but it is absent from the neuroblasts and INPs in these lineages. In addition, a single dorsal type I neuroblast expresses Optix. Inspection of mutant brains further confirmed the type II-biased expression of Optix, in that brat mutant brains exhibit a marked increase in Optix+ neuroblasts, and in lgl pins, the increase in Optix is almost exclusively in a Pros (type II-originating) region of the brain. These results indicate that the clustering relationships can be used to predict type I/type II expression bias with good accuracy. It is concluded that Optix is primarily expressed in type II but not type I neuroblasts, and that Optix and the other five genes in this cluster are excellent candidates for regulators of type II neuroblast identity (Carney, 2012).

It has previously been shown that co-clustering of genes in expression profiling data is likely to reflect physical or genetic interactions and participation in the same pathway. The current results are consistent with these conclusions. For example, a small group of 11 genes was identified containing the only two genes known to be expressed in type I but not type II neuroblasts, and a third gene was shown to have a similar pattern of expression — thus all genes in this cluster are likely to be expressed in type I but not type II neuroblasts. Furthermore, the strong enrichment of GO terms in small sub-clusters within both group A and group C indicates that genes within these clusters are likely to share similar functions or processes (Carney, 2012).

At the outset of this study, a large group of genes was expected to be differentially expressed in type II versus type I neuroblasts, because these neuroblasts have such strikingly different cell lineages. However, only a few gene clusters were identified that were differentially regulated in such a type I/type II consistent manner — the 11 genes in the pros/ase cluster depleted in type II neuroblasts and the 10 genes enriched in type II neuroblasts. This suggests that the small number of genes identified may play a disproportionately large role in generating differences between type I and type II neuroblasts. Might pros and ase be the only genes regulating type I/type II differences? Both Ase and Pros can promote cell cycle exit, which may result in the Ase+ Pros+ type I progeny taking a GMC identity and undergoing just one terminal division and the Ase Pros type II progeny taking an INP identity and continuing to proliferate. Indeed, the misexpression of either Ase or low levels of Pros in type II neuroblasts is sufficient to cause the loss of INPs and/or their premature cell cycle exit, thereby decreasing lineage size toward the size of type I neuroblasts. However, it is unclear what is required to fully transform these cells into type I neuroblasts; addressing this question will require additional molecular markers and tracing the axon projections of the progeny of these 'transformed' neuroblasts (e.g., do they now fail to make intrinsic neurons of the adult central complex?). The fact that mutants in ase and pros do not transform type I neuroblasts into type II neuroblasts indicates that other genes, perhaps some in the pros/ase cluster described here, are also important for specification of type I neuroblast identity (Carney, 2012).

It was found that the neuroblasts in each mutant have remarkably similar expression profiles, as shown by the extensive list of similarly expressed genes in group A and by the list of genes with depleted expression in mutant brains, represented by group C. It is believed that these categories provide lists of genes that are representative of those expressed in neuroblasts and neurons, respectively, based on all known neuroblast-specific genes showing up in group A and all known neuron- or glial-specific genes being excluded from group A (Carney, 2012).

Group B genes apparently are not expressed in all neuroblasts like the group A genes, nor in all neurons or glia like group C genes. However, group B genes are more likely to be expressed in subsets of neurons, not neuroblasts, because group B genes as a whole have an over-representation of GO terms more similar to group C than to group A. Why then are group B genes excluded from group C, the neuron cluster? One possible explanation is that different neuroblast lineages are affected in each mutant, and thus different subsets of neurons are missing in each mutant. If different neuroblast lineages express different genes (which seems likely), then each mutant would be missing a unique subset of neural differentiation genes, leading to the cluster being excluded from group C. This model raises the intriguing possibility that group B sub-clusters may represent lineage-specific genes (Carney, 2012).

It is also possible that the mutant genotypes themselves may cause unique transcriptional differences, leading to a cluster of genes in group B. For example, several small sub-clusters in group B are expressed differently only in aPKCCAAX brains. These transcriptional differences are not correlated with the number of type I or type II neuroblasts. Instead, these genes appear to be differentially expressed in response to elevated aPKC. Drosophila aPKC has been best studied as a component of the apical complex in mitotic neuroblasts, and its capacity for causing ectopic self-renewal has been shown to be reliant on both its catalytic activity and its membrane localizatio. However, aPKC has been ascribed a role in neuroblast proliferation as well as in polarit, and a vertebrate homolog, PKC-zeta, was shown to possess a nuclear role in both proliferation of neural progenitors and neuronal cell fate specification. These observations are consistent with a role of aPKC in causing transcriptional differences (Carney, 2012).

