castor

DEVELOPMENTAL BIOLOGY

Embryonic

See the embryonic expression pattern of cas at the Berkeley Drosophila Genome Project Patterns of Gene Expression Site

cas/ming is first expressed in lateral and dorso-lateral ectoderm of the procephalic neurogenic region (see Views of cephalic lobe neuroblasts). The most anterior staining marks progenitors of the larval eye known as Bolwig's organ. In the central nervous system, it is expressed first in midline glial precursors and only later in neuroblasts (Cui, 1992 and Mellerick, 1992).

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

cas/ming reaches its maximum level of expression during stages 11 and 12 [Image]. A reduction in size of cas/ming positive cells is noted with time in the cells lining the superesophagael ganglia.

Regulation of POU genes by castor and hunchback establishes layered compartments in the Drosophila CNS

In addition to its early regulatory functions during segmentation, Hunchback is also expressed in the developing nervous system. One possible CNS regulatory target for Hb is the POU gene pdm-1. Hb regulates pdm-1 expression at the cellular blastoderm stage, and may play a similar role in the CNS. Since Hb and Castor bind similar promoter target sequences, an exploration was carried out of the embryonic distribution of the three proteins using polyclonal antibodies. It is suggested that Hb and Cas act in a cooperative, non-overlapping manner to control POU gene expression during Drosophila CNS development. By silencing pdm expression in early and late NB sublineages, Hb and Cas establish three pan-CNS compartments whose cellular constituents are marked by the expression of either Hb, Pdm, or Cas. During the initial S1 and S2 waves of NB delaminations, Pdm-1 is expressed in most, if not all, neuroectoderm cells. However, no Pdm-1 is detected in fully delaminated NBs and during stage 9 only a small subset of ventral cord GMCs express detectable levels. At this time, Hb expression is detected in all fully delaminated NBs and in many of their GMCs but not in neuroectoderm cells. Starting at late stage 9, Hb immunoreactivity is progressively lost from NBs; by late stage 10 only a small subset of ventral cord NBs express Hb. However, Hb is detected in many GMC and in their progeny generated during the first rounds of GMC production. These early sublineages reside predominantly along the inner/dorsal surfaces of the developing ganglia (see Lateral views of Drosophila CNS). The reduction in Hb NB expression coincides with the activation of Pdm-1 NB expression; by late stage 10, Pdm-1 is detected in many cephalic lobe and ventral cord NBs and in GMCs. Similar to the dynamics of Hb expression, Pdm-1 NB expression is transient. However, many GMCs and their progeny arising from the Pdm-expressing NBs maintain high levels of Pdm-1 (Kambadur, 1998).

Onset of Cas expression in both ventral cord and cephalic lobe NBs parallels the loss of Pdm-1 NB expression, suggesting a transient overlap in their expression. NBs containing detectable levels of both Pdm-1 and Cas are observed during this period. However, no Pdm-1/Cas co-expression is detected in GMCs or in their progeny. Hb/Pdm-1 co-expression is also detected at a similar frequency in early S1 and S2 NBs but not in their progeny. By stage 11, ventral cord Pdm-1-expressing cells are juxtaposed to the more dorsal or internal Hb-positive sublineages and flanked on their ventral/ventral-lateral side by the superficially positioned Cas-positive NBs and GMCs. The same relative positioning of Hb, Pdm-1 and Cas subpopulations is also observed in the cephalic lobes, since Cas expressing NBs and their offspring predominantly cover the outer flanks of Pdm-1 sublineages while Hb positive cells occupy deeper internal positions. Although Hb and Cas immunopositive cells together make up >50% of the cells present in stage 12 ganglia, no Hb/Cas co-expressing cells are detected in NBs or in their progeny. In fact, no cell at any stage of embryonic development is observed co-expressing these Zn-finger proteins. Simultaneous labeling of Hb, Pdm-1 and Cas reveals that most, if not all, NB lineages express at least one of these transcription factors. The absence of prolonged overlap between Hb/Pdm-1 co-expression or Pdm-1/Cas co-expression in early and late sublineages respectively, suggests that Hb and Cas may control, via repression, the temporal boundaries of pdm expression during CNS development (Kambadur, 1998).

In situ mRNA localizations show that cas expression is predominantly restricted to CNS NBs, with little or no message detected in GMCs and no detectable mRNA observed in newly formed neurons or glia. However, Cas protein persists significantly longer than its message and is found in the nuclei of many cells generated during late sublineage development. Cas-positive nuclei in stage 14 and older embryos are detected in all CNS ganglia; many most likely belong to nascent neurons. Like Cas, Hb and Pdm-1 are also detected in all ganglia of stage 14 and older embryos, suggesting that the regulatory functions of all three transcription factors may be required in many neurons and glia until their functional phenotypes have been achieved (Kambadur, 1998).

The tightly choreographed NB expressions of Hb, Pdm, and Cas suggest temporally integrated processes participate in their formation. Clonal analysis of ventral cord NB lineages has revealed that many early delaminating NBs produce lineages that span most of the ventral cord's dorsal/ventral axis. For example the NB5-2, one of the first NBs to delaminate, generates a ventral-dorsal column of 17 to 26 cells. The dynamics of Hb, Pdm-1 and Cas expression in NBs indicates that many of the early S1 and S2 delaminating NBs may sequentially express all three and thereby produce lineages spanning all three compartments. Two such ventral cord candidates are the early NB5-2s and NB7-4s. Shortly after their delamination, during early stage 9, they activate Hb expression, while later, after several rounds of GMC divisions, they activate Cas expression. The fact that NBs co-expressing Hb/Pdm-1 or Pdm-1/Cas are detected (but never Hb/Cas) further suggests that at least some of the early NBs make the Hb->Pdm->Cas transition. However, not all NBs undergo these transitions. This is particularly evident in NBs that enter the proliferative zone during later delamination waves. For example, the first ventral cord NBs to express Cas, the S3 NB6-1s, activate Cas shortly after delaminating from the ectoderm and do not express Hb (Kambadur, 1998).

Programmed transformations in neuroblast gene expression during Drosophila CNS lineage development

During Drosophila embryonic CNS development, the sequential neuroblast (NB) expression of four proteins, Hunchback (Hb), Pou-homeodomain proteins 1 and 2 (referred to collectively as Pdm), and Castor (Cas), identifies a transcription factor network regulating the temporal development of all ganglia. The Zn-finger proteins Hb and Cas, acting as repressors, confine Pdm expression to a narrow intermediate temporal window; this results in the generation of three panneural domains whose cellular constituents are marked by expression of Hb, Pdm, or Cas. Seeking to identify the cellular mechanisms that generate these expression compartments, the lineage development of isolated NBs in culture was studied. The Hb, Pdm, and Cas expression domains are generated by transitions in NB gene expression that are followed by gene product perdurance within sequentially produced sublineages. These results also indicate that following Cas expression, many CNS NBs continue their asymmetric divisions and generate additional progeny, which can be identified by the expression of the bHLH transcription factor Grainyhead (Gh). Gh appears to be a terminal embryonic CNS lineage marker. Taken together, these studies indicate that once NBs initiate lineage development, no additional signaling between NBs and the neuroectoderm and/or mesoderm is required to trigger the temporal progression of Hb followed by Pdm and then Cas, and subsequently Gh expression during NB outgrowth (Brody, 2000).

Underpinning the formation of NB lineages are spatially and temporally regulated transcription factor networks that play pivotal roles in establishing the unique cellular identities of NBs and their progeny. Prior to NB delamination, during the initial specification of NBs, two spatially regulated transcription factor networks subdivide the ventral neuroectoderm along its anterior/posterior (A/P) x axis and dorsal/ventral (D/V) y axis. Later, during NB lineage development, at least one additional network, acting over several hours, gives rise to sequentially formed multilayered basal (inner or dorsal) to apical (outer or ventral) neuronal subpopulations. Along the basal/apical z axis, neuronal subpopulations in all ganglia can be identified by their expression of the transcription factors Hb, Pdm and Cas. Hb marks a deeper, basally distributed population of neurons that are born early, Cas marks a superficial, apically distributed population of neurons that are born late, and Pdm marks an intermediate population arrayed between the Hb- and the Cas-expressing cells. Both genetic and molecular analysis indicates that two Zn-finger proteins, Hb and Cas, act as repressors to silence pdm expression. By restricting pdm expression primarily to intermediate-born neuronal precursors these structurally different Zn-finger proteins help establish three pan-CNS neural subpopulations whose cellular constituents are marked by the expression of Hb, Pdm, or Cas (Brody, 2000).

