castor


DEVELOPMENTAL BIOLOGY

Embryonic

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 Katp 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).

Identity, origin, and migration of peripheral glial cells in the Drosophila embryo

Glial cells are crucial for the proper development and function of the nervous system. In the Drosophila embryo, the glial cells of the peripheral nervous system are generated both by central neuroblasts and sensory organ precursors. Most peripheral glial cells need to migrate along axonal projections of motor and sensory neurons to reach their final positions in the periphery. This paper studied the spatial and temporal pattern, the identity, the migration, and the origin of all peripheral glial cells in the truncal segments of wildtype embryos. The establishment of individual identities among these cells is reflected by the expression of a combinatorial code of molecular markers. This allows the identification of individual cells in various genetic backgrounds. Furthermore, mutant analysis of two of these marker genes, spalt major and castor, reveal their implication in peripheral glial development. Using confocal 4D microscopy to monitor and follow peripheral glia migration in living embryos, it was shown that the positioning of most of these cells is predetermined with minor variations, and that the order in which cells migrate into the periphery is almost fixed. By studying their lineages, the origin of each of the peripheral glial cells was uncovered and they were linked to identified central and peripheral neural stem cells (von Hilchen, 2008).

This study has characterized the expression of a collection of cell-specific molecular markers, which allows to identify and distinguish all glial cells in the embryonic peripheral nervous system. The reproducibility with which enhancer-trap lines and marker genes are expressed in the individual peripheral glial cells, indicates that these cells display unique identities. Furthermore, the spatial and temporal pattern of migration and the final arrangement of these cells are relatively stereotypic. This suggests that the specification of the unique identity of each cell does not only define a specific combination of genes to be expressed, but also includes the information about the timing of migration, the nerve tract it is associated with, and to some degree the final position to be occupied along the respective nerve. How the cell receives this information is still unknown. The individual characteristics could be determined (1) by lineage or (2) during migration by cell-cell interactions (between the glial cells or between the glia and other closely associated cells, e.g. neurons, tracheae), or (3) by a combination of both (von Hilchen, 2008).

The master regulatory gene glial cells missing (gcm) is required to induce the glial cell fate. Gcm as a transcription factor switches on downstream target genes, of which the gene encoding for the homeobox transcription factor Reversed polarity (Repo) is well described. As this cascade of gene activation is required for all glial cells in the Drosophila embryo (except the midline glia), it is unlikely to contribute to cell fate diversification among the glia. Whereas central glial cells migrate over rather short distances, in literally any possible direction, to finally occupy stereotypic positions within the CNS, the peripheral glial cells behave differently as they have to migrate over remarkable distances into the periphery. It has been recently shown that the migration of PGs depends on Notch signalling. In Notch mutants or in mutants where Notch signalling is altered in PGs, the migration is impaired or even completely blocked. However, this signalling does not appear to supply the cells with characteristics of their fate apart from the onset and/or maintenance of the migration itself. Sepp (2000) described the developmental dynamics and morphology of a subset of peripheral glial cells and could show that a signalling cascade mediated by the small GTPases RhoA and Rac1 influences the actin cytoskeleton of migrating PGs. Sepp further showed, that, within the analysed population of cells, a 'leading glia’ expresses filopodia-like structures whereas the ‘follower’ cells do not. Similar results were reported by Aigouy (2004). Aigouy established a 4D microscopy technique to record and analyse the developmental dynamics and migratory behaviour of PNS glia during pupal stages in the developing fly wing. In this system, differences between 'leading' and 'follower' glia cells were also observed. The glial cells in the wing PNS migrate along wing veins in a chain with one 'leading' cell in front. If this chain is interrupted by laser ablation of either the leading or intermediate cells, a new 'leading’ cell starts to form filopodia and explores the surrounding. Once this new 'leading' cell catches up with the previous chain or reaches its target area, the filopodia disappear and the cells' morphology changes again. Hence, these differences in glial cell morphology and behaviour in the wing PNS are based on interactions of the glial cells with each other rather than on a predetermined intrinsic cell fate (von Hilchen, 2008).

Findings for the embryonic PNS glia suggest that these cells are predetermined at least to a certain extent. The 4D microscopy approach allowed tracing of the migration of individually identified PGs in living embryos from the moment they leave the CNS until they reach their final position. Apart from the dorsal SOP-derived cells, which never change their position or behaviour, it is always the ePG9 that leaves the CNS first and 'leads' the track. This cell expresses filopodia-like structures, while the following cells do not, although it remains to be experimentally shown whether they can take over the leading function in the absence of ePG9. It is worth mentioning that the SOP-derived ePG12 migrates along trajectories of the ISN prior to ePG9. It is not clear whether ePG12 has any leading function for ePG migration or functions as a guidepost cell for axonal projections. It is the only cell, though, that swaps nerve tracts and finally associates with the TN. Most likely, cell-cell communication between ePG12 and axonal projections and/or neighbouring cells is required for proper pathfinding and positioning. It is always the ePG4 that migrates along and finally enwraps the segmental nerve. As this cell is the only cell associated with the distal part of the segmental nerve, it functions as 'leading' glia for this nerve and expresses filopodia-like structures at least in later stages when it enwraps the SN. This enwrapment occurs in a bidirectional fashion, i.e. the filopodia occur at both ends of the glial cell (von Hilchen, 2008).

Lineage analysis revealed that the PGs mentioned above originate from the CNS neuroblast NB 1-3 and a ventrally located SOP. Interestingly, the two NB 2-5 derived PGs (ePG6 and ePG8) differ from these cells with respect to both identity and behaviour. They express fewer of the analysed PG-specific markers (cas-Gal4 and mirr-lacZ) and it is not possible to distinguish between these two cells so far. Whether the lack of identifying markers is a consequence of or a prerequisite for their different identity and behaviour is not yet clear. The cells migrate along the ISN independently of the NB 1-3- and SOP-derived PGs and frequently overtake them (and occasionally even one another). The correlation of such characteristics with the different origin of these three subpopulations of PGs lends support to the hypothesis that some aspects of cell fate diversification among the PGs may be predetermined by lineage. It is likely, that such predetermined characteristics include the competence to respond to specific external signals that guide the respective cell along the correct nerve to its target position (von Hilchen, 2008).

One incidence for lineage-dependent cell fate determination comes from the analysis of the ladybird homeobox genes. The ladybird genes are expressed in the developing CNS in only few NBs including NB 5-6. The NB 5-6 lineage produces one of the proximal PGs (ePG2) which expresses the Ladybird early (Lbe) protein. It has been shown that a loss of ladybird gene function results in a loss of the ePG2 in a third of all analysed hemisegments, accompanied with a higher number of medially located glial cells in the CNS. An opposite phenotype with excessive cells in the transition zone was observed by ectopic expression of the ladybird genes throughout the CNS. Using an anti-Repo antibody as well as a subset specific reporter transgene (K-lacZ), De Graeve (2004) provided evidence suggesting that the ladybird genes play a role in glial subtype specification in particular NB lineages. Another factor shown to be required for the specification of a lineage-specific set of glial cells (NB1-1-derived subperineurial glia) is Huckebein, which interacts with Gcm to amplify its expression specifically in these cells (von Hilchen, 2008).

Furthermore, in cas mutants, it was shown that the two NB 2-5-derived glia (ePG6 and ePG8) do not migrate into the periphery but most likely stay at their place of birth, although they acquire glial cell fate (as can be deduced from Repo stainings). Thus, similar to Ladybird and Huckebein, Cas seems to be involved in lineage-dependent glial subtype specification rather than determination of glial fate in general. In contrast to ladybird (De Graeve, 2004), though, Cas is not sufficient to ectopically induce glial cell fate or PG subtype specification (von Hilchen, 2008).

This study shows that salm is a likely candidate participating in the control of glial development. Embryos homozygous for salm445 show a pleiotropic and variable phenotype affecting not only glial cells but also PNS neurons, sensory organs, and other tissues. Yet, nearly all ventrally derived PGs stall in the transition zone between CNS and PNS and do not migrate properly into the periphery. In about 50% of the analysed hemisegments, a variable number of one to three PGs are missing, even though these cells could remain in the CNS. salm-lacZ is expressed in the two ventral SOP-derived ePG4 and ePG5, as well as in the dorsal SOP-derived ePG11 along the DLN, and in some of the ligament cells of the lateral chordotonal organ. In salm445 mutants the ePG4 cell can sometimes be detected at its wildtypic position along the SN. If ePG4 is missing along the SN, it could well be a secondary effect, as the SN itself is affected with the SNc shortened or occasionally missing. The ePG5 however, cannot be unambiguously identified in Repo-staining within the group of cells stalling in the transition zone (von Hilchen, 2008).

It needs to be further shown whether the differences between the PGs derived from certain progenitor cells result in functional differences in the larva. The peripheral nerves of the larva are ensheathed by two distinct types of glial cells, the perineurial and the subperineurial glial cells. The subperineurial glia build septate junctions with each other (or themselves) and thereby form the blood-nerve barrier, whereas the perineurial glia form an outer layer and secrete the neural lemma. In order to allow proper electrical conductance, the peripheral nerves must be enwrapped and insulated at the end of embryogenesis when hatching of the larva requires coordinated muscle contractions. It is not known to date which of the embryonic PGs will become perineurial or subperineurial glia, or what other functions they might fulfill (von Hilchen, 2008).

The comprehensive description of the ancestry, identity and dynamics of the developing embryonic peripheral glia, and the molecular markers at hand, provide a crucial basis for further clarification of the mechanisms controlling development, migration, and function of peripheral glia on a single cell level (von Hilchen, 2008).

Segment-specific neuronal subtype specification by the integration of anteroposterior and temporal cues

The generation of distinct neuronal subtypes at different axial levels relies upon both anteroposterior and temporal cues. However, the integration between these cues is poorly understood. In the Drosophila central nervous system, the segmentally repeated neuroblast 5-6 generates a unique group of neurons, the Apterous (Ap) cluster, only in thoracic segments. Recent studies have identified elaborate genetic pathways acting to control the generation of these neurons. These insights, combined with novel markers, provide a unique opportunity for addressing how anteroposterior and temporal cues are integrated to generate segment-specific neuronal subtypes. Pbx/Meis, Hox, and temporal genes were found to act in three different ways. Posteriorly, Pbx/Meis and posterior Hox genes block lineage progression within an early temporal window, by triggering cell cycle exit. Because Ap neurons are generated late in the thoracic 5-6 lineage, this prevents generation of Ap cluster cells in the abdomen. Thoracically, Pbx/Meis and anterior Hox genes integrate with late temporal genes to specify Ap clusters, via activation of a specific feed-forward loop. In brain segments, 'Ap cluster cells' are present but lack both proper Hox and temporal coding. Only by simultaneously altering Hox and temporal gene activity in all segments can Ap clusters be generated throughout the neuroaxis. This study provides the first detailed analysis of an identified neuroblast lineage along the entire neuroaxis, and confirms the concept that lineal homologs of truncal neuroblasts exist throughout the developing brain. Also this study provides the first insight into how Hox/Pbx/Meis anteroposterior and temporal cues are integrated within a defined lineage, to specify unique neuronal identities only in thoracic segments. This study reveals a surprisingly restricted, yet multifaceted, function of both anteroposterior and temporal cues with respect to lineage control and cell fate specification (Karlsson, 2010).

To understand segment-specific neuronal subtype specification, this study focused on the Drosophila neuroblast 5-6 lineage and the thoracic-specific Ap cluster neurons born at the end of the NB 5-6T lineage. The thoracic appearance of Ap clusters was shown to result from a complex interplay of Hox, Pbx/Meis, and temporal genes that act to modify the NB 5-6 lineage in three distinct ways (see Summary of Hox/Pbx/Meis and temporal control of NB 5-6 development). In line with other studies of anterior-most brain development, it was found that the first brain segment (B1) appears to develop by a different logic. These findings will be discussed in relation to other studies on spatial and temporal control of neuroblast lineages (Karlsson, 2010).

In the developing Drosophila CNS, each abdominal and thoracic hemisegment contains an identifiable set of 30 neuroblasts, which divide asymmetrically in a stem-cell fashion to generate distinct lineages. However, they generate differently sized lineages -- from two to 40 cells, indicating the existence of elaborate and precise mechanisms for controlling lineage progression. Moreover, about one third of these lineages show reproducible anteroposterior differences in size, typically being smaller in abdominal segments when compared to thoracic segments. Thus, neuroblast-specific lineage size control mechanisms are often modified along the anteroposterior axis (Karlsson, 2010).