The findings of this study highlight the importance of expression profiling of multiple genotypes. This method gave a more reliable picture of the group A genes expressed in neuroblasts, because genes with lineage-specific or genetic background-specific changes in expression appeared to be focused into group B, where they do not interfere with the clustering of groups A and C. In addition, two small sub-clusters of genes were identified in group B that are excellent candidates for being preferentially expressed in type I or type II neuroblasts, for which there have been few examples to date. Finally, it is concluded that group A genes are likely to be expressed in neuroblasts, and functional studies have identified 84 genes that are conserved in mammals and required for regulating neuroblast numbers in Drosophila. Future phenotypic analysis in Drosophila will determine whether these genes regulate neuroblast survival, quiescence, asymmetric cell division, and/or self-renewal. Future studies on the expression and function of orthologous genes in mouse neural progenitors and human stem cells (IP or neural) will reveal whether they have conserved roles from flies to mammals (Carney, 2012).


The bristle mechanosensory organs of the adult fly are composed of four different cells that originate from a single precursor cell (pI) via two rounds of asymmetric cell division. The pattern of cell divisions in this lineage has been examined by time-lapse confocal microscopy using GFP imaging and by immunostaining analysis. pI divides within the plane of the epithelium and along the anteroposterior axis to give rise to an anterior cell, pIIb, and a posterior cell, pIIa. pIIb divides prior to pIIa (it has been previously reported that pIIa divides prior to pIIb) to generate a small subepithelial cell (not previously described) and a larger daughter cell, named pIIIb. This unequal division, oriented perpendicularly to the epithelium plane, has not been described previously. pIIa divides after pIIb, within the plane of the epithelium and along the AP axis, to produce a posterior socket cell and an anterior shaft cell. Then pIIIb divides perpendicular to the epithelium plane to generate a basal neuron and an apical sheath (glial) cell. The small subepithelial pIIb daughter cell (not previously described) has been identified as a sense organ glial cell: it expresses glial cell missing, a selector gene for the glial fate and migrates away from the sensory cluster along extending axons. It is proposed that mechanosensory organ glial cells, the origin of which has been until now unknown, are generated by the asymmetric division of pIIb cells. Both Numb and Prospero segregate specifically into the basal glial and neuronal cells during the pIIb and pIIIb divisions, respectively. This revised description of the sense organ lineage provides the basis for future studies on how polarity and fate are regulated in asymmetrically dividing cells (Gho, 1999).

The first detailed description of the sense organ lineage in the pupal notum of D. melanogaster had proposed that four cells are generated from a single pI cell via two rounds of asymmetric divisions. This first study also indicated the presence of a small BrdU-positive soma-sheath cell associated with the four BrdU-labelled sense-organ cells. Because this soma-sheath cell was often seen at some distance from the sensory cluster, it had been inferred that it derived from an unknown precursor, which also carried out its terminal DNA replication at approximately 16 hours APF. Soma sheath cells have previously been described in adult external sense organs as small, subepithelial, A101-positive cells found associated with the neuron and/or its axon. The data presented in the current study indicate that this soma-sheath cell most likely corresponds to the small pIIb daughter cell that differentiates as a glial cell. Earlier BrdU pulse-labelling experiments had indicated that the precursor of the shaft and socket cells, pIIa, replicated its DNA before the precursor of the neuron and sheath cells, named pIIb in this study. However, more recent studies indicate that the anterior Prospero-positive daughter cell, pIIb, enters mitosis prior to pIIa. The model proposed here suggests that pIIb does indeed divide prior to pIIa, while the precursor of the neuron and sheath cells, pIIIb, divides after pIIa (Gho, 1999).