To what extent are the z axis expression domains generated successively by invariant gene expression programs, maintained in different NBs, versus sequential gene expression programs within sublineages of single NBs? To better understand the nature of the temporal components regulating the CNS z axis network, attempts were made to determine if the sequential expression of Hb, Pdm-1, and Cas occurs during NB lineage development in vitro. In order to analyze the capacity of individual NBs to generate a full repertoire of Hb-, Pdm-, or Cas-expressing sublineages, overnight cultures were simultaneously immuno-stained for all three factors and the percentage of cells within a clone that were positive for each factor was subsequently determined. Not all clones contained cells expressing each of the transcription factors. The majority of clones containing Cas-expressing cells also contain additional NB descendants marked by the expression of Hb or Pdm-1. Triple-immunolabeling studies have revealed that clones expressing only Cas are the exception. Taken together the results indicate that many isolated S1 and S2 NBs, when maintained in culture, will generate neuronal descendants that are marked by Hb, Pdm, or Cas expression. Given that Hb and Cas are repressors of pdm gene NB expression, these observations also suggest that the overlapping Hb/Pdm and Pdm/Cas expressions, both in vivo and in culture represent transition states in NB gene expression. In other words, NBs undergo sequential transitions in gene expression, thus generating the multiple cell layers seen in vivo (Brody, 2000).

Triple-immunolabeling studies have revealed that many of the overnight NB clones contain a subset of cells that do not contain detectable levels of Hb, Pdm-1, or Cas. In many of these in vitro lineages the putative NB is also unstained. The bHLH transcription factor Gh is known to be expressed in CNS NBs but only after stage 14. In view of the late onset of Gh expression in NBs and the triple-staining results identifying cells in o/n clones that do not express Hb, Pdm-1, or Cas, it was hypothesized that these negative cells may represent an additional late NB expression window marked by Gh expression. To test this hypothesis, the spatial/temporal expression dynamics of Gh were compared to other members of the z axis network. Similar to its late activation during in vivo development, Gh expression was observed only in overnight cultures; when more than one Gh-positive cell was detected in a clone they were consistently found clustered together. Two-thirds of the Cas+ clones had at least one Gh+ cell and the average number of Gh+ cells in all clones was 2.3. Approximately 2/3 of the Gh+ clones also contained Hb-immunopositive cells. While no Hb-Gh coexpressing cells were observed, approximately 20% of the Gh+ cells also expressed Cas. Given the late onset of Gh expression in both the embryo and the cultured NB clones and the overlapping Cas and Gh expression, it is likely that Gh marks a fourth temporal window for NB transcription factor expression. In addition, because there was an average of more than one cell in an o/n clone that was immunopositive for Gh, it is likely that Gh is also expressed/maintained in a sublineage(s) born after the one marked by Cas expression (Brody, 2000).

The principle finding of this study is that built on top of the x and y axis neural identity systems is an additional temporal network that defines successive stages of lineage maturation in an apical/basal z axis. This global CNS network, identified by the temporal cascade of Hb followed by Pdm and subsequently Cas NB expression, most likely ensures in part that each NB generates a column of uniquely specified neuronal subtypes. The shared transcription factor expression within a given temporal layer also suggests that the cellular constituents of these expression domains may also have similar patterns of downstream target gene expression (Brody. 2000).

The following model for the origin of the layer sublineages marked by these transcription factors has been suggested. As each NB divides, generating a succession of GMCs, it undergoes multiple transitions in transcription factor expression. In succession, the NBs express Hb, Pdm, Cas, and Gh. The first progeny generated by the early S1 and S2 NBs express Hb, and the presence of Hb protein persists in their neural progeny. These early S1 and S2 NBs go on to activate the expression of the Pdms that, like Hb, persist in neural sublineages generated during this temporal window. Subsequently Cas is activated in NBs, represses Pdm transcription, and likewise persists in neural sublineages. After Cas expression, a fourth neural subpopulation, generated by dividing NBs, expresses Gh. This Gh subpopulation most likely represents the terminal sublineage of the embryonic NB. The data also reveal that not all NBs generate cells that occupy all four layers, a result that reflects the unique set of lineages, generated by each NB. Most likely, each NB has a preprogrammed time of delamination, but the timing of transitions is synchronized in a global fashion. The model further suggests that late delaminating NBs can be distinguished from early NBs by their inability to activate Hb. Although Hb is activated shortly after the S1s and S2s have delaminated, Hb is never seen in the proliferative zone during late delaminations (Brody, 2000).

What mechanism drives transitions in transcription factor expression in NBs and in their GMC progeny? It has been shown that Hb, Cas, and Pdm are involved in a regulatory circuit in which Hb and Cas repress Pdm in a cooperative, nonoverlapping fashion both early and late within NB lineages. In addition, Pdm is also required for the proper expression of Cas. It is likely, therefore, that this Hb to Pdm followed by Cas network is responsible for temporal transitions in transcription factors, related to the generation of multiple cellular layers. This conclusion must be tempered by the observation that less than 50% of the cells in clones and, by implication, in the CNS, are positive for even one of these transcription factors. There must be other factors involved in sublineage determination related to CNS layering. If the transitions observed are not caused by the partitioning of mRNA and proteins between NBs and their GMC, but by regulatory interactions within the cells themselves, then there must be additional mechanisms that are involved in the rapid disappearance of these molecules. Expression of transcription factors restricted to one or two generations of NB development could be accomplished if these transcription factors were autoregulatory, repressing their own expression in NBs and in their progeny (Brody. 2000).

The CNS midline cells coordinate proper cell cycle progression and identity determination of the Drosophila ventral neuroectoderm

Defects in single minded mutants are characterized by the loss of the gene expression required for the proper formation of the ventral neurons and epidermis, and by a decrease in the spacing of longitudinal and commissural axon tracks. Molecular and cellular mechanisms for these defects were analyzed to elucidate the precise role of the CNS midline cells in proper patterning of the ventral neuroectoderm during embryonic neurogenesis. These analyses have shown that the ventral neuroectoderm in the sim mutant fails to carry out its proper formation and characteristic cell division cycle. This results in the loss of the dividing neuroectodermal cells that are located ventral to the CNS midline. The CNS midline cells are also required for the cell cycle-independent expression of the neural and epidermal markers. This indicates that the CNS midline cells are essential for the establishment and maintenance of the ventral epidermal and neuronal cell lineage by cell-cell interaction. Nevertheless, the CNS midline cells do not cause extensive cell death in the ventral neuroectoderm. This study indicates that the CNS midline cells play important roles in the coordination of the proper cell cycle progression and the correct identity determination of the adjacent ventral neuroectoderm along the dorsoventral axis (Chang, 2000).

In order to show that the CNS midline cells control proliferation of the ventral neuroectoderm by activation of stg, stg expression was analyzed in both wild-type and sim mutant embryos. The stg expression profile almost completely matches that of phosphohistone H3 expression. stg expression in the medial, intermediate, and lateral neuroectoderm of wild-type embryos is abolished in sim embryos at stage 10. This indicates that the CNS midline cells promote mitosis of the ventral neuroectoderm by activation of stg expression through cell signaling (Chang, 2000).

To separate cell cycle-independent regulation of the sim gene from the effect on proper cell division in the ventral neuroectoderm, the stg mutant was employed in order to block cell division. The stg mutant is arrested at the G2 phase of cycle 14 since zygotic stg controls the G2/M transition at cell cycle 14. Therefore, analysis of the ventral neuroectodermal marker gene expression in stg and sim;stg double mutants allows one to determine whether sim regulates the cell cycle-independent expression of the genes that determine the identity of the ventral neural and ectodermal cells. The expression of neural (ac, castor, en) and ectodermal (BP28, otd, pnt) markers was analyzed in sim, stg, and sim;stg double mutants. ac gene is expressed in four ventral neuroectodermal clusters in each hemisegment and is successively maintained only in a single NB that is selected from each cluster: MP2, 3-5, 7-1, and 7-4. The expression of ac in S1 NBs is absent in 90% of the examined hemisegments of the sim and of sim;stg double mutant embryos. It is not, however, affected in stg embryos. This observation suggests that the CNS midline provides the ventral neuroectodermal cells with the extrinsic signal(s) that is required for the initial establishment of the ventral neuroectodermal cell fate (Chang, 2000).

Castor is expressed in the S3-S5 NBs 1-2, 2-1, 3-2, 3-3, 3-4, 4-1, 5-1, 5-2, 5-3, 6-1, 7-1, 7-2, and 7-4 of the wild-type embryos at stage 11. In sim embryos, its expression is absent in the medial NBs 1-2, 2-1, 4-1, and 5-1. Castor expression in most of the intermediate and lateral NBs is more severely reduced in the stg mutant than in the sim mutant embryos. This indicates that mitosis is required for the proper expression of Castor in the individual divided NBs. It is maintained in more than 95% of the NBs 2-1, 3-4, 4-1, and 6-1 of the stg mutant embryos. In sim;stg double mutant embryos, the expression of Castor disappears in all the medial NBs 2-1 and 4-1. This result indicates that the CNS midline cells are required for the identity determination of the medial NBs 2-1 and 4-1. It is also demonstrated that mitotic cell division is essential for the proper expression of Castor in order to establish the identity of the NBs 1-2 and 5-1, which undergo several rounds of cell division before Castor expression (Chang, 2000).

This analysis has demonstrated that the expression of neural (ac, castor/ming, en) and epidermal (BP28, otd) markers in the ventral neuroectodermal cells of the stg mutant disappears in the sim;stg double mutant. This indicates that the CNS midline cells also contribute to the establishment of NB identity by inducing the cell cycle-independent expression of NB, neural, and ectodermal marker genes by cell-cell interaction between the CNS midline and the ventral neuroectodermal (Chang, 2000).