Previous studies have shown that Hox input plays a key role in modulating segment-specific behaviors of neuroblast lineages. Recent studies have resulted in mechanistic insight into these events. For instance, in the embryonic CNS, Bx-C acts to modify the NB 6-4 lineage, preventing formation of thoracic-specific neurons in the abdominal segments. This is controlled, at least in part, by Bx-C genes suppressing the expression of the Cyclin E cell cycle gene in NB 6-4a. Detailed studies of another neuroblast, NB 7-3, revealed that cell death played an important role in controlling lineage size in this lineage: when cell death is genetically blocked, lineage size increased from four up to 10 cells. Similarly, in postembryonic neuroblasts, both of these mechanisms have been identified. In one class of neuroblasts, denoted type I, an important final step involves nuclear accumulation of the Prospero regulator, a key regulator both of cell cycle and differentiation genes. In 'type II' neuroblasts, grh acts with the Bx-C gene Abd-A to activate cell death genes of the Reaper, Head involution defective, and Grim (RHG) family, and thereby terminates lineage progression by apoptosis of the neuroblast. This set of studies demonstrates that lineage progression, in both embryonic and postembryonic neuroblasts, can be terminated either by neuroblast cell cycle exit or by neuroblast apoptosis. In the abdominal segments, it was found that the absence of Ap clusters results from a truncation of the NB 5-6 lineage, terminating it within the Pdm early temporal window, and therefore Ap cluster cells are never generated. These studies reveal that this truncation results from neuroblast cell cycle exit, controlled by Bx-C, hth, and exd, thereafter followed by apoptosis. In Bx-C/hth/exd mutants, the neuroblast cell cycle exit point is bypassed, and a thoracic sized lineage is generated, indicating that these genes may control both cell cycle exit and apoptosis. However, it is also possible that cell cycle exit is necessary for apoptosis to commence, and that Bx-C/hth/exd in fact only control cell cycle exit. Insight into the precise mechanisms of the cell cycle exit and apoptosis in NB 5-6A may help shed light on this issue (Karlsson, 2010).

Whichever mechanism is used to terminate any given neuroblast lineage -- cell cycle exit or cell death -- the existence in the Drosophila CNS of stereotyped lineages progressing through defined temporal competence windows allows for the generation of segment-specific cell types simply by regulation of cell cycle and/or cell death genes by developmental patterning genes. Specifically, neuronal subtypes born at the end of a specific neuroblast lineage can be generated in a segment-specific fashion 'simply' by segmentally controlling lineage size. This mechanism is different in its logic when compared to a more traditional view, where developmental patterning genes act upon cell fate determinants. But as increasing evidence points to stereotypic temporal changes also in vertebrate neural progenitor cells (Okano, 2009), this mechanism may well turn out to be frequently used to generate segment-specific cell types also in the vertebrate CNS (Karlsson, 2010).

These findings of Hox, Pbx/Meis, and temporal gene input during Ap cluster formation are not surprising -- generation and specification of most neurons and glia will, of course, depend upon some aspect or another of these fundamental cues. Importantly however, the detailed analysis of the NB 5-6T lineage, and of the complex genetic pathways acting to specify Ap cluster neurons, has allowed this study to pin-point critical integration points between anteroposterior and temporal input. Specifically, cas, Antp, hth, and exd mutants show striking effects upon Ap cluster specification, with effects upon expression of many determinants, including the critical determinant col. Whereas Antp plays additional feed-forward roles, and exd was not tested due to its maternal load, it was found that both cas and hth mutants can be rescued by simply re-expressing col. This demonstrates that among a number of possible regulatory roles for cas, hth, Antp, and exd, one critical integration point for these anteroposterior and temporal cues is the activation of the COE/Ebf gene col, and the col-mediated feed-forward loop. Both col and ap play important roles during Drosophila muscle development, acting to control development of different muscle subsets. Their restricted expression in developing muscles has been shown to be under control of both Antp and Bx-C genes. Molecular analysis has revealed that this regulation is direct, as Hox proteins bind to key regulatory elements within the col and ap muscle enhancers. The regulatory elements controlling the CNS expression of col and ap are distinct from the muscle enhancers, and it will be interesting to learn whether Hox, as well as Pbx/Meis and temporal regulatory input, acts directly also upon the col and ap CNS enhancers (Karlsson, 2010).

One particularly surprising finding pertains to the instructive role of Hth levels in NB 5-6T. At low levels, Hth acts in NB 5-6A to block lineage progression, whereas at higher levels, it acts in NB 5-6T to trigger expression of col within the large cas window. It is interesting to note that the hth mRNA and Hth protein expression levels increase rapidly in the entire anterior CNS (T3 and onward). In addition, studies reveal that thoracic and anterior neuroblast lineages in general tend to generate larger lineages and thus remain mitotically active for a longer period than abdominal lineages. On this note, it is tempting to speculate that high levels of Hth may play instructive roles in many anterior neuroblast lineages. In zebrafish, Meis3 acts to modulate Hox gene function, and intriguingly, different Hox genes require different levels of Meis3 expression. In the Drosophila peripheral nervous system, expression levels of the Cut homeodomain protein play instructive roles, acting at different levels to dictate different dendritic branching patterns in different sensory neuron subclasses. Although the underlying mechanisms behind the levels-specific roles of Cut, Meis3 or Hth are unknown, it is tempting to speculate that they may involve alterations in transcription factor binding sites, leading to levels-sensitive binding and gene activation of different target genes (Karlsson, 2010).

The vertebrate members of the Meis family (Meis1/2/3, Prep1/2) are expressed within the CNS, and play key roles in modulating Hox gene function. Intriguingly, studies in both zebrafish and Xenopus reveal that subsequent to their early broad expression, several members are expressed more strongly or exclusively in anterior parts of the CNS, in particular, in the anterior spinal cord and hindbrain. Here, functional studies reveal complex roles of the Meis family with respect to Hox gene function and CNS development. However, in several cases, studies reveal that they are indeed important for specification, or perhaps generation, of cell types found in the anterior spinal cord and/or hindbrain, i.e., anteroposterior intermediate neural cell fates. As more is learned about vertebrate neural lineages, it will be interesting to learn which Meis functions may pertain to postmitotic neuronal subtype specification, and which may pertain to progenitor cell cycle control (Karlsson, 2010).

In anterior segments -- subesophageal (S1-S3) and brain (B1-B3) -- a more complex picture emerges where both the overall lineage size and temporal coding is altered, when compared to the thoracic segments. Specially, whereas all anterior NB 5-6 lineages do contain Cas expressing cells, expression of Grh is weak or absent from many Cas cells. The importance of this weaker Grh expression is apparent from the effects of co-misexpressing grh with Antp -- misexpression of Antp alone is unable to trigger FMRFa expression, whereas co-misexpression with grh potently does so. It is unclear why anterior 5-6 lineages would express lower levels of Grh, since Grh expression is robust in some other anterior lineages (Karlsson, 2010).

In the B1 segment two NB 5-6 equivalents have been identified. However, the finding of two NB 5-6 equivalents is perhaps not surprising, since the B1 segment contains more than twice as many neuroblasts as posterior segments. Due to weaker lbe(K)-lacZ and -Gal4 reporter gene expression, and cell migration, these lineages could not be mapped. However, irrespective of the features of the B1 NB 5-6 lineages, bona fide Ap cluster formation could not be triggered by Antp/grh co-misexpression in B1. Together, these findings suggest that the B1 segment develops using a different modus operandi, a notion that is similar to development of the anterior-most part of the vertebrate neuroaxis, where patterning and segmentation is still debated. On that note, it is noteworthy that although Hox genes play key roles in specifying unique neuronal cell fates in more posterior parts of the vertebrate CNS, and can indeed alter cell fates when misexpressed, the sufficiency of Hox genes to alter neuronal cell fates in the anterior-most CNS has not been reported -- for instance, Hox misexpression has not been reported to trigger motoneuron specification in the vertebrate forebrain. Thus, in line with the current findings that Antp is not sufficient to trigger Ap cluster neuronal fate in the B1 anterior parts, the anterior-most part of both the insect and vertebrate neuroaxis appears to be 'off limits' for Hox genes (Karlsson, 2010).

The Hox, Pbx/Meis, and temporal genes are necessary, and in part sufficient, to dictate Ap cluster neuronal cell fate. However, they only do so within the limited context of NB 5-6 identity. Within each abdominal and thoracic hemisegment, each of the 30 neuroblasts acquires a unique identity, determined by the interplay of segment-polarity and columnar genes. In the periphery, recent studies demonstrate that anteroposterior cues, mediated by Hox and Pbx/Meis genes, are integrated with segment-polarity cues by means of physical interaction and binding to regulatory regions of specific target genes. It is tempting to speculate that similar mechanisms may act inside the CNS as well, and may not only involve anteroposterior and segment-polarity integration, but also extend into columnar and temporal integration (Karlsson, 2010).

A genetic cascade involving klumpfuss, nab and castor specifies the abdominal leucokinergic neurons in the Drosophila CNS

Identification of the genetic mechanisms underlying the specification of large numbers of different neuronal cell fates from limited numbers of progenitor cells is at the forefront of developmental neurobiology. In Drosophila, the identities of the different neuronal progenitor cells, the neuroblasts, are specified by a combination of spatial cues. These cues are integrated with temporal competence transitions within each neuroblast to give rise to a specific repertoire of cell types within each lineage. However, the nature of this integration is poorly understood. To begin addressing this issue, this study analyzed the specification of a small set of peptidergic cells: the abdominal leucokinergic neurons. The progenitors of these neurons were identified, along with the temporal window in which they are specified, and the influence of the Notch signaling pathway on their specification. The products of the genes klumpfuss, nab and castor were shown to play important roles in their specification via a genetic cascade (Benito-Sipos, 2010).

Recent findings on NB5-6 demonstrate that Cas and Grh act as crucial temporal genes to specify several cell fates at the end of this lineage. The current findings with NB5-5 reveal similar roles for Cas and Grh, and indicate that the ABLKs are specified in a Cas/Grh temporal window. It was observed that cas mutants generate no ABLKs, that cas misexpression leads to clusters of two to four ABLKs per hemisegment, and that Cas is expressed in all the ABLKs. Thus, these data confirm that cas plays a role as a temporal identity gene, which remains compatible with its proposed role as a switching temporal factor (Benito-Sipos, 2010).

The proposed role of grh as a temporal identity gene remains open to question. It has also been reported that it is required to regulate mitotic activity and apoptosis of post-embryonic NBs. However, recent evidence has emerged indicating that Grh also temporally regulates FMRFamide neuropeptide cell fate and can act in a combinatorial manner with dimm and apterous to trigger ectopic FMRFamide expression. Similarly, it was found that Grh is required for correct specification of the ABLKs. Together, these results suggest that Grh also plays an instructive role in ABLK specification. Thus, many neuropeptidergic neurons are generated late in several lineages, and depend upon the late temporal genes cas and grh for their specification (Benito-Sipos, 2010).

NB5-5 does not express the temporal genes hb and Kr, and genetic analysis confirms that these two genes are not required for specification of ABLK fate. It was observed that NB5-5 initially expresses Pdm at the time of delamination in late stage 11. Pdm is downregulated at early stage 12, when Cas is activated, and there is a brief period in which both proteins can be detected. The lack of molecular markers does not permit determination of whether the Pdm/Cas coexpression stage generates a GMC (Benito-Sipos, 2010).

It is of interest to note that the phenotype observed misexpressing cas with NB-specific drivers was very mildcompared with that obtained using a pan-neuronal driver. NB5-5 expresses cas soon after delamination and generates six to nine neurons. This suggests that NB5-5 probably has a broad Cas temporal window. Thus, the phenotype obtained upon misexpressing cas with elav-Gal4 indicates that Cas might have a later requirement in postmitotic cells that generates subtemporal windows. Consistent with this interpretation, cas misexpression rescues the grh phenotype of loss of ABLKs, which also suggests that Grh, in addition to being required as a temporal factor, would be indirectly required to activate cas expression in postmitotic cells (Benito-Sipos, 2010).

The Notch pathway is involved in many cell fate decisions in neural development. This study has shown that the ABLK and its sibling are equivalent cells committed to die, and that activation of the Notch pathway in the ABLK prevents its death. A similar situation has been described for specification of the anterior and posterior Corner Cells (CaCC/pCC) neurons in the grasshopper NB1-1 lineage, in which the siblings start as equivalent cells and interaction between them leads to different fates. By contrast, activation of Notch in the NB7-3 lineage drives PCD). Here, activation of the Notch pathway, or misexpression of p35 in the sibling cell, is sufficient to generate two ABLK neurons. A systematic analysis of the lineage of apoptotic cells in embryos in which apoptosis is prevented has shown that the lineage of abdominal NB5-5 contains twice the normal number of cells, but that they have wild-type-like axonal projections. It is concluded that in this lineage, Notch does not play an instructive role in specifying ABLK neuronal fate, but influences a fate decision by regulating the competence to respond to a program of cell death (Benito-Sipos, 2010).