This study confirms that pI and pIIa divide within the epithelial plane and along the AP axis. The orientation of the pI division is regulated by Frizzled signaling. By contrast, the orientation of the pIIa division relative to its sister cells does not require frizzled activity. The positioning of the mitotic spindle in pIIa might be influenced by cell signaling from anterior pIIb and/or pIIIb cells, or by cortical marks deposited during the previous pI division. Consistently, the mitotic spindle of pIIa is often tilted basally toward pIIIb. This study establishes that both pIIb and pIIIb divide perpendicular to the epithelial plane. This contrasts with a previous conclusion that pIIIb divides within the plane of the epithelium and perpendicular to the AP axis. Because horizontal sections were projected along the z-axis in the study, only mitotic spindles tilted relative to the apicobasal axis were recognized. This led to an erroneous conclusion. The previous observation that Numb localizes away from the midline, however, is consistent with the present finding that the most basal centrosome associated with the Numb crescent often occupies a lateral position (Gho, 1999 and references).

The current results confirm that Numb is asymmetrically distributed in dividing pI, pIIa and pIIIb cells, and is unequally inherited by the pIIb, shaft and neuron cells. It is also established that Numb forms a basal crescent in pIIb and segregates into the sense organ glial cell. In contrast with Numb, Prospero is not detected in dividing pI and pIIa. Prospero, like Numb, forms a basal crescent in pIIb and pIIIb, and preferentially segregates into the future glial cell and neuron. By contrast, two recent reports had indicated that Prospero is uniformly localized at the cell cortex in dividing pIIb. In these studies, the distribution of Prospero was examined in confocal sections perpendicular to the apicobasal axis of dividing pIIb. Therefore, it is possible that the basal distribution of Prospero could have escaped detection. A detailed co-localization analysis of Numb and Prospero in dividing pIIb and pIIIb has revealed that these two fate determinants do not strictly co-localize. In these cells, Prospero is mostly found at the basal pole, while Numb has also been found to accumulate in the cortical region of cell contact between sense organ cells. It will be interesting to examine how cell-cell interactions between sense organ cells regulate the activity of the protein complexes involved in the polar distribution of both Numb and Prospero (Guo, 1999).

The current analysis of the pIIb division reveals a striking analogy between the pIIb division in the notum and the neuroblast division in the embryo. (1) Both cells divide unequally to produce two cells of different size. (2) In both cases, the division is oriented along the apicobasal axis and the small daughter cell appears at the basal pole. (3) Numb and Prospero specifically localize at the basal pole and segregate into the small basal cell. It will thus be of interest to examine whether asymmetry is established by similar molecular mechanisms in both pIIb and neuroblast (Guo, 1999).

The basal pIIIb cell that inherits Numb and Prospero is proposed to be the neuron. As in dividing pIIb, Prospero has been found to localize asymmetrically at the basal pole of pIIIb, while Numb localizes in a basolateral crescent. Both proteins segregate preferentially into the basal daughter. Because Numb segregates into the basal daughter, it is proposed that the basal pIIIb daughter cell is the neuron. The apical pIIIb daughter must therefore be the sheath (glial) cell. This interpretation that the neuron corresponds to the basal pIIIb daughter cell implies that accumulation of Prospero in the neuron is only transient and that the high level accumulation of Prospero in the sheath cell is due to de novo synthesis. A transient accumulation of Prospero in the neuron would also be consistent with the hypothesis formulated by Manning (1999) that Prospero functions in the neuron to regulate axonal pathfinding (Guo, 1999).

Glial cells constitute a crucial component of the nervous system. They wrap the neuronal somata and axons and play a number of roles during normal neuronal activity and development, including axonal growth. Gliogenesis in the peripheral nervous system (PNS) of the adult fly has been best described in the wing. In this tissue, glial cells originate from regions of the ectoderm that also give rise to sense organs. Glial cells then migrate along the nerve following the direction taken by the axons. In addition, mutations that induce ectopic sense organs also lead to the emergence of ectopic glial cells. Conversely, mutations that reduce the number of sensory bristles result in a significant reduction of the number of glial cells. These observations have led to the hypothesis that gliogenesis is induced in the ectoderm by neighbouring sense organ cells. However, the exact origin of the glial cells is not known. The current finding that sense organ glial cells are produced by the asymmetric division of pIIb in the notum offers a novel interpretation for all these earlier observations and suggests that in the wing, glial cells originate from sensory lineages (Guo, 1999).