Molecular markers for identified neuroblasts in the developing brain of Drosophila

The Drosophila brain develops from the procephalic neurogenic region of the ectoderm. About 100 neural precursor cells (neuroblasts) delaminate from this region on either side in a reproducible spatiotemporal pattern. Neuroblast maps have been prepared from different stages of the early embryo (stages 9, 10 and 11, when the entire population of neuroblasts has formed), in which about 40 molecular markers representing the expression patterns of 34 different genes are linked to individual neuroblasts. In particular, a detailed description is presented of the spatiotemporal patterns of expression in the procephalic neuroectoderm and in the neuroblast layer of the gap genes empty spiracles, hunchback, huckebein, sloppy paired 1 and tailless; the homeotic gene labial; the early eye genes dachshund, eyeless and twin of eyeless; and several other marker genes (including castor, pdm1, fasciclin 2, klumpfuss, ladybird, runt and unplugged). Based on the combination of genes expressed, each brain neuroblast acquires a unique identity, and it is possible to follow the fate of individual neuroblasts through early neurogenesis. Furthermore, despite the highly derived patterns of expression in the procephalic segments, the co-expression of specific molecular markers discloses the existence of serially homologous neuroblasts in neuromeres of the ventral nerve cord and the brain. Taking into consideration that all brain neuroblasts are now assigned to particular neuromeres and individually identified by their unique gene expression, and that the genes found to be expressed are likely candidates for controlling the development of the respective neuroblasts, these data provide a basic framework for studying the mechanisms leading to pattern and cell diversity in the Drosophila brain, and for addressing those mechanisms that make the brain different from the truncal CNS (Urbach, 2003).

In the ventral nerve cord castor (cas), encoding a zinc-finger protein, has been shown to be expressed in 18 NBs per hemineuromere, including early (S1-S2) and late delaminating (S3-S5) NBs, and to be involved in cell fate control within NB lineages. In the procephalon, cas expression is not detectable before stage 10. It is dynamically expressed in the central and dorsal neuroectoderm of the ocular segment, in the median antennal segment, and, by stage 11, in the labral segment, which is surprising since cas is not expressed in the neuroectoderm of the trunk. A proportion of Cas-positive protocerebral and deutocerebral NBs are derive from these domains. Most NBs appear to delaminate from Cas-negative neuroectoderm, and start to express cas at the time of formation, or show a reproducible delay in the onset of cas expression. The latter may already have produced a part of their lineage, which likewise has been proposed for early trunk NBs (e.g. NB7-4). At late stage 11, Cas is expressed in about 60% of the total number of identified brain NBs (Urbach, 2003).

Regulation of temporal identity transitions in Drosophila neuroblasts

Temporal patterning is an important aspect of embryonic development, but the underlying molecular mechanisms are not well understood. Drosophila neuroblasts are an excellent model for studying temporal identity: they sequentially express four genes (hunchback → Krüppel → pdm1 → castor) whose temporal regulation is essential for generating neuronal diversity. hunchback → Krüppel timing is regulated transcriptionally and requires neuroblast cytokinesis, consistent with asymmetric partitioning of transcriptional regulators during neuroblast division or feedback signaling from the neuroblast progeny. Surprisingly, Krüppel → pdm1 → castor timing occurs normally in isolated or G₂-arrested neuroblasts, and thus involves a neuroblast-intrinsic timer. Finally, Hunchback potently regulates the neuroblast temporal identity timer: prolonged Hunchback expression keeps the neuroblast 'young' for multiple divisions, and subsequent downregulation allows resumption of Krüppel → pdm1 → castor expression and the normal neuroblast lineage. It is concluded that two distinct 'timers' regulate neuroblast gene expression: a hunchback → Krüppel timer requiring cytokinesis, and a Krüppel → pdm1 → castor timer which is cell cycle independent (Grosskortenhaus, 2004).

It is concluded that hb is regulated at the transcriptional level in neuroblasts, based on strong correlation with active transcription (intron probe) and protein levels (antibody probe). In addition, hb transcription in GMCs and differentiated neurons, but at this point it cannot be determine if the correlation between protein and transcription is as tight as in the neuroblasts. This does not rule out a role for posttranscriptional regulation, however, to ensure a very short half-life of both hb mRNA and protein. There are predicted miRNA binding sites in the hb 3′UTR and protein degradation (PEST) motifs in the Hb protein that may be necessary to restrict Hb protein to the early portion of neuroblast lineages. There is ample precedent for posttranscriptional regulation of hb in both Drosophila early embryos and C. elegans, but only for translational repression. In Drosophila, Nanos represses hb translation in the early embryo via binding to its 3′UTR. In C. elegans, the hb ortholog hbl-1 regulates temporal identity as part of the heterochronic pathway, and hbl-1 is a target of microRNA regulation through its 3′UTR. It is concluded that precise regulation of hb transcription, coupled with a short half-life of hb mRNA and protein, leads to the observed restriction of Hb protein to the initial cell cycles of neuroblast lineages. Identification of the hb cis-regulatory sequences necessary for proper hb CNS expression has been initiated, and it will be interesting to determine the associated factors that positively and negatively regulate hb transcription in neuroblasts (Grosskortenhaus, 2004).

Cell cycle-arrested neuroblasts maintain hb expression. However, a direct role of the cell cycle (e.g., counting S phases) or an indirect role (e.g., generation of a GMC which could signal back to the neuroblast) has not been distinguished. This study shows that hb transcription is maintained in pebble mutant neuroblasts, which lack cytokinesis but nevertheless go through repeated cell cycles including DNA replication, nuclear envelope breakdown, chromosome condensation, and spindle assembly. Thus, the timely downregulation of hb transcription requires cytokinesis. The requirement for cytokinesis is consistent with two quite different mechanisms: (1) feedback signaling from the GMC to the neuroblast to repress hb transcription, and (2) asymmetric partitioning of an hb transcriptional activator into the GMC to halt hb transcription (Grosskortenhaus, 2004).

Which mechanism is used has not yet been distinguished. Two candidate transcription factors have been tested for a role in hb regulation: Hb and Prospero. The Hb protein does not positively regulate its own transcription in the CNS -- neither hb mRNA nor protein is partitioned into the GMC during neuroblast cell division. The Prospero transcription factor is known to be partitioned into the GMC during neuroblast division, but Prospero protein is cytoplasmic in neuroblasts, and thus unlikely to positively activate hb transcription in this cell type. In addition, misexpression of Prospero in neuroblasts is unable to extend the window of hb transcription and prospero mutants have normal hb expression in neuroblasts, although there is reduced Hb protein in GMCs and neurons by stage 13 and beyond. Thus, Prospero may have a role in maintaining hb transcription in GMCs and neurons, consistent with its nuclear localization in these cell types, but it is not required for timing of hb transcription in neuroblasts (Grosskortenhaus, 2004).

To investigate the role of feedback signaling from the GMC, it would be ideal to do GMC ablations and assay for extended hb transcription in the parental neuroblast, but this experiment is technically very demanding, and even short GMC-neuroblast contact might be enough for the signaling to occur. Whether the feedback signal is mediated by the Notch pathway, which is active in all neuroblasts and GMCs examined to date was tested: blocking the pathway with a sanpodo mutant has no effect on the timing of hb → Kr → pdm1 → cas neuroblast expression. The identification of trans-acting factors that associate with the hb cis-regulatory DNA may be the best approach to distinguish between feedback signaling and transcription factor partitioning mechanisms (Grosskortenhaus, 2004).

Previous work provided strong hints that global extrinsic signals are not required for timing neuroblast temporal identity transitions. (1) Neuroblast lineages are asynchronous, with later-forming neuroblasts expressing hb at the same time adjacent early-forming neuroblasts are expressing cas, making it unlikely that global extrinsic signals trigger gene expression transitions. (2) In vitro culture experiments reported differentiated neuronal clones containing nonoverlapping populations of Hb⁺, Pdm1⁺, and Cas⁺ neurons, consistent with a normal progression of gene expression in the parental neuroblast over time, although gene expression timing was not assayed in neuroblasts. These observations have been confirmed and extended. Isolated neuroblasts progress from Hb⁺ to Kr⁺ to triple negative (presumptive Pdm1⁺) to Cas⁺ over time in culture, and clones in which the GMCs expressed a later gene than the neuroblast (e.g., Hb⁺ or Kr⁺ neuroblasts never had Pdm1⁺ or Cas⁺ GMCs). Thus, Hb → Kr → Pdm1 → Cas neuroblast gene expression timing occurs normally in isolated neuroblasts, demonstrating that lineage-extrinsic factors are not required for neuroblast temporal identity transitions. It is possible that extrinsic cues may still override or entrain an intrinsic program, however, which could be tested by heterochronic neuroblast transplants. In summary, in vitro and in vivo data show that timing of temporal identity transitions is regulated by a neuroblast lineage-intrinsic mechanism. For the latter genes in the cascade, it appears that the mechanism is actually intrinsic to the neuroblast itself (Grosskortenhaus, 2004).