A set of mutants were identified that produce an altered number of ABLKs. In most cases the effect is very mild. Among the mutants with the strongest phenotypes were jumu, nab and klu. The jumu phenotype was expected because it has been shown that Jumu is required in the NB4-2 lineage for normal segregation of Numb in the asymmetric cell divisions. Consistent with this interpretation, the fact that the phenotype of jumu in the NB5-5 lineage is similar to that seen in spdo explains its phenotype and indicates that in jumu embryos Notch is off in both siblings (Benito-Sipos, 2010).

nab and klu embryos display a strong reduction in the number of ABLKs, suggesting that both genes have direct roles in ABLK specification. Interestingly, Cas activates the expression of both genes via repression of Pdm. The lack of availability of markers for identifying ABLKs in earlier stages did not permit establishing whether Nab and Klu are required in the NB or in postmitotic cells (Benito-Sipos, 2010).

Misexpression of cas in nab embryos showed ectopic ABLKs, suggesting that Cas acts either parallel to, or downstream of, Nab. The lack of molecular markers specific to the NB5-5 lineage does not allow determination of whether all ectopic ABLKs are generated by the NB5-5 or by other lineages. Nevertheless, several results suggest that, most probably, all of them are produced by NB5-5. First, it has been observed that neurons that belong to one lineage form a coherent cluster. Second, all of them express gsb, which labels rows five and six NB, and do not express lbe, an NB5-6-specific marker. Third, ABLKs are the unique cells expressing Lk in the ventral ganglion. However, downregulation of cas was not observed in nab mutants, and the same has been reported in the better characterized lineages of NB3-3 and NB5-6; together, these results indicate that the molecular relationship between Cas and Nab requires a more complex interpretation than a linear genetic cascade (Benito-Sipos, 2010).

klu encodes a zinc-finger protein but does not appear to interact directly with Nab, and no evidence was found that nab and klu regulate each other. Surprisingly, nab misexpression rescues the phenotype of a lack of ABLKs observed in klu. By contrast, it was found that cas misexpression produces more ABLKs in grh, nab or klu than in wild-type background As proposed above, these results suggest that Cas plays a role in postmitotic cells that is crucial for ABLK specification (Benito-Sipos, 2010).

The sqz phenotype is epistatic over the nab phenotype. Thus, although nab embryos have no ABLKs, sqz and nab sqz show a normal pattern of ABLKs. It has been shown by pull-down assay that Nab physically interacts with Sqz, and in vertebrates the Nab homologs act as transcriptional co-factors. Since both genes, sqz and nab, are expressed in the ABLKs, it is proposed that the function of Sqz in NB5-5 lineage is to repress the ABLK fate. In normal development, as both genes are expressed in the ABLKs, Nab binds to Sqz and blocks its repressor activity; in nab embryos Sqz represses the ABLK fate, but in nab sqz the pattern is wild-type because there is no repression by Sqz. This intimate interplay between Sqz and Nab is also found in the NB 5-6 linage, in which sqz is first required to activate cell fate determinants, and then acts with nab to suppress the same determinants (Benito-Sipos, 2010).

The findings reported in this study extend understanding of the mechanisms of ABLK specification. However, more precise analysis of the genes and the mechanisms involved in specification of the different cell fates in the NB5-5 lineage will require additional molecular markers. This would permit identification of the different neurons generated from this NB and the genes required to specify their various fates (Benito-Sipos, 2010).

Origin and specification of the brain leucokinergic neurons of Drosophila: Similarities to and differences from abdominal leucokinergic neurons

The Drosophila central nervous system contains many types of neurons that are derived from a limited number of progenitors as evidenced in the ventral ganglion. The situation is much more complex in the developing brain. The main neuronal structures in the adult brain are generated in the larval neurogenesis, although the basic neuropil structures are already laid down during embryogenesis. The embryonic factors involved in adult neuron origin are largely unknown. To shed light on how brain cell diversity is achieved, a study was carried out of the early temporal and spatial cues involved in the specification of lateral horn leucokinin peptidergic neurons (LHLKs). The analysis revealed that these neurons have an embryonic origin. Their progenitor neuroblast were identified as Pcd6 in the Technau and Urbach terminology. Evidence was obtained that a temporal series involving the transcription factors Kr, Pdm, and Cas participates in the genesis of the LHLK lineage, the Castor window being the one in which the LHLKs neurons are generated. It was also shown that Notch signalling and Dimmed are involved in the specification of the LHLKs. It is concluded that serial homologies with the origin and factors involved in specification of the abdominal leucokinergic neurons (ABLKs) have been detected (Herrero, 2013).

Studies on neuroblast lineages in the developing ventral ganglia are numerous, but investigations of which lineages are present in cerebral ganglia and which are not have only just begun. Drosophila neurogenesis takes place at two stages: an embryonic stage, in which larval functions are established, and a larval stage, in which neurons involved in adult functions are added. Temporal genes regulating the postembryonic neuroblast lineages in the central brain and in the optic lobes have been identified, but little is known of brain neuroblast embryonic lineages. LHLK neurons offer the possibility of studying the embryonic origins of brain neurons and comparing them to the origins of other lineages including LK-expressing progeny. This study shows that LK-expressing neurons from different segments of brain and abdomen not only share neuropeptide expression but also cell number per hemisegment and neuronal cell appearance, characterized by long axons full of varicosities, large superficially located somas, but lack of coexpression of any small neurotransmitters. The results obtained above provide clues for defining the serial homology between neuroblasts from the protocerebrum and from the ventral ganglia, and for analyzing differences between the complex combinatorial code that defines the fates of LK-expressing neurons (Herrero, 2013).

The results suggest that the canonical temporal gene cascade Hb-Kr-Pdm-Cas-Grh is active in protocerebral neuroblasts as it is in thoracic and abdominal neuroblasts. Consequently, as in the VNC, temporal factors in the brain also activate the next gene and repress the 'next plus one' or the previous one. These factors, except for Hb and Kr, are weakly expressed in LHLK neurons at the early first instar larva, but the most important clues concerning their temporal implications are the effects of their loss and gain of function: LHLK specification is partially inhibited in kr and pdm mutants, and completely blocked in cas mutant. Only the grh mutant has no phenotypic effect on LHLKs, although its overexpression does have a phenotype, indicating that the Cas window is negatively regulated by Grh. On the other hand, svp is also involved in LHLK specification, probably not via its relation to hb but because it is expressed in another phase after the Cas window, as in many embryonic abdominal neuroblast lineages. Although the temporal factors implicated in the origin of LHLKs fit the model accepted for other NB lineages in the embryonic CNS, more studies are required to provide precise information about the timing of temporal factor expression and about the specification of the other progeny in the lineage and in other embryonic brain lineages (Herrero, 2013).

The results obtained in dimm overexpression experiments demonstrate the existence of other neurons with potential LK fates in the Drosophila brain. In this situation it seems that expression of the neuroendocrine differentiation gene dimm forces the 'almost' leucokinergic neurons to complete their differentiation. There are analogies with the results obtained for FMRFamide, where ectopic FMRFamide expression in Tv neurons is only observed when dimm is misexpressed. dimm is essential for transforming the synaptic vesicles of neurons into functional peptidergic vesicles. This study demonstrates that other neurons in the brain have the LK fate determinants but not the ability to adopt the neuropeptidergic cell fate. Interestingly, the ectopic LK neurons found in dimm overexpression correspond to different brain segments, namely deutocerebrum, tritocerebrum and protocerebrum. This could be pointing to serial homology in some brain lineages. Further analysis is needed to probe the LK fate in these segments (Herrero, 2013).

The two LK-expressing cell types share two main characteristics: the ventral-lateral location within their segments and their embryonic origin. LHLK neurons arise from a lineage located dorsally and near to the optic primordium, which corresponds to the protocerebral dorsal central lineage in Urbach (2003) terminology, or the basolateral dorsal lineage in Pereanu (2004) terminology. ABLKs arise from abdominal NB5-5, which is laterally located in the VNC, both are lateral in their respective segments, arise during embryonic neurogenesis and start expressing LK at the end of stage 17 (Herrero, 2013).

There are some differences in terms of temporal genes between LHLK and ABLK lineages. The analysis suggests that Cas is the temporal factor window specifying LHLK fate, whilea Cas/Grh temporal window has been proposed for ABLKs. There is evidence that the Cas window is long in some NBs of the trunk, and Cas has also been identified in postembryonic brain development. In the light of these findings it is proposed that, as in trunk neuroblasts, the Cas time window in the neuroblast Pcd6 lineage is extensive and the Cas inhibitory effect of Grh is delayed with respect to the abdominal segments. As a result, the LHLKs can be generated before grh expression; so that this factor is dispensable for the appearance of LHLKs. Hence, Grh effects on LHLKs are only observed when grh is overexpressed (Herrero, 2013).

Of the 27 genes, 7 were not expressed in either of the two types of LK neurons and their loss of function had different effects on their phenotypes. Three of these genes expressed in the ABLKs were hkb, gsb and ind, whose NB expression is weak in the protocerebrum. The expression of other two genes, also expressed in the ABLKs: unpg and runt, is sustained until the end of embryogenesis in the postmitotic cells. However ABLKs are controlled by the pair rule gene runt and the homeodomain gene unpg. It has been reported that runt regulates the expression boundaries of segment polarity genes in the VNC but not in the procephalon, while unpg, together with otd, is involved in the protocerebrum/deutocerebrum interface in the procephalic neuroctoderm. Hence these different functions could explain the different expression (Herrero, 2013).

Finally, ap and klu show extended brain expression in neuroblasts (klu) and in postmitotic neurons (ap) in the brain; however their effects are not the same in LHLKs and ABLKs: Ap regulates LK expression in LHLKs, while Klu does it in ABLKs. Xiao (2012) has shown that Klu is necessary in the brain for the renewal maintenance of type II neuroblasts, whereas VNC type I neuroblasts are probably not affected because other factors provide this function. Thus Klu has different functions in the brain and the VNC. In spite of these differences, ABLKs and LHLKs do share the presence or absence of expression of 19 genes, among which are not only the aforementioned temporal genes and postmitotic cofactors nab and sqz, but the segment polarity gene wg. Just as engrailed (en) marks the posterior border segment, wg marks the anterior one, as in the trunk segment, although less obviously. Four cephalic segments have been describe: intercalary, antennal, ocular and labral, the last two being part of the protocerebrum. The wg, en, gsb-d and hh segment polarity genes and the ind, msh, vnd columnar genes mark some of their boundary. The ocular segment contains the largest number of neuroblasts (60), and it is the most difficult to study because of its complexity. However it is clear that the anterior region of this neuromere is extended the most, with more than 25% of the wg-expressing neuroblasts at stage 11. On the other hand, the en expressing region is very much smaller (only 10 NBs). The LHLKs, like the ABLKs, belong to an anterior segment lineage. ABLK-progenitor neuroblast expresses ind but LHLKs cannot be assigned to a particular columnar neuroblast because the ocular segment has almost no ind identity. It may be concluded that the neuroblasts Pcd6 and NB5-5, from which the LK-expressing neurons arise in an equivalent temporal embryonic window, are serially homologous, although several individual characteristics distinguish their development. In some of the serially homologous neuroblast lineages of the VNC, there are differences between thoracic and abdominal neuromeres, and it is expected that such segment-specific differences would be more pronounced between the brain and the VNC where the genetic backgrounds are different, and the canonical orthogonal expression genes described in the VNC are mainly not conserved in the protocerebral neuromeres. Clarification of the progression of the Leucokinin-progenitor neuroblasts during brain development and comparison with the situation in the trunk could help in an understanding of what makes the brain different from the VNC (Herrero, 2013).

Global programmed switch in neural daughter cell proliferation mode triggered by a temporal gene cascade

During central nervous system (CNS) development, progenitors typically divide asymmetrically, renewing themselves while budding off daughter cells with more limited proliferative potential. Variation in daughter cell proliferation has a profound impact on CNS development and evolution, but the underlying mechanisms remain poorly understood. This study found that Drosophila embryonic neural progenitors (neuroblasts) undergo a programmed daughter proliferation mode switch, from generating daughters that divide once (type I) to generating neurons directly (type 0). This typeI> 0 switch is triggered by activation of Dacapo (mammalian p21CIP1/p27KIP1/p57Kip2) expression in neuroblasts. In the thoracic region, Dacapo expression is activated by the temporal cascade (castor) and the Hox gene Antennapedia. In addition, castor, Antennapedia, and the late temporal gene grainyhead act combinatorially to control the precise timing of neuroblast cell-cycle exit by repressing Cyclin E and E2f. This reveals a logical principle underlying progenitor and daughter cell proliferation control in the Drosophila CNS (Baumgardt, 2014).