The division of pIIb is intrinsically asymmetric. It produces a small subepithelial cell that will adopt a glial fate and a larger pIIIb cell. The intrinsic nature of this division suggests that expression of gcm in the small subepithelial is a consequence of the initial asymmetry established in pIIb. Two fate determinants, Numb and Prospero, are unequally inherited by the future glial cell. This raises the possibility that they participate in activating gcm expression in the small pIIb daughter and act upstream of gcm in establishing a glial fate (Guo, 1999).

Various cell markers to have been used to trace the development of the sensory cells of the thoracic microchaete. The results dictate a revision in the currently accepted model for cell lineage within the mechanosensory bristle. The sensory organ progenitor divides to form two secondary progenitors: PIIa and PIIb. PIIb divides first to give rise to a tertiary progenitor-PIII and a glial cell. This is followed by division of PIIa to form the shaft and socket cells as described before. PIII expresses high levels of Elav and low levels of Prospero and divides to produce neuron and sheath. Its sibling cell expresses low Elav and high Prospero and is recognized by the glial marker, Repo. This cell migrates away from the other cells of the lineage following differentiation (Reddy, 1999b).

Previous data had shown that Pros was expressed in PIIb and inherited by both the progeny following division. Shortly after division of PIIb, immunoreactivity becomes much more pronounced in the sub-epidermal cell and is weak in the larger cell. The latter cell can be seen to be in mitosis when stained with propidium iodide or antibodies against either beta-tubulin or phosphohistone. Since this cell was observed to undergo division in all clusters examined, it has been suggested that this cell is a tertiary progenitor which is denoted PIII. PIII can be identified by low Pros and high Elav immunoreactivity. Both markers become cytosolic during division and are probably inherited equally by both progeny. Sensory cells were examined in fully differentiated sensory clusters, after division of PIII, by staining with antibodies against Pros and Elav. Elav is expressed strongly in the differentiated neuron while Pros is expressed in the sheath cell. This observation implies that Pros is up regulated specifically in the sheath cell. The PIIb lineage gives rise to a tertiary progenitor (PIII) and a glial cell. Division of PIII would be expected to produce sensory clusters composed of five cells. Several such clusters were observed in nota from pupae 20-22 hours APF. The expression of Repo in the sub-epidermal cell was observed in most of the five cell clusters examined at 20-22 hours APF (71 out of 75 cases). The time course of Repo staining suggests that its expression begins in the sub-epidermal daughter of PIIb some time after its birth, indicating differentiation to the glial fate. At this time, the sibling cell (PIII) begins to undergo mitosis to give rise to neuron and sheath cell. Examination of several sensory clusters from 20-22 hour APF nota lead to the conclusion that the glial cell migrates away from the other cells of the cluster. These data convincingly demonstrate that the PIIb lineage undergoes two cell divisions and gives rise to three cells of different fates: a neuron, sheath and glial cell. In the adult the glial cell is not closely associated with the rest of the cells of the sense organ and apparently migrates away from these cells (Reddy, 1999b).

Specification of individual adult motor neuron morphologies by combinatorial transcription factor code

How the highly stereotyped morphologies of individual neurons are genetically specified is not well understood. This study identified six transcription factors (TFs; Ems, Zfh1, Pb, Zfh2, Pros and Toy) expressed in a combinatorial manner in seven post-mitotic adult leg motor neurons (MNs) that are derived from a single neuroblast in Drosophila. Unlike TFs expressed in mitotically active neuroblasts, these TFs do not regulate each other's expression. Removing the activity of a single TF resulted in specific morphological defects, including muscle targeting and dendritic arborization, and in a highly specific walking defect in adult flies. In contrast, when the expression of multiple TFs was modified, nearly complete transformations in MN morphologies were generated. These results show that the morphological characteristics of a single neuron are dictated by a combinatorial code of morphology TFs (mTFs). mTFs function at a previously unidentified regulatory tier downstream of factors acting in the NB but independently of factors that act in terminally differentiated neurons (Enriquez, 2015).