All available data suggest that Kr and Cas timing are regulated at the transcriptional level. Kr mRNA and protein are both detected in neuroblasts during embryonic stage 10 and subsequently maintained in a subset of neurons. Similarly, cas mRNA and protein are both widely detected in neuroblasts only at stage 12, and maintained in a subset of late-born neurons. In the future, it will be important to do mRNA/protein double labels for Kr, pdm1, and cas to determine the extent to which mRNA/protein levels are correlated at the single cell level. Unfortunately, it is not easy to assay for active transcription of Kr or cas due to the lack of large introns (Grosskortenhaus, 2004).

Surprisingly, it was found that cell cycle-arrested neuroblasts that lack Hb still express Kr → Pdm1 → Cas with the same timing as in wild-type embryos. What mechanism might time Kr → Pdm1 → Cas expression in the absence of cell division? Extrinsic cues can be ruled out, because isolated neuroblasts still undergo normal Kr → Pdm1 → Cas gene expression timing. A change in nucleo-cytoplasmic ratio, known to time certain early embryonic events, can be ruled out, because wild-type neuroblasts increase their nucleo-cytoplasmic ratio over time, but G₂-arrested neuroblasts decrease their nucleo-cytoplasmic ratio as they enlarge without dividing (Grosskortenhaus, 2004).

The most attractive model for Kr → Pdm1 → Cas in G₂-arrested neuroblasts is a cascade of transcriptional regulation between Kr, Pdm1, and Cas. Misexpression studies have shown that each gene can activate expression of the next gene in the series, and repress the 'next + 1' gene, which could account for the sequential activation of each gene. If each transcription factor can also repress its activator, similar to the known ability of Cas to negatively regulate pdm1 expression, it could explain the sequential downregulation of each gene as well. Currently, all misexpression data are consistent with this simple model. However, analysis of hb and Kr mutants reveals additional complexity. hb mutants show relatively normal Kr → Pdm1 → Cas timing, and Kr mutants show relatively normal Pdm1 → Cas timing. Thus, there must be at least one unidentified input that can activate Kr in the absence of Hb, and pdm1 in the absence of Kr. Regulation of Hb → Kr → Pdm1 → Cas appears to be primarily at the transcriptional level, and thus identification of the relevant cis-regulatory DNA and associated transcription factors should provide insight into the 'timer' mechanism that controls sequential gene expression in neuroblasts (Grosskortenhaus, 2004).

Hb seems to have a special role in advancing the temporal identity timer. It is the only factor in the cascade whose downregulation requires cytokinesis, and as long as it is present (either because of cell cycle arrest or misexpression) the timer is unable to advance. Misexpression of Hb beyond its normal expression window leads to generation of extra early cell types and blocks Kr → Pdm1 → Cas progression. However, these experiments do not reveal whether Hb generates these early fates by overriding Kr → Pdm1 → Cas neuronal identity while the temporal timer is advancing or if it arrests progress of the temporal timer. The results show that continuous expression of Hb blocks the advancement of the temporal identity timer, keeping the neuroblast in a 'young' state that is fully capable of resuming its normal cell lineage following downregulation of Hb. The ability of Hb to keep the neuroblast in a 'young' multipotent state, despite repeated rounds of cell division, raises the interesting question of how Hb acts at the mechanistic level. Transcriptional targets of Hb in the CNS are so far unknown. A mammalian homolog, Ikaros, is associated with chromatin and remodeling proteins and Drosophila Hb is thought to regulate chromatin-mediated heritable expression of homeotic genes. Thus, Hb might modulate chromatin structure in neuroblasts to prevent expression of later temporal identity genes, or to maintain plasticity of gene expression necessary for maintaining the multipotent state of the neuroblast (Grosskortenhaus, 2004).

Regulation of neuroblast competence: multiple temporal identity factors specify distinct neuronal fates within a single early competence window

Cellular competence is an essential but poorly understood aspect of development. Is competence a general property that affects multiple signaling pathways (e.g., chromatin state), or is competence specific for each signaling pathway (e.g., availability of cofactors)? This study has found that (1) Drosophila neuroblast 7-1 (NB7-1) has a single early window of competence to respond to four different temporal identity genes (Hunchback, Krüppel, Pdm, and Castor); (2) each of these factors specifies distinct motor neuron identities within this competence window but not outside it, and (3) progressive restriction to respond to Hunchback and Krüppel occurs within this window. This work raises the possibility that multiple competence windows may allow the same factors to generate different cell types within the same lineage (Cleary, 2006).

To determine whether NB7-1 undergoes progressive restriction in competence to respond to Kr, similar to that observed for Hb, pulses of Kr were generated at progressively later points in the NB7-1 lineage. Both hsp70-Kr and hsp70-hb were used to allow precise comparison of the effects of both genes. Progressively later pulses of Hb produce a decreasing frequency of U1/U2 neurons. Similarly, progressively later Kr pulses generate decreasing frequencies of extra U3 at each subsequent stage, with the exception of the earliest portion of the lineage, where Hb is known to be dominant to Kr. Thus, NB7-1 shows progressive restriction in competence to respond to both Hb and Kr, and competence to respond to both Hb and Kr is lost at the same point in the lineage (after five divisions) (Cleary, 2006).

An independent method was used to measure the competence window in the NB7-1 lineage. prospero-gal4 was used to induce expression of Kr within the NB7-1 lineage from the fourth division onward. When one copy of UAS-Kr was used at 22°C, which provides relatively low levels of Kr, only five to six Eve⁺ U neurons were observed, mostly U1, U2, and three U3 neurons (91%), but also U1, U2, and four U3 neurons (9%). Thus, NB7-1 loses competence to respond to prolonged Kr expression after five to six cell divisions, similar to results from the Kr pulse experiments described above. Prolonged expression of Hb using the same conditions (prospero-gal4, one copy of UAS-Hb, 22°C) also results in just five to six Eve⁺ U neurons. It is concluded that NB7-1 has a single competence window for generating U1-U3 neurons in response to Hb and Kr (Cleary, 2006).

Next to be tested was whether the later-expressed temporal identity factors Pdm and Cas share the same early competence window with Kr, or if they have distinct competence windows. Pdm specifies the U4 neuronal identity, while Pdm/Cas together specify U5 neuronal identity. scabrous-gal4 was used to prolong Kr expression for a variable length of time within the NB7-1 lineage (two copies of UAS-Kr at 29°C), which delayed but did not prevent the sequential expression of Kr, Pdm, and Cas. This experiment allowed NB7-1 competence to be assayed when presented with Kr, Pdm, or Cas at different times in its lineage (Cleary, 2006).

It was found that the scabrous-gal4 UAS-Kr embryos always had a total number of seven to eight Eve⁺ U neurons, although ectopic U3 neurons ranged from two to six in number. Interestingly, hemisegments with only two ectopic U3 neurons typically had U4/U5 neurons; those with three ectopic U3 neurons had only a U4 neuron, and those with four or more ectopic U3 neurons lacked both U4/U5 neuronal fates. These data are interpreted in the following way: in segments where Kr declines the fastest (fewest ectopic U3 neurons), there is time for Pdm to induce U4 fate and Pdm/Cas to induce U5 fates prior to loss of competence; however, in segments where Kr lasts the longest, both Pdm and Cas expression occur after the competence window and no U4/U5 fates are produced. Taken together, this experiment allows several conclusions to be drawn: (1) prolonged Kr expression can partially extend the neuroblast competence window (from five to six divisions to seven to eight divisions); (2) competence to respond to Kr, Pdm, and Cas is simultaneously lost at the end of this competence window, suggesting that there is a single competence window for responding to multiple temporal identity factors, and (3) each temporal identity factor specifies different U1-U5 motor neuron identities within the competence window, but not outside it. It is currently an open question as to how prolonged expression of one factor (Kr or Hb) can extend the competence window to respond to three distinct factors (Kr, Pdm, and Cas) (Cleary, 2006).

The previous experiment showed that prolonging Kr expression (scabrous-gal4 UAS-Kr) in NB7-1 lineage can only partially extend neuroblast competence. Interestingly, similar experiments prolonging Hb expression (scabrous-gal4 UAS-hb) revealed that the neuroblast maintains full competence for as long as Hb is expressed, in some cases over 15 divisions, with normal U3-U5 fates appearing after Hb levels decline. Thus, extended Hb expression (but not extended Kr expression) can maintain the neuroblast in a young, fully competent state. This raised the possibility that down-regulation of Hb is required for loss of neuroblast competence; alternatively, Hb may be more potent than Kr in maintaining neuroblast competency (Cleary, 2006).

