prospero

DEVELOPMENTAL BIOLOGY

Embryonic

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).

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).

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).

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).

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).

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 (N^ts) mutant. N^ts flies were first crossed to an allelic series that included N^55e11, N^264.47, N^ts1 and N^nd.1. The strongest loss of function combinations (N^ts/N^55e11 and N^ts/N^264.47) failed to generate viable adult flies even at the permissive temperature, often dying as pharate adults. N^ts/N^ts flies produced viable adults at the permissive temperature with midguts similar to wild type. N^ts/N^ts flies shifted to the non-permissive temperature led to a mild increase in the number of small cells. The weakest allelic combination, N^ts/N^nd.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, Gal80^ts was combined with the esg-Gal4 transcriptional activator. To verify that the Gal80^ts transgene functions in the midgut, the temporal and spatial induction of a UAS-GFP transgene was characterized. Adult esg-Gal4,UAS-GFP, tub-Gal80^ts 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-N^RNAi transgene, to reduce Notch signalling. In control experiments, UAS-N^RNAi; esg-Gal4,UAS-GFP, tub-Gal80^ts 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-N^RNAi; esg-Gal4,UAS-GFP, tub-Gal80^ts 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 N^intra, a constitutively active form of Notch. In control experiments, esg-Gal4,UAS-GFP, tub-Gal80^ts; UAS-N^intra 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-Gal80^ts; UAS-N^intra 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 incenp^EC3747 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 incenp^EC3747 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).

Larvae

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).

Pupal

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).

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