Proliferation analysis of the developing Drosophila VNC reveals that most, if not all, lateral NBs initially divide in the type I proliferation mode, generating daughters that divide once. Three specific lineages, as well as many other NBs, subsequently switch to generating daughters that do not divide (type 0 mode). The full extent of the typeI>0 switch is currently difficult to precisely assess for several reasons. One such complicating issue pertains to possible developmental changes in daughter cell-cycle length over time. On this note, however, no obvious change was found in NB5-6T daughter divisions prior to the switch. In addition, if the accelerated decline in daughter division was indeed caused by a lengthening of the cell cycle rather than a typeI>0 switch, a long 'tail' of daughter divisions would be expected, perduring into St16-17. This is not the case; rather, daughter proliferation drops down to almost zero by St16-17. Similarly, no evidence was found for changes in NB cell-cycle length over time in the three specific NB lineages. Another complicating issue pertains to the fact that NBs differ in their time point of delamination, number of division rounds, and time point of switching, so that even if all NBs switched, only a fraction of the NBs would be in their type 0 window at the same time. However, these complications are more likely to lead to under- rather than overappreciation of the extent of the typeI>0 switch, and it is tempting to speculate that it may indeed involve the vast majority of NBs (Baumgardt, 2014).

The typeI>0 switch is triggered by the onset of Dap expression in NBs at precise stages of lineage progression. The mammalian Dap orthologs, p21CIP1/p27KIP1/p57Kip2, can act as inhibitors of the CycE/Cdk2 complex. By analogy, the mechanism behind the typeI>0 switch is, presumably, that type 0 daughters are prevented from entering the cell cycle by the presence of Dap at the G1/S checkpoint. The onset of Dap expression already in the NB suggests that Dap needs to be present at an early stage in newborn daughters to block their entry into S phase. These findings are also in line with the emerging role for the Cip/KIP family and cell-cycle exit in the mammalian CNS, although there has been no report of a connection to changes in daughter proliferation mode (Baumgardt, 2014).

No evidence was found for a role of pros in the type 0 mode, and, conversely, no evidence was found for a role of dap in the type I mode. The distinct roles of pros and dap in control of the type I versus 0 modes is further underscored by the expression of E2f, CycE, and Dap. In type I daughters (GMCs), E2f and CycE are rapidly repressed, by pros, and Dap is only weakly expressed at a later stage, around the time point of mitosis. The short window of E2f and CycE expression is still sufficient for the GMC to enter another cell cycle, since Dap expression is absent. As each GMC divides, the postmitotic cells (neurons/glia) are prevented from entering the cell cycle by the lack of E2f and CycE. In type 0 daughters, on the other hand, E2f and CycE expression is robust, but daughters still fail to enter the cell cycle due to the presence of high levels of Dap. These findings point to strikingly different strategies in daughter proliferation control: pros repression of E2f/CycE in type I, and Dap overriding E2f/CycE/Cdk2 in type 0 daughters (Baumgardt, 2014).

Changes in daughter cell proliferation could perhaps have been envisioned to merely result from a gradual loss of the proliferative potential of each progenitor, as a result of its undergoing many rapid cell cycles. If so, typeI>0 switches could have been predicted to occur somewhat stochastically toward the end stage of each lineage, perhaps loosely linked to the last NB division. In contrast to such simplified models, this study found that the typeI>0 switch can occur many divisions prior to NB exit and that it is programmed to occur at a precise stage during each lineage development. In the thorax, it was found that the precise timing of typeI>0 switches is controlled by the temporal gene cas and the Hox gene Antp, which are expressed at a late stage within NBs. Remarkably, in cas mutants, most, if not all, thoracic typeI>0 lineages fail to enter the type 0 mode. The primary mechanism by which cas and Antp control the switch appears to be by activating the expression of Dap, evident by the reduction of Dap in cas and Antp mutants; by the finding that cas-Antp co-misexpression triggers ectopic Dap expression; and by the finding that cas can be cross-rescued by elav>dap (Baumgardt, 2014).

The finding that the timing of the typeI>0 switch is scheduled by a temporal gene cascade points to an intriguing regulatory model where daughter cell proliferation mode switches are executed at stereotyped positions within the lineage tree by the activity of specific temporal genes. Since temporal genes also control the progression of NB competence, evident by their roles in cell fate specification, the temporal cascade can act to simultaneously control both cell fate and cell number, thereby ensuring that precise number of each neural cell subtype is produced (Baumgardt, 2014).

After a stereotyped number of divisions, each NB subtype stops proliferating. This study found that, for many NBs, this is a G1/S decision influenced by the activities of E2f, CycE, and dap. The nuclear localization of Pros was previously identified to be associated with cell-cycle exit in postembryonic NBs. However, previous studies of NB5-6T, and the current study on NB7-3A, do not indicate a general role for pros in NB cell-cycle exit in the embryonic CNS. Instead, in the thorax, the expression levels of E2f, CycE, and Dap are gradually modulated during lineage progression, by the temporal genes cas and grh as well as Antp. Because Cas, Grh, and Antp are progressively activated in thoracic NBs, this brings into view a logical model for timely NB cell-cycle exit where sequential activation of temporal and Hox genes act combinatorially to push E2f, CycE, and/or Dap to limiting levels after a determined number of divisions (Baumgardt, 2014).

For the majority of NBs in the thorax, cell-cycle exit is followed by quiescence until larval stages. In contrast, for the majority of abdominal NBs, cell-cycle exit is followed by apoptosis. However, for some NBs, such as NB7-3A, apoptosis is the functional exit mechanism. Thus, three general strategies for lineage stop are emerging: (1) cell-cycle exit > quiescence (most thoracic NBs), (2) cell-cycle exit > apoptosis (NB5-6T), and (3) lineage stop by apoptosis (NB7-3A). The balance of E2f, CycE, and Dap is involved in the first two strategies, while the balance of apoptosis gene expression presumably is at the core of the latter strategy (Baumgardt, 2014).

In addition to the type I and type 0 daughter proliferation modes described here in the embryo, recent studies of Drosophila larval CNS development have identified a third, more prolific, proliferation mode: the type II mode, identified in a small number of larval brain. Type II NBs divide asymmetrically, renewing themselves while budding of daughters that, in turn, undergo multiple rounds of proliferation before finally differentiating. This allows for the generation of very large lineages (some 500 cells) from each individual type II NB (Baumgardt, 2014).

In mammals, the most obvious equivalent of Drosophila NBs is the radial glia cell (RG), which divides asymmetrically to generate neurons and. During these RG asymmetric divisions, studies have identified several different division modes; RGs dividing asymmetrically to bud off a neuron, to bud off a daughter cell that divides once to generate two neurons, or to bud off daughter cells that themselves divide multiple times before generating neurons. Although mammalian CNS development likely will involve more complex and more elaborate lineage variations, there is, nevertheless, a striking similarity between these alternate mammalian daughter proliferation modes and the type 0, I and II modes now identified in Drosophila. Intriguingly, in line with these analogies between Drosophila and mammals, recent time-lapse studies on the developing primate cortex have revealed a global temporal switch in the proliferation profiles of daughter cells (Betizeau, 2013). It will be interesting to learn if such temporal proliferation changes are intrinsically controlled and if they are stereotypically linked to changes in neural subtype specification also in mammals (Baumgardt, 2014).

Neuronal cell fate diversification controlled by sub-temporal action of Kruppel

During Drosophila CNS development, neuroblasts express a programmed cascade of five temporal transcription factors that govern the identity of cells generated at different time-points. However, these five temporal genes fall short of accounting for the many distinct cell types generated in large lineages. This study finds that the late temporal gene castor sub-divides its large window in neuroblast 5-6 by simultaneously activating two cell fate determination cascades and a sub-temporal regulatory program. The sub-temporal program acts both upon itself and upon the determination cascades to diversify the castor window. Surprisingly, the early temporal gene Kruppel acts as one of the sub-temporal genes within the late castor window. Intriguingly, while the temporal gene castor activates the two determination cascades and the sub-temporal program, spatial cues controlling cell fate in the latter part of the 5-6 lineage exclusively act upon the determination cascades (Stratmann, 2016).

This study, along with previous work, found that the temporal gene cascade results in the expression of Cas in the latter part of NB5-6T. cas acts together with spatial input, provided by Antp, hth, exd and lbe to activate col in the NB. col in turn activates ap and eya in the early postmitotic cells, which represents a transient and generic Ap cluster cell fate. col subsequently acts in a feedforward loop of col>ap/eya>dimm>Nplp1 to determine Tv1 cell fate. However, in addition to col, cas activates five other genes, including the last temporal gene grh, and the sub-temporal genes sqz, nab, svp and, as shown in this study, Kr. These five genes engage in a postmitotic cross-regulatory interplay, unique to each of the three cell types, which results in the propagation of the col>ap/eya>dimm>Nplp1 terminal selector cascade exclusively in Tv1, and the ap/eya/dac>dimm/BMP>FMRFa cascade in Tv4, while the Tv2/3 cells acquire a non-peptidergic interneuron identity. The role of Kr is to suppress the sub-temporal gene svp, in order to safeguard the expression of col and dimm, and thereby ensures the propagation of the col>ap/eya>dimm>Nplp1 terminal selector cascade, crucial for specification of the Tv1 cells. The other four genes (grh, sqz, nab, svp) each have unique roles, and act as sub-temporal micromanagers to ensure high fidelity and precision in the sub-division of the cas temporal window (Stratmann, 2016).

The temporal gene cas plays a pivotal role in the specification process of the different Ap cluster cells due to its activator role on a number of downstream regulators; col, a terminal selector in Tv1 specification, the sub-temporal genes sqz, nab, svp and Kr, as well as the temporal gene grh. Strikingly, cas thus activates both of the two terminal selector feedforward loops (FFLs), and the genes required to refine both FFLs (Stratmann, 2016).

cas activates Kr and svp, but how is Kr expression then restricted to only Tv1 and svp expression to Tv2/3? For Kr, restricted expression of sqz, nab and svp in Tv2-Tv4, all of which suppress Kr, can explain the confined expression pattern of Kr to Tv1. The gradually restricted expression of svp in Tv2-3 is in turn explained by Kr repressing svp in Tv1, and by grh repressing svp in Tv4. However, because grh misexpression is not sufficient to repress svp, it is tempting to speculate that there exists a similar factor to Kr, being exclusively expressed in the Tv4 cell, acting to suppress svp expression in a highly confined manner to ensure FMRFa/Tv4 specification (Stratmann, 2016).

Besides its activation by cas, col activation requires additional spatial information, provided by lbe, Antp, hth and exd, which subsequently initializes the generic Ap cluster program, by activating ap and eya. In contrast, cas alone activates grh and the sub-temporal factors, which are then important for the cell diversification, whether by activating or repressing each other's actions, or the FFLs, or partake in the FFL (grh), in order to allocate the correct cell fate to the four Ap cluster neurons. Remarkably, the four spatial inputs (lbe, Antp, hth and exd) act only on col, while the temporal input (cas) acts both on col, as well as the temporal and sub-temporal factors (sqz, nab, svp, Kr and grh). It is tempting to speculate that this may point to a general role for spatial versus temporal cues, and may be logically explained by the fact that spatial cues generally do not display the highly selective temporal expression profile necessary for sub-temporal cell diversification (Stratmann, 2016).

An unexpected finding in this study pertains to the dual role of Kr, first acting early in the canonical temporal cascade and subsequently late in the sub-temporal cascade, to ensure the specification of the Tv1 cell. The main role of Kr in Tv1 cells is to suppress svp, hence allowing for the maintenance of Col, which itself is critical for the propagation of the terminal FFL, fundamental for Tv1 cell fate. Interestingly, dual expression of Kr, first in the neuroblast and subsequently in neurons, was previously observed in NB3-3, but the functional role of the second Kr expression pulse was not addressed. svp itself also displays a dual expression and function, being expressed early in many NB lineages to suppress hb, then being re-expressed in several lineages, and in NB5-6T it acts to suppress col and dimm. With regards to postmitotic activity, another example of a temporal gene acting postmitotically applies to the role of the last temporal gene, grh, which is necessary and sufficient for FMRFa expression in Tv4 cells, and can trigger ectopic FMRFa in Ap neurons when misexpressed postmitotically. Yet in contrast to Kr, grh does not experience a dual expression profile. Hence, with several examples of dual (Kr and svp) and progenitor versus postmitotic roles of temporal genes (Kr and grh), it is tempting to speculate that this type of temporal multi-tasking may indeed be a common feature for many temporal genes, both in Drosophila and in higher organisms (Stratmann, 2016).