To distinguish these models, the effect was tested of high-level Hb or Kr expression beginning at the fourth neuroblast division (prospero-gal4, 2x UAS-hb or UAS-Kr, 29°C), which would allow Hb down-regulation and permit comparison of the efficacy of Hb versus Kr in extending neuroblast competence. Performing this experiment with Hb resulted in a partial extension of neuroblast competence and the production of an average of 9.1 Eve⁺ U neurons: U1-U3, 6.1 extra U1, and no U4/U5. Performing the experiment with Kr resulted in an almost identical phenotype of 9.8 Eve⁺ U neurons: U1/U2, 7.8 U3s, and no U4/U5. Thus, Hb and Kr appear equally efficient at extending neuroblast competence; this is supported by their equivalent effect when expressed under heat shock or lower level prospero-gal4 control (competence lost after five divisions). More importantly, a comparison of the scabrous-gal4 UAS-hb and prospero-gal4 UAS-hb experiments shows that Hb down-regulation is critical for loss of neuroblast competence. When Hb is maintained from the beginning of the lineage (scabrous-gal4 UAS-hb), competence persists for the length of Hb expression, in some cases over 15 divisions; when Hb down-regulation occurs followed by permanent Hb re-expression one division later (prospero-gal4 UAS-hb), then competence is lost after approximately nine divisions. It is concluded that down-regulation of Hb, but not Kr, initiates progressive restriction in neuroblast competence that is normally complete after five divisions (Cleary, 2006).

Thus far, how neuroblast competence changes over multiple rounds of cell division was investigated. Now, how competence changes during neuronal differentiation is considered. Kr was expressed in high levels in the newborn post-mitotic U1-U5 neurons (eve-gal4 UAS-Kr). In these embryos, Kr is first detected just as the U1-U5 neurons are born. Despite high levels of Kr protein, no change in U1-U5 fate was ever detected. Conversely, transient expression of Kr in NB7-1/GMCs can occasionally generate ectopic U3 neurons that do not maintain Kr expression, despite the ability of Kr to positively autoregulate within the CNS. Thus, mitotic progenitors but not post-mitotic neurons are competent to respond to Kr. Similar results have been observed for competence to respond to Hb (Cleary, 2006).

These experiments, combined with previous studies, allow four major conclusions to be drawn:

1. A single early competence window is used by multiple temporal identity factors. The molecular basis for the early competence window is unknown, but it must be general enough to modulate response to four distinct transcription factors rather than being factor specific. Perhaps loss of competence leads to restricted access of Hb, Kr, Pdm, and Cas to target loci involved in U1-U5 neuronal specification; other loci may remain unaffected, allowing these four transcription factors to induce different cell fates later in the neuroblast lineage. Identifying Hb and Kr target genes, and investigating how or whether they undergo chromatin modifications during the process of progressive restriction will help resolve this question, and may provide insight into the mechanism of progressive restriction in mammalian neural progenitors (Cleary, 2006).
   
2. Each temporal identity factor specifies distinct motor neuron fates within the competence window, but not outside of it. Within the early competence window, each temporal identity factor specifies a unique U1-U5 neuronal identity: high Hb, U1; low Hb, U2; Kr, U3; Pdm, U4; Pdm/Cas, U5. The loss of competence to generate U1-U5 fates may allow each of these transcription factors to be 'reused' later in the NB7-1 lineage to generate different subsets of neurons. This model is supported by the fact that a second round of Kr and Cas neuroblast expression is observed later in embryonic development. These findings suggest that neuroblasts have the potential for cycling through distinct competence windows, and may provide a model for understanding how successive competency states are established (e.g., in vertebrate retinal progenitors) (Cleary, 2006).
   
3. NB7-1 undergoes progressive restriction in competence to respond to both Hb and Kr. Competence to respond to both Hb and Kr is progressively restricted early in the lineage, then completely lost after five divisions of NB7-1. Progressive restriction may be regulated autonomously in the neuroblast or by changing environmental cues, such as inhibitory feedback from GMC or neuronal progeny. A lineage-intrinsic mechanism is favored because different neuroblasts lose competence to respond to Hb at different times (e.g., NB7-1 remains competent to respond to Hb for five divisions, whereas the adjacent NB1-1 is only competent to respond to Hb for two to three divisions). A feedback inhibition model would have parallels with vertebrate retinal progenitors, where differentiated amacrine cells send an inhibitory feedback signal to terminate amacrine cell production. In this case, the signal would likely depend on the number of progeny produced rather than the type of progeny, because loss of competence can occur without production of the last-born neurons in the competence window (U4/U5) (Cleary, 2006).
   
4. Down-regulation of Hb but not Kr initiates progressive restriction and loss of competence. Neuroblast competence is maintained if Hb is expressed from the beginning of the lineage. However, neuroblast competence is not maintained when Kr is expressed from the beginning of the lineage (where Hb is down-regulated normally) or when Hb or Kr are expressed later in the lineage after normal Hb down-regulation. It is proposed that Hb down-regulation initiates progressive restriction in neuroblast competence, ultimately leading to a complete loss of competence (Cleary, 2006).
   

The origin of islet-like cells in Drosophila identifies parallels to the vertebrate endocrine axis

Single-cell resolution lineage information is a critical key to understanding how the states of gene regulatory networks respond to cell interactions and thereby establish distinct cell fates. This study identified a single pair of neural stem cells (neuroblasts) as progenitors of the brain insulin-producing neurosecretory cells of Drosophila, which are homologous to islet β cells. Likewise, a second pair of neuroblasts was identified as progenitors of the neurosecretory Corpora cardiaca cells, which are homologous to the glucagon-secreting islet α cells. Both progenitors originate as neighboring cells from anterior neuroectoderm, which expresses genes orthologous to those expressed in the vertebrate adenohypophyseal placode, the source of endocrine anterior pituitary and neurosecretory hypothalamic cells. This ontogenic-molecular concordance suggests that a rudimentary brain endocrine axis was present in the common ancestor of humans and flies, where it orchestrated the islet-like endocrine functions of insulin and glucagon biology (Wang, 2007).

The principal insulin producing-cells (IPCs) in higher metazoans, such as flies and mammals, direct organismal growth, metabolism, aging, and reproduction via a conserved signal transduction pathway. Gut- or pancreas-based IPCs, with endodermal origin, emerged as the principal IPC locus with the evolution of lower vertebrates such as the jawless fish. In contrast, the principal IPCs of invertebrates are found in the nervous system and are likely of ectodermal origin. Despite this difference, the possibility that gene regulatory modules may be conserved for cell fate programming the principal IPCs of all higher animals, irrespective of germ layer origin, has led the development of islet-like cells to be addressed in Drosophila (Wang, 2007).

Brain IPCs in Drosophila were first recognized by their expression of insulin (Drosophila insulin-like peptide, Dilp2) at the end of embryonic development. The goal of this work was to understand the developmental origin of these cells. The absence of morphological and vital markers for identifying brain neuroblasts for dye-labeled lineage tracing necessitated the combined use of mosaic analysis to demonstrate lineage relationships and immunohistology to follow cell identities. In this study, 16 molecular lineage markers corresponding to conserved genes were used to follow cells in fixed embryos. To identify genes involved in early IPC lineage development, before the differentiation of IPCs, 650 transposable GAL4-transgene insertions, obtained from public collections, that reported gene enhancer activity (GAL4 enhancer traps) in the CNS, were screened. Enhancer-driven GAL4 activity was used to trigger heritable and irreversible lineage labeling, which was assayed for coexpression with Dilp2 in late larval brains, thereby identifying lineage markers and potential developmental determinants. It was found that enhancers near the genes dachshund (dac), eyeless (ey), optix, and tiptop (tio) each triggered IPC lineage labeling by the time of Dilp2 expression onset just before hatching (late-stage 17). tio enhancer-triggered labeling was highly specific to the IPCs within the pars intercerebrallis (PI), the dorsomedial brain region harboring the IPCs and other neurosecretory cells. Antibody staining of Dac, Ey, and Optix proteins recapitulated enhancer reporter labeling and revealed expression in the tio⁺ cell cluster in late-stage embryos just after IPC differentiation, and before IPC differentiation at early-stage 17. Thus, a bilateral cluster of 10-12 Dac⁺ Ey⁺ cells were identified, 6-8 of which expressed tio before continuing on to express insulin (Dilp2) slightly later in development (Wang, 2007).

The hypothesis was tested that the Dac⁺ Ey⁺ cluster is generated by the proliferation of a single neuroblast. The pre-Dilp2 Dac⁺ Ey⁺ cluster comprised 10-12 cells at stage 17, but only a single Dac⁺ cell at stage 12, suggesting that a lineage expanded from a single progenitor beginning at stage 12. The Dac⁺ cluster maintains a posterior and lateral position within the anterior PI, identified by dChx1 expression, which allows following it during the morphogenetic changes in the developing brain. To mark progenitors and their lineage descendants, stage 11-12 embryos harboring both a heat-shock promoter-flip recombinase (hsp70-flp) transgene and an FRT-mediated flip-out Actin promoter-LacZ reporter were heat-shocked to induce random clone marking events in cell lineages. After aging embryos for 6 h at 25°C to reach stage 16-17, marked clusters of clonally related cells were occasionally recovered that comprised the 10-12 cell Dac⁺ Ey⁺ cluster. Clones that partly labeled the Dac⁺ Ey⁺ cluster, which were posterior in the cluster, were interpreted as being labeled by a lineage marking event induced after the neuroblast had divided one or more times. It was unlikely that multiple marking events accounted for the apparent clonal labeling of IPCs because the frequency of marked clone induction was extremely low (tens per brain). Clones were also found that labeled neighboring cells, but do not label Dac⁺ Ey⁺ cells, suggesting there is a lineage restriction that defined the Dac⁺ Ey⁺ cluster. Thus, all data are consistent with a lineage model whereby one neuroblast produced 10-12 Dac⁺ Ey⁺ cells, 6-8 of which were IPCs (Wang, 2007).