Neurons are the most morphologically diverse cell types in the animal kingdom, providing animals with the means to sense their environment and move in response. In Drosophila, neurons are generated by neuroblasts (NBs), specialized stem cells dedicated to the generation of neurons and glia. As they divide, NBs express a temporal sequence of transcription factors (TFs) that contribute to the generation of neuronal diversity. For example, in the embryonic ventral nerve cord (VNC), most NBs express a sequence of five TFs (Hunchback, Krüppel, Pdm1/Pdm2, Castor, and Grainyhead), while in medulla NBs and intermediate neural progenitors of the Drosophila larval brain a different series of TFs have been described. In vertebrates, analogous strategies are probably used by neural stem cells, e.g., in the cerebral cortex and retina, suggesting that this regulatory logic is evolutionarily conserved. Nevertheless, although temporally expressed NB TFs play an important role in generating diversity, this strategy cannot be sufficient to explain the vast array of morphologically distinct neurons present in nervous systems. For example, in the Drosophila optic lobe there is estimated to be ~40,000 neurons, classified into ~70 morphologically distinct types, each making unique connections within the fly's visual circuitry neurons (Enriquez, 2015).

A second class of TFs has been proposed to specify subtypes of neurons. For example, in the vertebrate spinal cord, all motor neurons (MNs) express a common set of TFs at the progenitor stage (Olig2, Nkx6.1/6.2, and Pax6) and a different set of TFs after they become post-mitotic (Hb9, Islet1/2, and Lhx3). Hox6 at brachial and Hox10 at lumbar levels further distinguish MNs that target muscles in the limbs instead of body wall muscles. Subsequently, limb-targeting MNs are further refined into pools, where all MNs in a single pool target the same muscle. Each pool is molecularly defined by the expression of pool-specific TFs, including a unique combination of Hox TFs. In Drosophila embryos, subclasses of MNs are also specified by unique combinations of TFs: evenskipped (eve) and grain are expressed in six MNs that target dorsal body wall, and Hb9, Nkx6, Islet, Lim3, and Olig2 are required for ventral-targeting MNs. However, each neuronal subtype defined by these TFs includes multiple morphologically distinct neurons, leaving open the question of how individual neuronal morphologies are specified neurons (Enriquez, 2015).

A third class of TFs suggested to be important for neuronal identity is encoded by terminal selector genes. Initially defined in C. elegans, these factors maintain a neuron's terminally differentiated characteristics by, for example, regulating genes required for the production of a particular neurotransmitter or neuropeptide. Consequently, these TFs must be expressed throughout the lifetime of a terminally differentiated neuron. Notably, as with neurons that are from the same subtype, neurons that share terminal characteristics, and are therefore likely to share the same terminal selector TFs, can have distinct morphological identities. For example, in C. elegans two terminal selector TFs, Mec-3 and Unc-86, function together to maintain the expression of genes required for a mechanosensory fate in six morphologically distinct touch sensitive neurons neurons (Enriquez, 2015).

In contrast to the logic revealed by these three classes of TFs, very little is known about how individual neurons, each with their own stereotyped dendritic arbors and synaptic targets, obtain their specific morphological characteristics. This paper addresses this question by focusing on how individual MNs that target the adult legs of Drosophila obtain their morphological identities. The adult leg MNs of Drosophila offer several advantages for understanding the genetic specification of neuronal morphology. For one, all 11 NB lineages that generate the ~50 leg-targeting MNs in each hemisegment have been defined. More than two-thirds of these MNs are derived from only two lineages, Lin A (also called Lin 15) and Lin B (also called Lin 24), which produce 28 and 7 MNs, respectively, during the second and third larval stages. Second, each leg-targeting MN has been morphologically characterized-both dendrites and axons-at the single-cell level. In the adult VNC, the leg MN cell bodies in each thoracic hemisegment (T1, T2, and T3) are clustered together. Each MN extends a highly stereotyped array of dendrites into a dense neuropil within the VNC and a single axon into the ipsilateral leg, where it forms synapses onto one of 14 muscles in one of four leg segments: coxa (Co), trochanter (Tr), femur (Fe), and tibia (Ti). Not only does each MN target a specific region of a muscle, the pattern of dendritic arbors of each MN is also stereotyped and correlates with axon targeting. The tight correlation between axon targeting and dendritic morphology has been referred to as a myotopic map. The stereotyped morphology exhibited by each MN suggests that it is under precise genetic control that is essential to its function neurons (Enriquez, 2015).