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 liodrlP;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).

Subtypes of glial cells in the Drosophila embryonic ventral nerve cord as related to lineage and gene expression

In the Drosophila embryonic CNS several subtypes of glial cells develop, which arrange themselves at characteristic positions and presumably fulfil specific functions. The mechanisms leading to the specification and differentiation of glial subtypes are largely unknown. By DiI labelling in glia-specific Gal4 lines the lineages of the lateral glia in the embryonic ventral nerve cord were clarified and each glial cell was linked to a specific stem cell. For the lineage of the longitudinal glioblast, it was shown to consist of 9 cells, which acquire at least four different identities. A large collection of molecular markers (many of them representing transcription factors and potential Gcm target genes) reveals that individual glial cells express specific combinations of markers. However, cluster analysis uncovers similar combinatorial codes for cells within, and significant differences between the categories of surface-associated, cortex-associated, and longitudinal glia. Glial cells derived from the same stem cell may be homogeneous (though not identical; stem cells NB1-1, NB5-6, NB6-4, LGB) or heterogeneous (NB7-4, NB1-3) with regard to gene expression. In addition to providing a powerful tool to analyse the fate of individual glial cells in different genetic backgrounds, each of these marker genes represents a candidate factor involved in glial specification or differentiation. This was demonstrated by the analysis of a castor loss of function mutation, which affects the number and migration of specific glial cells (Beckervordersandforth, 2008).

This report provides a comprehensive description of marker gene and enhancer trap expression in CNS glial cells of late Drosophila embryos. The markers include many transcription factors known to be involved in cell fate specification, as well as a number of still unknown factors. They were chosen for this analysis either because they were known to be expressed in subsets of glial cells or because they were known to be involved in cell fate determination in the nervous system. All together, more than 50 markers were tested, 39 of which showed expression in glial cells and hence were described in detail. Their specific expression patterns, though in many cases not restricted to glia, enable identification of groups of cells, as well as individual cells (Beckervordersandforth, 2008).

The lateral CNS glial cells have been assigned to three categories, according to their spatial distribution and morphology: the surface-, the cortex-, and the neuropile-associated glial cells. Categories were further divided into subgroups, as for example the surface-associated glial cells into subperineural glial cells and channel glia. Several of the molecular markers described in this study exhibit expression patterns, which correspond to the spatial/morphological definition of glial categories or subgroups. For example, moody or svp-lacZ are expressed in all surface-associated glial cells, whereas P101-lacZ is only expressed in the SPG-subgroup and engrailed only in the CG-subgroup. In addition, nearly each individual glial cell expresses a specific combination of markers indicating that they develop unique identities. Yet, nothing is known about how these identities are acquired. Comparing subtype affiliation or lineage ancestry of all glial cells with their respective marker gene expression patterns, it becomes obvious that glial cell specification is a process occurring on the level of individual cells. Cells might have a predisposition for a particular subtype laid down by lineage (e.g. NB5-6 derived cells to become subperineurial glia). In contrast, a temporal cascade within a lineage could determine individual cell identities (as it might be the case for the NB7-4 lineage) (Beckervordersandforth, 2008).

DiI labelling of the lineages of various progenitor cells in combination with cell-specific enhancer trap lines revealed that the composition of glial progeny within the lineages is invariant. Clonally related glia cells often express similar combinations of marker genes. The LGs, a prominent subgroup of the neuropile-associated glia and the only interface glia in the embryonic VNC, have been defined as the progeny of the LGB, which become aligned along the longitudinal connectives. However, there has been confusion about the size and composition of the LGB-lineage. By means of DiI labelling and marker gene expression, the size of this lineage was determined to be 9 cells. Although all cells of the LGB-lineage express a similar set of markers, a few markers are restricted to only parts of the lineage. Based on such markers, as well as positional criteria, the group of LGs was further subdivided. One of the cells, the LP-LG, is located slightly more lateral than the other LGs, and seems to be geared towards the ISN. Since it lies close to, and expresses a similar combination of markers as the M-ISNG, it would be justified to assign this cell to the group of nerve root glia; however, this was not done in order to avoid conflicts with the established nomenclature. Despite of their similarities, these two cells are of different origin: the LP-LG derives from the LGB, and the M-ISNG is generated by NB1-3 (Beckervordersandforth, 2008).

Most of the NBs that arise at corresponding positions and times in thoracic and abdominal segments (called serially homologous NBs) acquire the same fate, i.e. they generate the same lineages expressing corresponding sets of markers. However, some of these serially homologous lineages develop characteristic, tagma-specific differences with regard to cell number and/or cell types. Tagma-specific characteristics of these lineages have been shown to be under the control of Hox genes (Beckervordersandforth, 2008).

Although the total number of CNS glial cells is identical in thoracic and abdominal neuromeres, there are some differences in their origin and distribution of subtypes. This is due to tagma-specific differences among serially homologous lineages of NBs 1-1, 2-2, 5-6, and 6-4, which give rise to CBGs and SPGs. NB6-4A (A, abdominal) generates only two CBGs: MM-CBG and M-CBG, whereas the NB6-4T (T, thoracic) lineage comprises an additional MM-CBG2 and a neuronal sublineage. NB1-1T generates only neurons, whereas NB1-1A produces three SPGs (A-SP-G, B-SPG, and LV-SPG) in addition to neurons. In the thorax, the LV-SPG is presumably generated by NB5-6T, a cell at the position of A-SPG is produced by NB2-2T, and a cell at the B-SPG position is missing. Despite their different origin, the NB1-1A- and NB2-2T-derived SPGs specifically express hkb-lacZ and mirr-lacZ. Furthermore, the NB1-1A- and the NB2-2T-derived A-SPG appear to assume the same identity, as they express the same set of markers (including castor, which is not found in the abdominal B-SPG. Taken together, the differences between thorax and abdomen are restricted to only few glial cells, most of which acquire similar cell fates in thorax and abdomen (as judged by marker gene expression) irrespective of their progenitor (Beckervordersandforth, 2008).

The collection of marker genes and enhancer trap lines presented in this study provides powerful tools for the identification of specific glial cells in different genetic backgrounds. Each of markers also represents a candidate factor involved in glial subtype specification and/or differentiation. Many of these genes encode transcription factors known to be involved in cell fate specification, like fushi tarazu, mirror, and muscle segment homeobox. Other genes encode factors involved in cell signalling, e.g moody and CG11910, or enzymes like CG7433 and CG6218 (Beckervordersandforth, 2008).

moody is expressed in all cells belonging to the surface-associated glia. At the end of embryogenesis, surface-associated glial cells form a thin layer ensheathing the entire CNS, thereby establishing the blood-brain barrier. Moody is a G-protein coupled receptor, which acts in a complex pathway to regulate the cortical actin, thereby stabilizing the extended morphology of the surface-glia. This is necessary for the formation of septate junctions to achieve proper sealing of the nerve cord (Bainton, 2005; Daneman, 2005; Schwabe). Moody therefore represents a protein, which is essential for establishing and maintaining a specific function of surface glia (Beckervordersandforth, 2008).

Two of the markers analyzed represent metalloproteases: Neprilysin4 (Nep4) and Invadolysin. Invadolysin has been described to play a role in mitotic progression and in migration of germ cells (McHugh, 2004), but as for Nep4, its function in the nervous system is unknown. In vertebrates it has been shown that metalloproteases are involved in various processes in the CNS: they are associated with neurite outgrowth, migration of neurons and myelination of axons. For one matrix-metalloprotease, MMP-12, it has been shown that it is expressed in oligodendrocytes, where it functions in maturation and morphological differentiation of OL lineages (Larsen, 2004; Larsen, 2006). It has been postulated that LGs are analogous to vertebrate oligodendrocytes (Hidalgo, 2000), as both groups of cells enwrap axonal projections in the CNS, although to different degrees (no myelination in Drosophila). The two metalloproteases analysed are exclusively expressed in neuropile- (LGs) and cortex-associated glial cells (CBGs). Thus, both Nep4 and Invadolysin may possibly be involved in the differentiation of LGs. In invadolysin loss of function mutants, the specification of lateral glial cells does not seem to be affected, but the LGs show a very subtle phenotype in their positioning (data not shown). An explanation for the subtle phenotype may be redundant function of both enzymes. Indeed, in vertebrates it has been shown that metalloproteases have many overlapping substrates in vitro, and redundancy and compensation has been shown for matrix-metalloproteases (MMPs) in double mutants. Furthermore, it has been shown for members of the neprilysin family of metalloendopeptidases in Caenorhabditis elegans and Drosophila melanogaster, that many of the enzymatic properties have been conserved during evolution (Beckervordersandforth, 2008).

Making use of the molecular markers, this study characterized the phenotype of a cas loss of function mutation. Cas is a transcription factor, which acts in temporal cell fate specification. Together with Pdm, Cas is involved in the determination of late progeny cells in CNS lineages. In late embryonic stages, cas is specifically expressed in four glial cells per hemisegment, the V-CG and D-CG, the A-SPG and the LV-SPG, as well as in many neurons. The A- and LV-SPG, which are late progeny of the NB1-1A, are not affected in cas mutants, whereas the NB7-4-derived CGs seem mislocalized, with the medial migration of both CGs being impaired in cas mutants. This points to different functions of Cas in distinct NB lineages. As can be deduced from Repo stainings, general aspects of glial differentiation do not seem to be affected in cas mutants. Further analysis will have to clarify whether the role of Cas in NB7-4 derived glial cells is on the level of cell fate determination and/or whether it directly acts on specific aspects of differentiation (migration, motility). It also remains to be shown whether Cas acts cell-autonomously in this process (Beckervordersandforth, 2008).

Neuroblast entry into quiescence is regulated intrinsically by the combined action of spatial Hox proteins and temporal identity factors.

Neural stem cell quiescence is an important feature in invertebrate and mammalian central nervous system development, yet little is known about the mechanisms regulating entry into quiescence, maintenance of cell fate during quiescence, and exit from quiescence. Drosophila neural stem cells (neuroblasts or NBs) provide an excellent model system for investigating these issues. Drosophila NBs enter quiescence at the end of embryogenesis and resume proliferation during larval stages; however, no single neuroblast lineage has been traced from embryo into larval stages. This study establishes a model NB lineage, NB3-3, which allows reproducibly observation of lineage development from NB formation in the embryo, through quiescence, to the resumption of proliferation in larval stages. Using this new model lineage, a continuous sequence of temporal changes is shown in the NB, defined by known and novel temporal identity factors, running from embryonic through larval stages; quiescence suspends but does not alter the order of neuroblast temporal gene expression. NB entry into quiescence is regulated intrinsically by two independent controls: spatial control by the Hox proteins Antp and Abd-A, and temporal control by previously identified temporal transcription factors and the transcription co-factor Nab (Tsuji, 2008).

This study has revealed for the first time the temporal changes in a Drosophila NB lineage from embryonic NB formation, through entry into quiescence, to resumption of proliferation in larval stages. Using a model NB system with which the complete lineage formation can be reproducibly traced at the resolution of individual cell divisions, it was shown that despite considerable differences in extracellular environment the temporal changes (as defined by the switching of transcription factor/co-factor expression) proceeded continuously in each NB throughout the embryonic and larval stages. Moreover, mutual regulation was found between quiescence and the series of the temporal transcription factors/co-factor; the temporal transcription factors/co-factor endogenously control the timing of triggering NB quiescence, which in turn suspends the switching of late temporal transcription factor expression (Tsuji, 2008).

In the Antp mutant and following ectopic expression of Abd-A there was a lack of NB quiescence, and consequently what appeared to be a precocious generation of larval neurons during embryogenesis was observed. This strongly supports the notion that temporal changes in NBs actually continue in sequence before and after quiescence, i.e., through embryogenesis and larval stages, and in the absence of quiescence the changes occur precociously. In addition, this indicates that spatial and temporal factors control NB quiescence through independent routes (Tsuji, 2008).

Antp mutants did not exhibit NB3-3T quiescence in all thoracic T1-T3 segments. In Antp mutants, epidermis in T2 and T3 segments transform into that in the T1 segment, and some thoracic NB lineages retain thoracic-specific features. These facts indicate that the inhibition of NB3-3T quiescence by Antp mutation is not just a consequence of global transformation of thoracic-to-abdominal segments but rather results from specific effects on individual NBs. NB-specific misexpression of Abd-A also repressed Antp and inhibited NB3-3T quiescence. This also provides evidence that the effect is specific to NBs. Furthermore, because the effect could be observed even when Abd-A was induced after several divisions of the NB, thoracic NBs probably maintain plasticity of their identities during lineage formation (Tsuji, 2008).