Whether the single Dac⁺ cell progenitor of IPCs seen at stage 12 was indeed a neuroblast was further tested by using markers of neuroblast lineage development. Asymmetrically dividing neuroblasts can be identified by nuclear expression of the pan-neuroblast marker Deadpan (Dpn) and Prospero (Pros) localization to the plasma membrane. It was found that the single Dac⁺ cell expressed Dpn and also showed Pros localization at the plasma membrane, which indicated that it was a neuroblast. As the Dac⁺ cluster increased in cell number with age, it was found that Pros was present in the nucleus of Dac⁺ cells anterior to the Dac⁺ neuroblast, which indicated that these were the neuroblast daughter cells, or ganglion mother cells (GMCs) generated by asymmetric neuroblast divisions. By stage 14, the most anterior Dac⁺ cells in the cluster lacked Dpn and Pros, suggesting that they were early, undifferentiated neurons or neurosecretory cells generated by GMC cell divisions. It was also found that tio expression occurs in the most anterior Dac⁺ cells of the lineage group, furthest from the posterior-located Dac⁺ neuroblast, suggesting that the six to eight IPCs are the products of the first three to four GMCs to be generated by asymmetric neuroblast division. This observation confirmed the interpretation of the marked clone data that showed partial labeling by a clone occupies the posterior, more recently formed region of the Dac⁺ Ey⁺ cluster, near the IPC neuroblast. Thus, a histological pattern of cell identities and divisions within the Dac⁺ IPC lineage group was observed that was consistent with the generic lineage development of a single neuroblast, with the IPCs being produced from the first three to four GMCs formed (Wang, 2007).

Further attempts were made to identify the precise origin of the IPC neuroblast within the neuroectoderm epithelium and the blastoderm embryo to place this lineage in the context of early axial patterning. The IPC neuroblast was first recognized by Dac expression only after neuroblast formation, but before its first division. However, preceding the formation of the IPC neuroblast, the markers Castor (Cas) and dChx1 and the proneural factor Lethal of Scute (L'Sc) showed coexpression in eight nearby cells of the neuroectoderm epithelium. Cas and dChx1 were maintained in all neuroblast lineages that delaminated from this group, as indicated by coexpression of Dpn. The IPC neuroblast was the only neuroblast from this group to express Dac, and it was always the first Dpn⁺ neuroblast to delaminate, becoming the most posterior in a chain of delaminating Cas⁺ dChx1⁺ neuroblasts. The Cas⁺ dChx1⁺ L'Sc⁺ proneural group lies within a 'gap gene' head stripe corresponding to the Bicoid responsive giant head stripe 1 (gt1), which suggested that the IPC neuroblast, or its earliest progenitor, arose from this pattern element of the precellular blastoderm (Wang, 2007).

β Cell and α cell development in mammals shares a largely common pathway. Thus attempts were made to study the origin of the α-like cells in Drosophila and their development relative to the IPC lineage. Corpora cardiaca (CC) cells are analogous in function to islet α cells. These neuroendocrine cells reside in the endocrine ring gland, just dorsal to the brain. CC cells produce and secrete a glucagon-like peptide, adipokinetic hormone, in response to circulating glucose levels, via a conserved K_atp sensor. The gene glass (gl) is a marker of CC cells and their precursors that specifically labels the CC lineage beginning at stage 10. The Gl⁺ group of cells expands in number to form a bilateral pair of six to eight cell clusters, aligned at the border of the brain and the developing foregut (stage 13). The Gl⁺ clusters then migrated out of the protocerebrum (stage 14), and posterior along the roof of the pharynx, to ultimately coalesce at the midline within the prospective ring gland (stage 16). Remarkably, the first Gl⁺ cells appeared a single cell diameter apart from the dChx1⁺ cluster containing the IPC neuroblast, also within the gt1 stripe (Wang, 2007).

These results suggested that the CC cell lineage, like the IPC lineage, is also generated from a progenitor within the gt1⁺ dorsal neuroectoderm. Indeed, a neuroblast progenitor for CC cells was suggested by expression of a Kruppel reporter (Kr-GFP) found to specifically label the Gl⁺ cells and an adjacent cell that both was Dpn⁺ and showed membrane localized Pros, indicating that it was a neuroblast. As for IPCs, tests were made to see if CC cells are derived from a single progenitor, perhaps the Kr-GFP⁺ neuroblast. Gl⁺ β-gal⁺-marked clones were recovered that comprised all or part of a CC cell cluster, after their migration to the prospective ring gland at stage 16. Because labeled CC cells had moved from their point of origin in the developing PI, it could not be determine whether a progenitor also produced other cells besides the CC cells, which did not similarly migrate. Together, these observations suggest that the CC cells are related by lineage to a neuroblast progenitor (Wang, 2007).

Typically, neuroblasts inherit the expression of cell specification factors from their point of origin in the patterned neuroectoderm before the neuroblast forms. It was found that this was the case with the IPC neuroblast, which retains dChx1 and Cas expression from the neuroectoderm. It was therefore hypothesized that this may also be the case for the CC cell neuroblast. CC cell specification was shown to require the function of gt, sine oculis (so), twist (twi), and snail (sna). Indeed, it was found that all of these factors are expressed in the Gl⁺ CC cell lineage. Moreover, the Kr-GFP⁺ cell group, containing the neuroblast and CC cell precursors, also expressed Eyes absent (Eya), the cognate protein tyrosine phosphatase of So. It was subsequently found that at stage 10, the time that Gl⁺ cells are first detected, a region of gt1⁺ neurectoderm shows expression of So. It was also found that one to two So⁺ gt1⁺ neuroblasts can be detected by labeling with Dpn at this stage. Thus, it is proposed that the So⁺ Eya⁺ gt1⁺ neuroectoderm gives rise to the Kr-GFP⁺ So⁺ Eya⁺ gt1⁺ neuroblast, which is the single progenitor of the CC cells (Wang, 2007).

The model of a dorsal neurectoderm origin for CC cells is in disagreement with another extant model. The anterior ventral furrow (AVF) epithelium was suggested to be the CC cell origin based on gene expression and function studies implicating So, Gt, Twi, and Sna in CC cell formation. To distinguish between the AVF and dorsal neuroectoderm as possible origins of CC cells, two newly available gt promoter fragment reporters were used whose expression persists late enough in development, beyond endogenous protein and transcript expression, to serve as a coarse-grain lineage marker of CC cells. The AVF is marked by the gt23 reporter, whose expression is limited to the two gt head stripes posterior to gt1 at the blastoderm stage. This reporter does not label the Gl⁺ cells. However, as has been shown, the Gl⁺ cells arise in the context of the most anterior gt head stripe, gt1, which reaffirms the proposed origin from the gt1⁺ neuroectoderm (Wang, 2007).

The organization of this gt1⁺ segment-derived proendocrine neuroectoderm was investigated with respect to the conserved factors Optix, So, Eya, and dChx1. Optix and Eya expression aligned with the gt1 reporter expression domain. The D-six4 gene also shows expression specific to this domain. Labeling studies showed that this domain is subdivided into several small compartments of 2-12 cells with discrete gene expression profiles. The data indicate that the IPC neuroblast was derived from compartment B (Optix⁺, dChx1⁺, Cas⁺, So^-, low-level Eya) and the CC cell neuroblast arose from the adjacent compartment C (Optix⁺, So⁺, Eya⁺, dChx1^-). This somewhat surprising finding suggests that the largely common developmental pathway of β and α cells may be partly conserved in Drosophila, perhaps with respect to a domain of Sine oculis/Six family and Eya gene expression (Wang, 2007).

The early expression of the mouse ortholog of the Drosophila homeodomain gene optix, Six6, demarcates the hypophyseal placode and infundibular region, which give rise to the anterior pituitary and neurosecretory hypothalamus, respectively. Mutation of the Six6 gene leads to reduction of the pituitary in mice and humans. The hypophyseal placode and adjacent ectoderm also expresses the other so-called 'placode genes,' Six1, Six4, and Eya, and this coexpression pattern is conserved in amphibians, fish, and lower chordates such as ascidians. In mice, the anterior pituitary is reduced in size in the double mutant of Eya1 and Six1, and in zebrafish, Eya1 is essential for differentiation of all pituitary cell types except for prolactin-expressing cells. In Drosophila, So and Eya are essential for CC cell formation. Thus, there is a striking conservation of the molecular signature of tissues that give rise to elements of the brain endocrine axis in flies, mammals, lower vertebrates, and lower chordates (Wang, 2007).