This study demonstrates that individual post-mitotic MNs express a unique combination of TFs that endows them with their specific morphological properties. Focus was placed on Lin B, which generates seven MNs, and six TFs were identified that can account for most of the morphological diversity within this lineage. Interestingly, these TFs do not cross-regulate each other and are not required for other attributes of MN identity, such as their choice of neurotransmitter (glutamine) or whether their axons target muscles in the periphery, i.e., they remain terminally differentiated leg motor neurons. Consistent with the existence of a combinatorial code, when two or three, but not individual, TFs were simultaneously manipulated nearly complete transformations in morphology were observed. However, removing the function of a single TF, which is expressed in only three Lin B MNs, resulted in a highly specific walking defect that suggests a dedicated role for these neurons in fast walking. Together, these findings reveal the existence of a regulatory step downstream of temporal NB factors in which combinations of morphology TFs (mTFs) control individual neuron morphologies, while leaving other terminal characteristics of neuronal identity unaffected neurons (Enriquez, 2015).

Inherent in the concept of a combinatorial TF code is the idea that removing or ectopically expressing a single TF will only generate a transformation of fate when a different wild-type code is generated. Consistent with this notion, only when the expression of two or three mTFs were simultaneously manipulated was it possible to partially mimic a distinct mTF code and, as a result, transform the identity of one Lin B MN into another. In contrast, manipulating single TFs typically resulted in aberrant or neo-codes that are not observed in wild-type flies. For example, removing pb function from Lin B resulted in two MNs with a code (Ems+Zfh1) and MN morphology that are not observed in wild-type Lin A and Lin B lineages. Analogously, ectopic Pb expression in Lin A, which normally does not express this TF, generated aberrant codes and MN morphologies. This latter experiment was particularly informative because although Pb redirected a subset of Lin A dendrites to grow in an anterior region of the neuropil, it did not alter the ability of these dendrites to cross the midline. Thus, the dendrites of these MNs had characteristics of both Pb-expressing Lin B MNs (occupying an antero-ventral region) and Pb-non-expressing Lin A MNs (competence to cross the midline). Axon targeting of these MNs was also aberrant: although they still targeted leg muscles, Pb-expressing Lin A MNs frequently terminated in the coxa, which is not a normal characteristic of Pb-expressing Lin B MNs or of any Lin A MN. These observations suggest that the final morphological identity of a neuron is a consequence of multiple TFs executing functions that comprise a complete morphological signature. Some functions, such as the ability to occupy the antero-ventral region of the neuropil, can be directed by a single TF (e.g., Pb), while other functions, such as the ability to accurately target the distal femur, require multiple TFs (e.g., Pb+Ems). Further, because it was possible to generate MNs that have both Lin B and Lin A morphological characteristics, hte results argue against the idea that there are lineage-specific mTFs shared by all progeny derived from the same lineage. Instead, the data are more consistent with the idea that the final morphological identity of an MN depends on its mTF code neurons (Enriquez, 2015).

Drosophila NBs, and perhaps vertebrate neural stem cells, express a series of TFs that change over time and have therefore been referred to as temporal TFs. For Lin B, the sequence of these factors is unknown, in part because the Lin B NB is not easily identified in the second-instar larval VNC, the time at which it is generating MNs. Nevertheless, each MN derived from Lin B and Lin A has a stereotyped birth order, consistent with the idea that temporal TFs play an important role in directing the identities of MNs derived from these lineages and, therefore, the mTFs they express. For Lin B, this birth order is Co1->Tr1->Fe1->Tr2->Co2->Co3->Co4. Interestingly, according to the mTF code proposed in this study, each of these MNs differs by at most two mTFs in any successive step. For example, Tr1 has the code [Zfh1, Ems, Pb, Zfh2] while Fe1, the next MN to be born, has the code [Zfh1, Ems, Pb]. Thus, it is posited that the sequence of temporal TFs acting in the NB is responsible for directing each successive change in mTF expression in postmitotic MNs (e.g., in the Tr1->Fe1 step, repression of zfh2). Although a link between temporal TFs and TFs expressed in postmitotic neurons has been proposed in Drosophila, the role of these TFs in conferring neuron morphologies is not known. Further, there may be additional diversity-generating mechanisms in lineages that produce many more neurons than the seven MNs generated by Lin B. One additional source of diversity may come from NB identity TFs, which distinguish lineages based on their position. Such spatial information could in principle allow the same temporal TFs to regulate different sets of mTFs in different NB lineages. It is also likely that differences in the levels of some mTFs may contribute to neuronal identities. Consistent with this idea, the levels of Zfh2 and Pros differ in the Lin B MNs expressing these TFs, differences that are consistent in all three thoracic segments and between animals. Further, Zfh1 levels vary between Lin B MNs and its levels control the amount of terminal axon branching. Previous studies also demonstrated that TF levels are important for neuron morphology, including Antp in adult leg MNs derived from Lin A and Cut in the control of dendritic arborization complexity in multidendritic neurons. If the levels of mTFs are important, it may provide a partial explanation for why the transformations of morphological identity generated in this study with the MARCM technique, which cannot control levels, are typically only partially penetrant neurons (Enriquez, 2015).