It was shown that the temporal transcription factors/co-factor Pdm, Cas, Sqz and Nab play a role in triggering NB quiescence intrinsically in NBs. All of these factors also controlled temporal specification within late lineages of embryonic NBs in both thoracic and abdominal segments. This was confirmed by further examining the relationships of the temporal factors. For example, the loss of Pdm function in NB3-3T resulted in precocious transcription factor switching and precocious quiescence. Conversely, in cas mutant embryos, in which Pdm expression was de-repressed, quiescence was inhibited and expression of late-stage-specific temporal factors disappeared. Similar to Pdm upregulation, loss of nab function resulted in loss of both transcription factor switching and quiescence (Tsuji, 2008).

Although Nab and Sqz can form a complex, nab and sqz mutants displayed very different phenotypes. Both mutants showed de-repression of Kr expression; however, sqz mutants showed no other abnormality in transcription factor switching, whereas nab mutants showed the above-mentioned defects in transcription factor switching and timing of quiescence. These mutant phenotypes revealed that regulation of late temporal events is distributed into multiple pathways. Pdm probably coordinately regulates all factors involved in the timing of NB quiescence, because the loss of Pdm alone is sufficient to cause precocious entry into quiescence (Tsuji, 2008).

Nab and Sqz were shown to work for NB quiescence in NBs. The Nab/Sqz-mediated repression of Kr may be controlled in NBs due to changes in NB temporal identity, or in both NBs and their neurons. Nab might inhibit transcription by recruiting the nucleosome remodeling and deacetylase chromatin remodeling complex as does mammalian Nab (Srinivasan, 2006). Mammalian Nab acts with EGR-1, EGR-2 to determine the fate of cells in hematopoiesis (Laslo, 2006; Svaren, 1996), but whether it can act with the mammalian homolog of LIN-29/Sqz has not been reported. Loss of lin-29, a C. elegans homolog of sqz, causes a heterochronic phenotype in which adulthood is not reached and molting is repeated (Ambros, 1984; Rougvie, 1995). C. elegans has a nab homolog gene, mab-10, that acts with lin-29 in a heterochronic genetic cascade (Tsuji, 2008).

It is unclear what molecular mechanisms enable NBs to suspend the switching of transcription factor expression and maintain temporal identity during quiescence. It is known that the mechanisms for switching expression of early temporal transcription factors can be either cell division dependent or independent. Irrespective of the mechanism used, a NB can 'memorize' its temporal state before quiescence and resume the intrinsic temporal changes once cell cycle progression is reactivated. Embryonic stem cells may maintain multipotency during a slow proliferation state by staying in S phase. When quiescent NBs re-entered the cell cycle, their initial progeny incorporated BrdU fed since hatching, indicating that quiescent NBs stay either prior to S phase or early in S phase. It will be important to identify the point in the cell cycle at which NB enters quiescence (Tsuji, 2008).

Another well-established mechanism that governs temporal aspects of lineage formation is the heterochronic gene cascade in C. elegans. This cascade contains one each of the hunchback homolog and lin-29 genes and generates five distinct temporal cell identities within a single cell lineage. Drosophila NB lineage formation uses two types of Zn-finger proteins, namely the Hb/Cas class [Cas shares DNA-binding characteristics with Hb and the Kr/LIN-29 class. These pairs are expressed three times in NB lineages in the following order: (1) Hb and Kr-> (2) Cas, Kr and Sqz--> Cas and DmLin-29-->end of lineage. This sequence seems to produce at least ten distinct temporal identities within an NB lineage. The repetitive use of these temporal transcription factors in three distinct phases appears to have made the NB lineage longer and more diverse. Lack of Hb also generates NB lineage variety; the NB3-3 and NB2-1 lineages lack Hb and initiate their lineage with Kr. Although the model NB employed in this study lacks Hb, the sequence and entry into quiescence described in this study are common to many typical NB lineages that start with Hb. Interesting questions from the perspective of evolution are how do the three phases combine to form a single lineage and how has NB quiescence evolved in the middle of the NB lineages (Tsuji, 2008)?

Neural stem cells in the mouse cerebral cortex go through ~11 divisions and some enter quiescence in late embryo. The possibility has to be considered that mammalian neural stem cell and Drosophila NB share a similar intrinsic mechanism that induces quiescence (Tsuji, 2008).

Pdm and Castor close successive temporal identity windows in the NB3-1 lineage

Neurogenesis in Drosophila and mammals requires the precise integration of spatial and temporal cues. In Drosophila, embryonic neural progenitors (neuroblasts) sequentially express the transcription factors Hunchback, Kruppel, Pdm1/Pdm2 (Pdm) and Castor as they generate a stereotyped sequence of neuronal and glial progeny. Hunchback and Kruppel specify early temporal identity in two posterior neuroblast lineages (NB7-1 and NB7-3), whereas Pdm and Castor specify late neuronal identity in the NB7-1 lineage. Because Pdm and Castor have only been assayed in one lineage, it is unknown whether their function is restricted to neuronal identity in the NB7-1 lineage, or whether they function more broadly as late temporal identity genes in all neuroblast lineages. This study identified neuronal birth-order and molecular markers within the NB3-1 cell lineage, and then used this lineage to assay Pdm and Castor function. Hunchback and Kruppel were shown to specify first and second temporal identities, respectively. Surprisingly, Pdm does not specify the third temporal identity, but instead acts as a timing factor to close the second temporal identity window. Similarly, Castor closes the third temporal identity window. It is concluded that Hunchback and Kruppel specify the first and second temporal identities, an unknown factor specifies the third temporal identity, and Pdm and Castor are timing factors that close the second and third temporal identity windows in the NB3-1 lineage. The results provide a new neuroblast lineage for investigating temporal identity and reveal the importance of Pdm and Cas as timing factors that close temporal identity windows (Tran, 2008).

Pdm expression follows Hb and Kr in most neuroblasts, and thus is an excellent candidate for specifying the third temporal identity. Indeed, Pdm is necessary and sufficient to specify the third temporal identity (U4 neuron) within the NB7-1 lineage. To determine whether Pdm is a multi-lineage temporal identity gene, its loss-of-function and misexpression phenotype was examined in the NB3-1 lineage. Embryos homozygous for the deficiency Df(2L)ED773, which eliminates both pdm1 and pdm2 (henceforth referred to as pdm mutant embryos), were examined. In pdm mutant embryos, normal timing of Hb expression was observed in NB3-1 and other neuroblasts, a modest extension of Kr expression, and a similar delay in Cas expression. Consistent with this change in neuroblast gene expression, pdm mutant embryos showed normal specification of the early-born Hb+ RP1 and RP4 neurons, but possessed extra Kr+ RP3 neurons, followed by an apparently normal Cut+ late-born RP5. It is concluded that Pdm is not required to specify the third temporal identity (the Cut+ RP5 neuron), but is required to limit Kr expression in the neuroblast and thus close the second temporal identity window after the birth of just one Kr+ RP3 neuron (Tran, 2008).

Next it was determined whether the continuous expression of Pdm in NB3-1 was sufficient to induce ectopic RP5 neurons (i.e. extend the third temporal identity window). insc-gal4 UAS-pdm2 was used to generate continuous Pdm expression in neuroblasts, and normal timing of Hb expression in NB3-1 and other neuroblasts was observed, but premature loss of Kr expression and precocious Cas expression. Consistent with this change in neuroblast gene expression, normal specification was observed of the early-born Hb+ RP1 and RP4 neurons, but a lack of Kr+ RP3 neurons; there was also a loss of the Cut+ late-born RP5 neuron. It is concluded that Pdm is not sufficient to specify the third temporal identity (RP5), but rather it acts as a timer element to define the window of Kr expression and thus the length of the second temporal identity window. The precocious expression of Cas in these Pdm misexpression embryos may result in the precocious formation of Cas+ interneurons at the expense of the RP5 neuron (Tran, 2008).

Cas is expressed in NB3-1 following Hb, Kr and Pdm, but is not detected in any of the post-mitotic RP1-RP5 motoneurons. In addition, flies were examined carrying the cas-lacZ reporter transgene, and no residual β-galactosidase expression was observed in any NB3-1-derived RP neurons. This suggests that Cas expression is initiated after NB3-1 has made its fourth GMC, at the time when it shifts to producing local interneurons. Thus, although it was possible to test whether Cas is important for closing the third (RP5) temporal identity window, owing to the lack of interneuronal markers, it was not possible to assay for a Cas function in specifying the fourth (interneuron) temporal identity (Tran, 2008).

To test whether Cas is required to close the third temporal identity window, cas-null mutant embryos were examined. It was found that cas mutants have normal Hb and Kr expression in neuroblasts, but prolonged Pdm expression, consistent with previous work showing that Cas is required to repress pdm. At the neuronal level, it was found that cas mutants have normal early-born RP1, RP4 and RP3 neurons but possess ectopic RP5 neurons, consistent with a prolonged third temporal identity window. The ectopic RP5 neurons are not specified by the persistent Pdm protein because pdm mutants still formed apparently normal RP5 neurons and pdm cas double mutants still formed Cut+ RP5 neurons. Interestingly, cas mutants had a few RP-like (Islet+ HB9+) neurons that lacked expression of the motoneuron marker Late Bloomer and thus might have a mixed interneuron/RP motoneuron identity. Next insc-gal4 UAS-cas embryos, which have continuous expression of Cas in NB3-1, were examined. It was found that RP5 was often missing, but the early-born RP1, RP4 and RP3 were normal. It is concluded that the precocious expression of Cas is sufficient to close the third temporal identity window, in which RP5 is specified. Taken together, these results suggest that Cas is necessary and sufficient to close the third temporal identity window in the NB3-1 lineage (Tran, 2008).

This study has characterized the neuronal birth-order of the first four motoneurons within the NB3-1 lineage, described the temporal identity gene expression pattern within NB3-1 and its motoneuronal progeny, and performed a functional analysis of the four known and of candidate temporal identity genes. The results confirm and extend previous conclusions that Hb and Kr are multi-lineage temporal identity genes, and reveal novel aspects regarding the role of Pdm during the specification of temporal identity. It was found that both Pdm and Cas play essential roles as part of the neuroblast gene expression timer, Pdm closing the second temporal identity window and Cas closing the third (Tran, 2008).

It was shown that Hb and Kr are necessary and sufficient to specify the first and second temporal identities, respectively, in the NB3-1 lineage. It can now be concluded that Hb and Kr function as temporal identity factors in many spatial domains of the CNS [anterior-medial (NB3-1), posterior-medial (NB7-1) and posterior-lateral regions (NB7-3)], showing that temporal identity and spatial identity are independent with regards to Hb and Kr. Furthermore, Hb and Kr maintain similar functions in neuroblasts that form at distinct times during embryogenesis [early (NB7-1), middle (NB3-1) and late (NB7-3)], thus confirming that temporal identity is a lineage-autonomous event that is not coordinated by embryo-wide timing events. Overall, the data strongly support the conclusion that Hb and Kr are multi-lineage temporal identity genes (Tran, 2008).

The data also provide insight into neuroblast competence. When Hb was misexpressed in the NB3-1 lineage, it was possible to generate up to nine RP motoneurons; if each has a non-RP sibling, it would be close to the expected number of cells for the entire lineage. Thus, Hb seems capable of maintaining at least three very different neuroblast lineages (NB3-1, NB7-1 and NB7-3) in a 'young' state for their entire lineage. By contrast, misexpression of Kr produces only a few RP3 motoneurons before NB3-1 proceeds to make the later-born neurons. The inability of Kr to maintain a second temporal identity state might be due to the initiation of progressive restriction in neuroblast competence in NB3-1, as occurs in NB7-1 (Tran, 2008).

The findings show that Pdm is not required to specify the third temporal identity in the NB3-1 lineage, but rather that Pdm is a timer element that represses Kr expression and closes the second temporal identity window. Loss of Pdm allows for a transient extension of the Kr expression window, leading to the generation of a few ectopic Kr-specified RP3 neurons followed by a Cut+ RP5. It is hypothesized that the production of the RP5 cell is possible because Kr is not permanently maintained in the neuroblast. By contrast, permanent expression of Kr in NB3-1 (insc-gal4 UAS-Kr) also leads to extra RP3 neurons but does not allow production of a Cut+ RP5, perhaps owing to the continuous expression of Kr. Pdm is not the first transcription factor known to act as a timing element. The orphan nuclear hormone receptor Seven up (Svp) is required for repressing Hb in order to close the first temporal identity window in the NB7-1 and NB7-3 lineages and in the NB3-1 lineage. It should be noted that Svp represses Hb expression in all neuroblasts tested to date, whereas Pdm represses Kr expression in some but not all neuroblasts (Tran, 2008).