There are also parallels between vertebrate and fly with respect to tissue morphogenesis within the developing brain endocrine system and adjacent oral ectoderm, although there appears to be considerable variation on a general theme. For example, in mouse, the progenitors of the anterior pituitary and neurosecretory hypothalamus appear to arise respectively from Rathke's pouch, an invagination of the oral ectoderm, and the neurectoderm, which do not start as neighboring regions, but come into direct contact only after neurulation. However, in the zebrafish, which does not form a Rathke's pouch, the progenitors of the anterior pituitary and neurosecretory hypothalamic cells (GnRH1⁺) arise from neighboring regions of the hypohyseal placode, which is situated directly dorsal to the stomodeal ectoderm. In Drosophila, the ventral cells of the gt1⁺ Optix⁺ Eya⁺ ectoderm invaginate to form the roof of the pharynx, the fly's oral ectoderm, whereas the dorsal cells contribute to the endocrine axis. Therefore, there is considerable evidence for evolutionarily conservation of the close relationship between the oral ectoderm and the developing compartments of the endocrine axis, all of which express the hypophyseal placode genes. The gene expression profile and specification of endocrine cell functions from the anterior ectoderm appears to be more 'fixed' across the bilateria, whereas the pattern of accompanying tissue morphogenesis and diversity of cell types is more variable, just as has been demonstrated for the specification of the bilaterian CNS, eye, gut, and heart (Wang, 2007).

The model proposed in this study contrasts with the prior suggestion, based on the proximity of developing CC cells to the posterior foregut in the moth, Manduca, that CC cells originate from neurogenic placodes of the foregut that engender the stomatogastric nervous system. Because CC cell progenitors were not identified in those studies, and subsequent mutational analysis in Drosophila demonstrated that the CC cells develop independently of the stomatogastric nervous system and posterior foregut, it is suggested that the current model of CC cell origin is the most strongly supported (Wang, 2007).

It is proposed that the brain endocrine systems of invertebrates and vertebrates are derived from a common ancestry because they both develop from a domain of Eya and sine oculis/Six family gene expression that comprises the anterior neuroectoderm and adjacent oral ectoderm. Indeed, these results extend prior observations that the neurosecretory cells of the PI and ring gland show other aspects of homology to the hypothalamic-pituitary axis. The specification of islet-like cells within a conserved brain endocrine axis raises the intriguing possibility that islet organogenesis, which is a derived feature of vertebrates, may have coopted brain endocrine cis-regulatory modules for specification of islet fates in endoderm. Indeed, the ectopic expression of the nominal rat insulin promoter reporter in anterior pituitary and hypothalamus underscores the similar gene regulatory state of these endocrine tissues. It is expected that further genetic analysis of endocrine cell fate specification within the gt1 domain of Drosophila will lead to insights into the patterning and organogenesis of endocrine compartments and provide the basis for identifying conserved pan-IPC regulatory modules with relevance to mammalian systems (Wang, 2007).

Effects of Mutation or Deletion

Loss of cas/ming function results in precise alterations in CNS gene expression, defects in axonogenesis and embryonic lethality. In cas/ ming mutants posterior commissures have only half the diameter of those in wild type embryos. One Fasciculin III positive fascicle is missing, and engrailed expression in the CNS is abnormal (Cui, 1992 and Mellerick, 1992).

CAS/Ming regulates late neuroblast development. Two observations support this notion. Consider first that Engrailed is a neuroblast marker, involved in directing neuroblast fate. Early engrailed expression appears to be normal in cas/ming mutants. Second, there is only a partial disruption of axonal tracts in cas/ming mutants and an absence of neuroblasts generated late in neurogenesis (Mellerick, 1992).

castor encodes a zinc finger protein expressed in a subset of Drosophila embryonic neuroglioblasts where it controls neuronal differentiation. cas is expressed at larval and pupal stages in brain cell clusters where it participates in the elaboration of the adult structures. In particular using the MARCM system (mosaic analysis with a repressible cell marker), it has been shown that cas is required postembryonically for correct axon pathfinding of the central complex (CX) and mushroom body (MB) neurons. The derailed gene, alternatively termed linotte (lio) in this study, encodes a transmembrane protein expressed at larval/pupal stage in a glial structure, the TIFR, and interacts with the no-bridge (nob) gene. cas interacts genetically with derailed and nob. These interactions do not involve direct transcription regulation but probably cellular communication processes (Hitier, 2001).

Derailed/Linotte is expressed at the embryonic stage in neurons of the VNC and of the procephalic region, and in the late third instar larvae in a glial transient interhemispheric fibrous ring (TIFR) that persists at the early pupal stages and disappears before adulthood. drl null mutants are viable and drl has been implicated in axon pathway selection in the embryonic VNC, and in adult brain development at metamorphosis. The no-bridge mutant, which exhibits adult brain defects, interacts with drl via the TIFR. Using a genetic screen designed to isolate mutations interacting with drl from a collection of Gal4 lines, a new hypomorphic cas allele (cas³⁹²¹) has been identified. Cas protein is expressed in larval and pupal brain in cell clusters. Analysis of mutant clones generated with the MARCM method demonstrates that cas expression is required during larval life to control axonal outgrowth in CX and MB neurons. Although single mutants show only weak brain defects, double mutants lio;cas³⁹²¹ and lio^drlP;cas³⁹²¹ exhibited strong defects in MB and CX indicating that drl and cas interact to build up the adult brain. cas expressing cells are disorganized in the third instar larva brain of drl mutants, whereas no defect is detected in drl embryos. Moreover, nob also interacts genetically with cas. Altogether these data indicate that cas is involved in postembryonic brain development where it interacts with drl and nob, these interactions probably involve cell/cell communication (Hitier, 2001).

Anti-Cas antibodies have revealed that the Cas protein is present in the CNS during larval and pupal stages confirming the cas³⁹²¹ enhancer trap expression pattern. In larva, cas was found expressed in disseminated cells on the ventral side of the VNC. In the dorsal part of the larval brain, cas is found expressed in five linearly organized cells clusters on both sides of the interhemispheric junction. Twenty-four hours after pupariation, expression progressively disappears and no clear signal is detected in the adult brain. The cas³⁹²¹ line led to adult expression in a subset of ellipsoid body and fan-shaped body fibers, and in the pars intercerebralis. Since the enhancer trap expression of cas³⁹²¹ is very similar to that of Cas expression during embryonic, larval and pupal stages, it is speculated that the adult CX expression displayed by cas³⁹²¹ actually reflects cas expression. The stability of Gal4 and ß-gal protein might allow detection in the adult where Cas might be present at weak level. Alternatively the adult Cas product might not be recognized by the antibody because the protein is modified. Cas expression appears normal in the cas³⁹²¹ mutant, confirming that cas³⁹²¹ is a weak hypomorph allele (Hitier, 2001).

Since viable cas mutants are hypomorphic, to fully assess the postembryonic role played by cas in adult brain development the MARCM method was used in combination with a cas null mutation and the UAS-cd8-GFP reporter. Clones were analyzed in paraffin section with anti-GFP antibody rather than with confocal microscopy in whole amount preparations to allow for the detection of non-autonomous effect of the mutant clones on adjacent brain structures. Mutant cas clones induced during larval stages lead to EB and MB defects, indicating that the postembryonic cas expression is required for CX and MB development. No obvious defects are observed with cas^- clones in the lobula, the medulla and the antennal lobes, all regions, where no cas expression is detected. Multi-cellular clones were observed in the CX and in the MBs, indicating that the cas null mutation is not lethal for neuroblasts or ganglion mother cells. Nevertheless, large cas^- clones in the central brain lead to the death of the individual. This probably prevents the observation of wider brain defects. In particular this could explain why the cas clone experiment did not lead to the defect observed in the brain of cas³⁹²¹/cas²⁹⁰ individuals. When only a small subset of EB neurons are mutant for cas, neither cas^- fibers nor the EB complete structure exhibit any obvious defects. However when larger cas^- clones occur in neurons presumably identified as large field R2 or R4 neurons, the EB exhibits a ventral cleft. Interestingly the defect also affects EB neurons that are not mutant for cas, showing that cas is locally non-autonomous. In the MBs non-autonomous defects are also observed: when a subset of ß or ß' neurons lack the Cas product, MBs exhibit a severe fusion of ß and/or ß' lobes. The fusion comprises cas^- fibers but also cas⁺ fibers. Moreover, although cas clones occurring in gamma neurons do not lead to any obvious intrinsic defects, they nevertheless induce cas⁺ ß lobes to fuse. Since gamma lobes differentiate before ß lobes, one can hypothesize that cas controls the expression of a gamma lobe signal that guides ß fibers during pupal differentiation. Alternatively, since a Elav-gal4 driver was used to detect cas clones, only the neuronal component of clones was observed (Hitier, 2001).

The possibility cannot be excluded that glial cas^- cells are responsible for cas⁺ fibers misrooting. This idea is supported by the fact that during embryogenesis Cas is expressed in midline glial precursor cells (Hitier, 2001 and references therein).

cas is shown to interact genetically with drl to build up the adult brain and drl is required for the correct organization of cas cell clusters. Neither drl nor cas control the expression of the other gene, and neither mutation affects the correct development of the cells expressing the second gene during embryogenesis. However, subtle defects might have escaped analysis. At the third instar larvae the situation is different. The five clusters of Cas positive cells linearly organize in a wild-type context but appear disorganized in derailed mutants. Cluster positioning is disturbed and some clusters appeared 'fused' together. Analysis of cas and drl expression in the double mutant third instar larvae CNS suggests that drl and cas are not expressed in the same cells. In particular cas expressing fibers in the central brain did not include the TIFR where drl is expressed. These results suggest that the drl/cas post embryonic interaction does not involve direct transcriptional regulation but rather cellular interactions, between cas expressing clusters and interhemispheric glial cells expressing Drl (Hitier, 2001).