Another distinction between temporal TFs and mTFs is that no evidence has been observed of cross-regulation between mTFs. In situations when mTFs were either removed (e.g., pb-/-; emsRNAi) or ectopically expressed (e.g., UAS-pb + UAS-ems) in postmitotic Lin B MARCM clones, the expression of the remaining mTFs was unchanged. In contrast, when an NB lineage is mutant for a temporal TF, the prior TF in the series typically continues to be expressed. These observations suggest that the choice of mTF expression is made in the NB and that once the postmitotic code is established, it is not further influenced by coexpressed mTFs neurons (Enriquez, 2015).

The data further suggest that mTFs are distinct from terminal selector TFs. In mutants for the mTFs studied here, the resulting neurons remain glutamatergic leg motor neurons: they continue to express VGlut, which encodes a vesicular glutamate transporter, expressed by all Drosophila MNs, and they still exit the VNC to target and synapse onto muscles in the adult legs. Thus, whereas terminal selector TFs maintain the terminal characteristics of fully differentiated neurons, mTFs are required transiently to execute functions required for each neuron's specific morphological characteristics. Together, it is suggested that the combined activities of terminal selector TFs and mTFs specify and maintain the complete identity of each post-mitotic neuron neurons (Enriquez, 2015).

Although the mTFs defined in this study, e.g., Ems, Pb, and Toy, do not fit the criteria for a terminal selector TF, it is plausible that some TFs function both as mTFs and terminal selector TFs. One example may be Apterous, a TF that is expressed in six interneurons in the thoracic embryonic segments and that functions with other TFs to control the terminal differentiation state of these neuropeptide-expressing neurons. In addition to the loss of neuropeptide expression, these neurons display axon pathfinding defects in the absence of apterous. Despite the potential for overlapping functions, it is conceptually valuable to consider the specification of neuronal morphologies as distinct from other terminal characteristics, as some mTFs regulate morphology without impacting these other attributes. It is also plausible that some of the TFs that have been previously designated as determinants of subtype identity may also be part of mTF codes. For example, eve is required for the identity of dorsally directed MNs inDrosophila embryogenesis, but the TFs required for distinguishing the individual morphologies of these neurons are not known. It may be that Eve is one component of the mTF code and that it functions together with other mTFs to dictate the specific morphologies of these neurons neurons (Enriquez, 2015).

Flies containing a single pb mutant Lin B clone exhibited a highly specific walking defect: when walking at high speed, these flies were significantly more unsteady compared to control flies. The restriction of this defect to high speeds suggests that the Pb-dependent characteristics of these MNs may be specifically required when the walking cycle is maximally engaged, raising the possibility that Tr1, Tr2, and Fe1 are analogous to so-called fast MNs described in other systems. Further, these data support the idea that the highly stereotyped morphology of these MNs is critical to the wild-type function of the motor circuit used for walking. In particular, the precise dendritic arborization pattern exhibited by these MNs, which is disrupted in the pb mutant, is likely to be essential for their function. Although it cannot be excluded that other pb-dependent functions contribute to this walking defect, these observations provide strong evidence that the myotopic map, in which MNs that target similar muscle types have similar dendritic arborization patterns, is important for the fly to execute specific adult behaviors neurons (Enriquez, 2015).

prospero: Biological Overview | Evolutionary Homologs | Regulation | Protein Interactions | Effects of Mutation | References

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