Pdm does not act as a timer element in all neuroblast lineages. For example, pdm mutants do not show extended Kr expression in the NB7-1 or NB7-3 lineages, as judged from the lack of ectopic Kr+ neurons in these lineages. These results suggest that the spatial identity of a neuroblast can alter its response to timing factors such as Pdm. Although this is counter to the simple model that spatial and temporal factors are independent and act combinatorially to specify birth-order identity within each lineage, it is consistent with the finding that spatial identity occurs at the time of neuroblast formation, prior to the expression of temporal factors. Taken together, these data suggest that spatial cues allow individual neuroblasts to respond differently to a temporal identity factor expressed at a similar time in all lineages (Tran, 2008).

The prior expression of early temporal identity factors is also likely to alter the response of a neuroblast to later temporal identity factors. Previous work has shown that misexpression of later temporal factors such as Kr, Pdm or Cas, has no detectable effect on the fate of first-born Hb+ neurons in the NB7-1 lineage. Consistent with these results, it was found that in the NB3-1 lineage, Pdm misexpression cannot repress Kr or activate Cas during the early Hb+ expression window. Just as prior spatial patterning cues may alter the response to a later temporal identity factor, so too may prior temporal identity factor expression alter the response of a neuroblast to later temporal identity factors. The mechanism by which spatial and temporal factors confer heritable changes to neuroblasts remains a mystery. An entrypoint into this mechanism could be the investigation of how Hb blocks Pdm from repressing Kr gene expression (Tran, 2008).

If Pdm does not specify temporal identity in NB3-1, what is the third temporal identity factor in this lineage? It has recently been reported that the SoxB family member Dichaete is expressed immediately prior to Cas in many embryonic neuroblast lineages (Maurange, 2008). However, Dichaete is only transiently expressed in medial column neuroblasts, such as NB3-1, at their time of formation and thus does not have the proper timing for a third temporal identity factor in this lineage. Alternatively, absence of Hb, Kr and Cas might specify the third temporal identity, with Pdm acting solely as a timing factor to establish a gap between Kr and Cas expression. Another possibility is that an as yet unknown factor specifies the third temporal identity in the NB3-1 lineage. Finally, Pdm might specify aspects of RP5 identity that was not possible to detect with the limited number of markers available; unfortunately, owing to severe morphological defects in late-stage pdm mutant embryos, it was not possible to assay the RP5 axon projection to its target muscle, which would provide a sensitive read-out of its neuronal identity (Tran, 2008).

Cas is expressed right after Pdm in most neuroblasts, and at the time NB3-1 is generating its fourth temporal identity (interneurons). cas mutants were found to have an extended window of Pdm neuroblast expression and exhibit production of ectopic RP5 neurons. Thus, Cas is required to close the third (RP5) temporal identity window. In addition, it was found that precocious expression of Cas can prematurely close the third temporal identity window and repress the specification of RP5. Comparable phenotypes were observed in the NB7-1 lineage, in which loss of Cas leads to ectopic U4 formation and gain of Cas results in the repression of the U4 identity. Based on these observations, it is predicted that Cas functions in multiple neuroblast lineages to close the third temporal identity window. Does Cas specify the fourth temporal identity? It was not possible to answer this question in the NB3-1 lineage owing to a lack of interneuron markers, but Cas does specify the fourth temporal identity (together with Pdm) in the NB7-1 lineage. In the future, the role of Cas in the NB3-1 lineage could be examined by making CD8::GFP-marked cas mutant clones and assaying neuronal identity by axon projections, or by developing molecular markers for interneurons within the lineage (Tran, 2008).

It is proposed that there are two classes of genes that regulate neuroblast temporal identity. One class, of which Hb and Kr are good examples, encodes temporal identity factors that are necessary and sufficient to directly specify a particular temporal identity in multiple neuroblast lineages. A second class encodes timing factors that establish the timing of temporal identity gene expression, but do not directly specify temporal identity. Timing factors, however, may indirectly influence the specification of temporal identities as seen in NB3-1, in which pdm is required to restrict the specification of RP3 and properly advance the neuroblast to the Cas-positive state. Seven up, the one timing factor identified previously, downregulates Hb protein levels and, along with cytokinesis, closes the first temporal identity window to facilitate the Hb --> Kr transition. The Kr --> Pdm --> Cas transitions are independent of cell-cycle progression. This study has shown that Pdm closes the second temporal identity window by repressing Kr expression and activating Cas in NB3-1. Taken together, these observations suggest that Kr and Pdm are involved in a negative-feedback loop in which Kr activates Pdm, which in turns represses Kr and activates Cas to advance neuroblast timing independent of cell-cycle progression. Through its role as a regulator of Kr and Cas timing, Pdm can restrict the production of neuronal cell types and advance the NB3-1 lineage (Tran, 2008).

Lineage and birth date specify motor neuron targeting and dendritic architecture in adult Drosophila

Locomotion in adult Drosophila depends on motor neurons that target a set of multifibered muscles in the appendages. This study describes the development of motor neurons in adult Drosophila, focusing on those that target the legs. Leg motor neurons are born from at least 11 neuroblast lineages, but two lineages generate the majority of these cells. Using genetic single-cell labeling methods, the birth order, muscle targeting, and dendritic arbors for most of the leg motor neurons were analyze. The results reveal that each leg motor neuron is born at a characteristic time of development, from a specific lineage, and has a stereotyped dendritic architecture. Motor axons that target a particular leg segment or muscle have similar dendritic arbors but can derive from different lineages. Thus, although Drosophila uses a lineage-based method to generate leg motor neurons, individual lineages are not dedicated to generate neurons that target a single leg segment or muscle type (Baek, 2010).

To study the development of the Drosophila leg motor neurons, a clonal analysis was performed using a modified version of mosaic analysis with a repressible cell marker (MARCM) method. The Vglut-Gal4 (also called OK371-Gal4) driver was used to positively label clones. This Gal4 driver, which is inserted into the Vglut gene, is expressed in all neurons that use glutamate as a neurotransmitter, including all motor neurons. As can be seen in adult leg preparations in which Vglut-Gal4 was used to express a membrane-tagged version of green fluorescent protein (CD8GFP), motor neurons innervating all of the muscles in the coxa (co), trochanter (tr), femur (fe), and tibia (ti) were labeled by this driver. In addition, a subset of sensory neurons, whose cell bodies reside in the tibia and tarsal segments, were labeled by Vglut-Gal4. Except for the tarsus, each leg segment has a stereotyped set of multifibered muscles that are labeled by the MHC-tauGFP reporter gene. This reporter gene was used to identify each of the muscles innervated by the leg motor neurons. In the adult CNS, Vglut-Gal4 labeled groups of neurons in each thoracic hemisegment. In addition to motor neuron cell bodies, the dendritic arbors of these neurons were observed in densely packed neuropils in each thoracic hemisegment. This study focused on the motor neurons innervating the first thoracic (T1) legs. The axons of these motor neurons fasciculate and exit the CNS through a large nerve that extends into the ipsilateral leg (Baek, 2010).

Drosophila NBs are born during embryogenesis and undergo two waves of neurogenesis, one during embryogenesis and one during larval development. During the first, embryonic wave of NB divisions, the majority of the embryonically born neurons are dedicated to larval motor and sensory functions and die during metamorphosis. To determine how many independent NB lineages give rise to the leg motor neurons, positively labeled MARCM clones were induced during embryogenesis and analyzed in the adult. Because these clones were generated infrequently and early in development, entire NB lineages were labeled. These data revealed that the leg motor neurons are derived from at least 11 independent lineages. Strikingly, two of these lineages, Lin A and Lin B, give rise to the majority of the leg motor neurons. Embryonically induced clones of Lin A innervated the muscles of the femur and tibia but did not include any motor neurons that targeted the coxa or trochanter. Moreover, the tibia is only targeted by Lin A-derived motor neurons. Thus, Lin A motor neurons generally target distal, but not proximal, leg segments (Baek, 2010).

The second major lineage defined by these experiments is Lin B, which gives rise to seven leg motor neurons. In contrast to Lin A, Lin B motor neurons target the three most proximal leg segments, the coxa, trochanter, and femur, but does not generate any motor neurons that target the tibia. Thus, Lin B motor neurons generally target proximal, rather than distal, leg segments (Baek, 2010).

Embryonically induced MARCM clones revealed that another 12 Vglut-Gal4+ leg motor neurons are generated from nine additional lineages, Lin C to Lin K. These 12 motor neurons target the coxa (six), the trochanter (one), and the femur (five), but not the tibia. In contrast to Lin A and Lin B, these lineages give rise to only one or two Vglut-Gal4-expressing leg motor neurons. Lin E is distinctive because, in addition to generating a single motor neuron targeting the coxa, it also gives rise to ~25 Vglut-Gal4-expressing interneurons. Five of these lineages (C to G) were labeled frequently, by both embryonic and postembryonic heat shocks. In contrast, four of these lineages, Lin H to Lin K, were labeled infrequently and only by embryonic heat shocks. These findings suggest that these motor neurons, which target the coxa (one) and femur (five), are born during embryogenesis and persist to the adult stage in which they contribute to the adult leg nervous system (Baek, 2010).

In total, 53 neurons were identified, derived from 11 independent NBs, that innervate the T1 leg. Two of these lineages give rise to 35 of these 53 motor neurons. By characterizing individually labeled motor neurons, the birth dates, muscle targets, and dendritic arbors for most of these motor neurons were determined. These results show that, although each motor neuron is born from a specific lineage, and at a specific time during development, individual lineages give rise to motor neurons that target multiple leg segments and multiple muscles within these leg segments (Baek, 2010).

Accurate motor neuron development in the fly requires that axons target the correct muscles along the PD axis of the leg. This axis has several levels of refinement. The first level is the global PD axis of the leg. Lin A only generates motor neurons that target the two more distal leg segments, the tibia and the femur. In addition, Lin A is the only lineage that produces motor neurons that target the tibia. In contrast, the seven Lin B motor neurons target all leg segments except the tibia. Thus, there is a PD bias built into these lineages (Baek, 2010).

A second level of refinement within the PD axis is targeting the correct muscle in individual leg segments. Among the Lin A-derived motor neurons, a PD bias was observed within the tibia and within the femur that correlates with birth date: the first half of the motor neurons born from Lin A have a strong bias for targeting proximal positions in these segments, whereas the later-born half of the motor neurons target distal muscles in these segments (Baek, 2010).

Third, for muscles that are targeted by multiple motor neurons (e.g., ltm1 in the tibia), it was found that more distal projecting motor neurons are born before those that target more proximal positions in the same muscle. The differential targeting of axons to unique positions within the same muscle suggests the existence of high-resolution topographic maps that match specific motor neurons to specific muscle compartments, as has been observed in mouse skeletal muscles (Baek, 2010).

Most of the leg motor neurons are born within a narrow window of development. The NB that gives rise to Lin A, for example, switches into a phase that is dedicated to generating leg motor neurons at ~50 h AEL. At that time, this NB begins to produce its 28 motor neurons for the next ~40 h. Presumably, this NB gives rise to nonmotor neuron progeny before this time and possibly after it completes this motor neuron generating phase. This scenario shares some similarities with the lineages that give rise to postembryonic neurons in the fly brain. For example, the entire mushroom body of Drosophila, the portion of the fly brain used in olfactory learning and memory, is derived from only four NBs that each give rise to one of four nearly identical anatomical units. Interestingly, there is a temporal switch in the types of neurons that these NBs generate at specific times of development. Thus, like Lin A, mushroom body NBs switch the type of neuron they generate at specific times. However, unlike the leg motor neuron NBs, those that generate the mushroom body are dedicated to forming this brain structure. In contrast, it was found that functionally related leg motor neurons, for example those that target a specific leg segment, muscle, or muscle type, are often derived from several different NB lineages. This logic is reminiscent of that used to generate olfactory projection neurons in the fly, in which three neuroblasts each give rise to different numbers and types of projection neurons (Baek, 2010).

The temporal control of NB identity in Drosophila is directed by transcription factors that are sequentially expressed as NBs age. During embryogenesis, progeny postmitotic neurons inherit the transcription factor expressed in the NB at the time it was born. This temporal information works in combination with positional information that makes each NB unique, providing progeny neurons their individual identities. Although the specific factors are not yet known, a similar transcription factor code may exist for leg motor neurons. Two of the temporal control genes that are used during Drosophila embryogenesis, seven-up (svp) and castor (cas), are also important for controlling postembryonic neural fates. Interestingly, some NBs switch from expressing cas to svp at ~50 h AEL, similar to the time that the leg NBs begin to generate their leg motor neuron progeny. It will be interesting to determine whether this or other changes in transcription factors are responsible for initiating the production of leg motor neurons in the lineages defined here (Baek, 2010).