Pdm and Castor specify late-born motor neuron identity in the NB7-1 lineage

Embryonic development requires generating cell types at the right place (spatial patterning) and the right time (temporal patterning). Drosophila neuroblasts undergo stem cell-like divisions to generate an ordered sequence of neuronal progeny, making them an attractive system to study temporal patterning. Embryonic neuroblasts sequentially express Hunchback, Krüppel, Pdm1/Pdm2 (Pdm), and Castor (Cas) transcription factors. This study shows that Pdm and Cas regulate late-born motor neuron identity within the NB7-1 lineage: Pdm specifies fourth-born U4 motor neuron identity, while Pdm/Cas together specify fifth-born U5 motor neuron identity. It is concluded that Pdm and Cas specify late-born neuronal identity; that Pdm and Cas act combinatorially to specify a temporal identity distinct from either protein alone, and that Cas repression of pdm expression regulates the generation of neuronal diversity (Grosskortenhaus, 2006).

This study shows that Pdm and Cas are required for specifying the late-born U4 and U5 neuron fates within the NB7-1 lineage. Thus, all four transcription factors are required to specify sequential temporal identities within the NB7-1 lineage: High Hb gives U1 fate, low Hb gives U2 fate, Kr gives U3 fate, Pdm gives U4 fate, and Pdm/Cas gives U5 fate. Cas is then transiently expressed in the lineage during the window of interneuron production, and it remains possible that Cas alone specifies one or more interneuron identities later in the lineage (Grosskortenhaus, 2006).

Pdm is detected in neuroblasts during the window that NB7-1 is generating the GMC progenitors of the U4/U5 neurons, and pdm mutant embryos lack the U4/U5 neurons. What happens in the NB7-1 lineage following production of the U3 progenitor in pdm mutant embryos? It is unlikely that NB7-1 dies or is cell cycle arrested, because Cas+ neuroblasts can be observed well after the time of pdm expression. It is more likely that the U4/U5 neurons undergo cell death or that NB7-1 'skips' production of U4/U5 neurons and goes directly to the interneuron phase of its lineage. Independent of the mechanism used, it is clear that Pdm is required for the proper development of the late-born U4 and U5 neurons (Grosskortenhaus, 2006).

If Pdm is required for both U4 and U5 fates, what distinguishes these neuronal identities? The Cas transcription factor is detected in U5 but not U4, leading to a model in which Pdm alone specifies U4 identity and Pdm/Cas specifies U5 identity. The data fully support such a model. First, cas mutant embryos have an extended window of Pdm only expression, and the formation of supernumerary U4 neurons is observed during this window of Pdm expression. Furthermore, pdm cas double mutants lack these ectopic U4 neurons, showing that the extended window of Pdm is required for specifying the ectopic U4 neurons. These data provide strong support for the conclusion that Pdm without Cas specifies U4 neuronal identity (Grosskortenhaus, 2006).

If Pdm specifies U4 neuron identity, then why are ectopic U4 neurons not observed following Pdm misexpression? The answer to this apparent paradox is that Pdm misexpression induces Cas expression, resulting in the Pdm/Cas double-positive state that specifies U5 identity. Precocious expression of Pdm also results in repression of Kr, and the occasional loss of the U3 neuron. Finally, misexpression of Pdm can result in the absence of U5 at very low frequency. One possible explanation is that in these segments, Pdm induces sufficiently high levels of Cas to trigger production of the Cas+ interneuron identity that normally occurs after U5 production (Grosskortenhaus, 2006).

The proposal that Pdm and Cas together specify U5 neuronal identity is supported by several observations: (1) Both pdm and cas single mutants lack U5 neuronal identity; (2) misexpression of Pdm can extend the window of Pdm/Cas coexpression and generate ectopic U5 neurons; (3) misexpression of Pdm in a cas mutant background generates U4 neurons but not U5 neurons; and (4) misexpression of Pdm and Cas together results in ectopic U5 neurons. How might the combination of Pdm and Cas specify a unique neuronal identity, different from either factor alone? It is possible that Pdm and Cas form a heterodimer with a different set of target genes than either factor alone; POU domain proteins such as Pdm are known to heterodimerize with a wide range of transcription factors, including zinc finger transcription factors. However, there are no reported Pdm1/Cas or Pdm2/Cas interactions in a genome-wide yeast two-hybrid screen, and it has not been possible to coimmunoprecipitate HA:Pdm2/Cas after co-overexpression. It is also possible that genes differ in the composition of Pdm- and Cas-binding sites, some genes having sites for Pdm, others having Cas sites, and yet others having coclusters of both sites. Testing this hypothesis using bioinformatics is currently not possible due to the low information content of the Pdm DNA-binding motif (Neumann and Cohen 1998) (Grosskortenhaus, 2006).

It is clear that Pdm and Cas specify late-born U4/U5 motor neuron fates within the NB7-1 lineage. If they specify late-born neuronal fates in other lineages, they would be temporal identity genes; if they only have this function in the NB7-1 lineage, then they would be better defined as having a U4/U5 cell type specification function. Currently, not enough information exists to distinguish these two possibilities. Besides NB7-1, the only other neuroblast lineage where birth-order data exists is NB7-3, but that lineage is short -- just three GMCs -- and it does not express cas. In the future, it will be important to determine birth-order relationships in additional embryonic neuroblast lineages, and then test Pdm and Cas for a role in specifying late-born neuronal identity in these lineages. Pdm is known to specify the first-born GMC in the NB4-2 lineage, which shows Pdm is not restricted to specifying late-born temporal identity, but this does not preclude it from specifying late-born cell fates in NB4-2 or other neuroblast lineages (Grosskortenhaus, 2006).

NB7-1 has the longest embryonic lineage of any neuroblast, producing ~40 neurons (U1-U5 motor neurons, five U siblings, and 30 interneurons). It has been shown that pulses of low levels of Hb or Kr can induce one to three extra Eve+ early-born neurons only during the first five cell cycles of the NB7-1 lineage, and misexpression of high levels of Hb or Kr can generate an average of only 4.1 and 4.8 extra Eve+ U neurons, respectively. Thus, NB7-1 has a single early competence window to respond to Hb and Kr. Interestingly, in this study it was found that high levels of Pdm or Pdm/Cas also induced approximately four extra Eve+ U neurons. Thus, NB7-1 may lose competence to respond to all four temporal identity factors at the same time - after nine to 10 cell cycles. These data support the conclusion that NB7-1 has a single competence window for all four temporal identity factors. Alternatively, Pdm or Pdm/Cas may induce levels of Cas that exceed a threshold for inducing Eve minus interneuron identity (Grosskortenhaus, 2006).

Prolonged Pdm or Pdm/Cas coexpression generates more U4 or U5 neurons in thoracic segments than in abdominal segments. One explanation might be the effect of homeotic gene expression on the NB7-1 lineage. Homeotic genes are known to regulate the length of neuroblast lineages, the type of neurons generated within neuroblast lineages, and the timing of neuroblast apoptosis. Thus, it is possible that homeotic genes also regulate the ability of Pdm or Cas to induce late-born neuronal identity, the length of the competence window, or the survival/proliferation state of NB7-1 (Grosskortenhaus, 2006).

The results provide new information on the "gene expression timer" that regulates sequential hb, Kr, pdm, and cas expression in embryonic neuroblasts. Previous studies showed that loss of Hb or Kr in neuroblasts did not significantly alter the timing of hb, Kr, pdm or cas neuroblast expression; the current study confirms and extends these conclusions. pdm or cas mutants have no effect on the timing of transcriptional initiation of hb, Kr, pdm, or cas within neuroblast lineages. Thus, expression of hb, Kr, pdm, cas must be induced by one or more unknown transcriptional activators. This highlights the importance of identifying the relevant cis-regulatory region controlling the timing of hb, Kr, pdm, and cas expression, and characterizing the trans-acting factors that initiate temporally accurate neuroblast gene expression (Grosskortenhaus, 2006).

Although mutant analysis reveals the presence of unknown transcriptional activators of hb, Kr, pdm, and cas neuroblast expression, misexpression experiments reveal regulatory interactions between each of these genes. Each temporal identity factor is capable of activating transcription of the next gene in the pathway. The ability of each transcription factor to activate the next gene in the cascade may act redundantly with the unknown transcriptional activators to maintain the linear cascade of gene expression. In addition, many repressive interactions occur between the temporal identity genes, which may serve to maintain distinct temporal windows of expression. It is unknown whether these regulatory interactions are direct or indirect; this is a question actively being investigated (Grosskortenhaus, 2006).

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date revised: 15 July 2008

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