These results demonstrate that adult motor neurons in the fly come from identifiable lineages that give rise to stereotyped progeny with defined birth dates. Importantly, however, of the 11 lineages that give rise to leg motor neurons in the fly, only one of these, Lin A, appears to be dedicated to producing these neurons. Even this restriction only occurs during the ~50 to ~90 h AEL time window. Although most of the progeny produced by the other lineages were not marked in these experiments (except for Lin E, which generates ~25 Vglut-Gal4+ interneurons), it is likely that these lineages also produce nonmotor neuron progeny. Thus, although seemingly invariant lineages are used in the fly, the closest relatives for many leg motor neurons are not other leg motor neurons. This conclusion is similar to the picture that emerged from lineage analyses performed in the vertebrate spinal cord showing that cell lineages are not dedicated to the production of motor neurons. As in the fly, closely related cells in the spinal cord may have distinct fates. Conversely, although adult fly motor neurons are born from stereotyped lineages, position within the CNS determines NB identities and, consequently, the progeny they generate. Although C. elegans has a more extreme version of a lineage-based mechanism, even in this case cell-cell signaling plays an important role in specifying identities. These considerations blur the distinction between lineage and position-based mechanisms and suggest that both play a role in vertebrates and invertebrates (Baek, 2010).

Consistent with the idea that lineage may play a role in vertebrates, the transcription factor Coup-TF acts as a temporal switch between neurogenesis and gliogenesis in the vertebrate brain. Interestingly, Coup-TF is a relative of Drosophila svp, which encodes one of the temporal transcription factors used in postembryonic fly neuroblasts. The use of Coup-TF/Svp for executing a temporal switch in both flies and vertebrates suggests the existence of a conserved molecular mechanism for controlling developmental timing in neural lineages (Baek, 2010).

Because motor neurons receive complex inputs from interneurons and sensory neurons, the architecture of their dendritic arbors is critical for forming the circuitry that is required for locomotion. An initial analysis of the dendritic arbors of the leg motor neurons suggests that, as in other systems, they exhibit a functional organization in the thoracic neuromere. For example, nine leg motor neurons, targeting two different reductor muscles in different leg segments (coxa and femur), have overlapping dendritic arbors. That these nine motor neurons have similar dendritic architectures suggests that they share presynaptic inputs, perhaps allowing these two reductor muscles to contract in synchrony. Similarly, all eight motor neurons that have dendrites that cross the midline of the CNS, and thus probably make contacts with neurons in the contralateral neuromere, send their axons to one of two long tendon muscles, one in the tibia and one in the femur. These two examples suggest that the organization of motor neuron dendrites may be important for muscle synergies as described in vertebrate locomotion (Baek, 2010).

In vertebrate motor systems, motor neuron cell bodies are organized in columns and pools that correlate with their muscle targets. This organization implies that many of the presynaptic inputs into the motor neurons within individual pools will be similar. Consistently, in some cases, the dendritic arbors of motor neurons have been shown to correlate with motor neuron targeting. In these examples, the arborization patterns are controlled by the transcription factor Pea3, which requires a specific Hox code to be activated, but is only induced after motor axons invade the limb target. In contrast, the myotopic map exhibited by the dendrites of the fly larval motor neurons does not need target muscles to form. In the fly olfactory system, the dendrites of projection neurons form a map in the antennal lobe before the arrival of olfactory receptor neurons (ORNs), suggesting that this map forms independently of ORNs. It remains unclear whether the characteristic dendritic arbors of the fly's leg motor neurons require muscle targeting or whether they form independently of their targets using local cues in the CNS and the identities they acquire at birth (Baek, 2010).

Developmental transcriptional networks are required to maintain neuronal subtype identity in the mature nervous system

During neurogenesis, transcription factors combinatorially specify neuronal fates and then differentiate subtype identities by inducing subtype-specific gene expression profiles. But how is neuronal subtype identity maintained in mature neurons? Modeling this question in two Drosophila neuronal subtypes (Tv1 and Tv4), tests were performed to see whether the subtype transcription factor networks that direct differentiation during development are required persistently for long-term maintenance of subtype identity. By conditional transcription factor knockdown in adult Tv neurons after normal development, it was found that most transcription factors within the Tv1/Tv4 subtype transcription networks are indeed required to maintain Tv1/Tv4 subtype-specific gene expression in adults. Thus, gene expression profiles are not simply 'locked-in,' but must be actively maintained by persistent developmental transcription factor networks. The cross-regulatory relationships were examined between all transcription factors that persisted in adult Tv1/Tv4 neurons. Certain critical cross-regulatory relationships that had existed between these transcription factors during development are no longer present in the mature adult neuron. This points to key differences between developmental and maintenance transcriptional regulatory networks in individual neurons. Together, these results provide novel insight showing that the maintenance of subtype identity is an active process underpinned by persistently active, combinatorially-acting, developmental transcription factors. These findings have implications for understanding the maintenance of all long-lived cell types and the functional degeneration of neurons in the aging brain (Eade, 2012).

The data provide novel insight supporting the view of Blau and Baltimore (1991) that cellular differentiation is a persistent process that requires active maintenance, rather than being passively 'locked-in' or unalterable. Two primary findings are made in this study regarding the long-term maintenance of neuronal identity. First, all known developmental transcription factors acting in postmitotic Tv1 and Tv4 neurons to initiate the expression of subtype terminal differentiation genes are then persistently required to maintain their expression. Second, it was found that key developmental cross-regulatory relationships that initiated the expression of certain transcription factors were no longer required for their maintained expression in adults. Notably, this was found to be the case even between transcription factors whose expression persists in adults (Eade, 2012).

In this study, all transcription factors implicated in the initiation of subtype-specific neuropeptide expression in Tv1 and Tv4 neurons were found to maintain subtype terminal differentiation gene expression in adults (see Summary of changes in subtype transcription network configuration between initiation and maintenance of subtype identity). In Tv1, col, eya, ap and dimm are required for Nplp1 initiation during development. In this study, knockdown of each transcription factor in adult Tv1 neurons was shown to dramatically downregulate Nplp1. In Tv4 neurons, FMRFa initiation during development requires eya, ap, sqz, dac, dimm and retrograde BMP signaling. Together with previous work showing that BMP signaling maintains FMRFa expression in adults (Eade, 2009), this study now demonstrates that all six regulatory inputs are required for FMRFa maintenance. Most transcription factors, except for dac, also retained their relative regulatory input for FMRFa and Nplp1 expression. In addition, individual transcription factors also retained their developmental subroutines. For example, as found during development, dimm was required in adults to maintain PHM (independently of other regulators) and FMRFa/Nplp1 expression (combinatorially with other regulators) (Eade, 2012).

The few genetic studies that test a persistent role for developmental transcription factors support their role in initiating and maintaining terminal differentiation gene expression. In C. elegans, where just one or two transcription factors initiate most neuronal subtype-specific terminal differentiation genes, they then also appear to maintain their target terminal differentiation genes. In ASE and dopaminergic neurons respectively, CHE-1 and AST-1 initiate and maintain expression of pertinent subtype-specific terminal differentiation genes. In vertebrate neurons, where there is increased complexity in the combinatorial activity of transcription factors in subtype-specific gene expression, certain transcription factors have been demonstrated to be required for maintenance of subtype identity. These are Hand2 that initiates and maintains tyrosine hydroxylase and dopa ß-hydroxylase expression in mouse sympathetic neurons, Pet-1, Gata3 and Lmx1b for serotonergic marker expression in mouse serotonergic neurons, and Nurr1 for dopaminergic marker expression in murine dopaminergic neurons (Eade, 2012).

However, while these studies confirm a role for certain developmental transcription factors in subtype maintenance, it had remained unclear whether the elaborate developmental subtype transcription networks, that mediate neuronal differentiation in Drosophila and vertebrates, are retained in their entirety for maintenance, or whether they become greatly simplified. This analysis of all known subtype transcription network factors in Tv1 and Tv4 neurons now indicates that the majority of a developmental subtype transcription network is indeed retained and required for maintenance. Why would an entire network of transcription factors be required to maintain subtype-specific gene expression? The combinatorial nature of subtype-specific gene expression entails cooperative transcription factor binding at clustered cognate DNA sequences and/or synergism in their activation of transcription. In such cases, the data would indicate that this is not dispensed with for maintaining terminal differentiation gene expression in mature neurons (Eade, 2012).

How the transcription factors of the subtype transcription networks are maintained is less well understood. An elegant model has emerged from studies in C. elegans, wherein transcription factors stably auto-maintain their own expression and can then maintain the expression of subtype terminal differentiation genes. The transcription factor CHE-1 is a key transcription factor that initiates and maintains subtype identity in ASE neurons. CHE-1 binds to a cognate DNA sequence motif (the ASE motif) in most terminal differentiation genes expressed in ASE neurons, as well as in its own cis-regulatory region. Notably, a promoter fusion of the che-1 transcription factor failed to express in che-1 mutants, indicative of CHE-1 autoregulation, and for the cooperatively-acting TTX-3 and CEH-10 transcription factors in AIY neurons. Thus, subtype maintenance in C. elegans is anchored by auto-maintenance of the transcription factors that initiate and maintain terminal differentiation gene expression (Eade, 2012).

In contrast, all available evidence in Tv1 and Tv4 neurons fails to support such an autoregulatory mechanism. An ap reporter (apC-t-lacZ) is expressed normally in ap mutants, and in this study apdsRNAi was not found to alter apGAL4 reporter activity. Moreover, col transcription was unaffected in col mutants that express a non-functional Col protein. This leaves unresolved the question of how the majority of the transcription factors are stably maintained. For transcription factors that are initiated by transiently expressed inputs, a shift to distinct maintenance mechanisms have been invoked and in certain cases shown. In this study, this was found for the loss of cas expression in Tv1 (required for col initiation) and the loss of cas, col and grh in Tv4 (required for eya, ap, dimm, sqz, dac initiation). However, it was surprising to find that the cross-regulatory relationships between persistently-expressed transcription factors were also significantly altered in adults. Notably, eya initiated but did not maintain dimm in Tv4. In Tv1, col initiated but did not maintain eya, ap or dimm. This was particularly unexpected as eya remained critical for FMRFa maintenance and col remained critical for Nplp1 maintenance. Indeed, although tests were performed for cross-regulatory interactions between all transcription factors in both the Tv1 and Tv4 subtype transcription networks, only Dimm was found to remain dependent upon its developmental input; Eya and Ap in Tv1 as well as Ap in Tv4. However, even in this case, the regulation of Dimm was changed; it no longer required eya in Tv4, and in Tv1 it no longer required col, in spite of the fact that both col and eya are retained in these neurons. It is anticipated that such changes in transcription factor cross-regulatory relationships will be found in other Drosophila and vertebrate neurons, which exhibit high complexity in their subtype transcription networks. Indeed, recent evidence has found that in murine serotonergic neurons, the initiation of Pet-1 requires Lmx-1b, but ablation of Lmx-1b in adults did not perturb the maintenance of Pet-1 expression (Eade, 2012).

The potential role of autoregulation for the other factors in the Tv1/Tv4 subtype transcription networks is being pursued. However, there are three additional, potentially overlapping, models for subtype transcription network maintenance. First, regulators may act increasingly redundantly upon one another. Second, unknown regulators may become increasingly sufficient for transcription factor maintenance. Third, transcription factors may be maintained by dedicated maintenance mechanisms, as has been shown for the role of trithorax group genes in the maintenance of Hox genes and Engrailed. Moreover, chromatin modification is undoubtedly involved and likely required to maintain high-level transcription of Tv transcription factors as well as FMRFa, Nplp1 and PHM. However, the extent to which these are instructive as opposed to permissive has yet to be established. In this light, it is intriguing that MYST-HAT complexes, in addition to the subtype transcription factors Che-1 and Die-1, are required for maintenance of ASE-Left subtype identity in C. elegans (Eade, 2012).

Taken together, these studies have identified two apparent types of maintenance mechanism that are operational in adult neurons. On one hand, there are sets of genes that are maintained by their initiating set of transcription factors. These include the terminal differentiation genes and the transcription factor dimm. On the other, most transcription factors appear to no longer require regulatory input from their initiating transcription factor(s). Further work will be required to better understand whether these differences represent truly distinct modes of gene maintenance or reflect the existence of yet unidentified regulatory inputs onto these transcription factors. One issue to consider here is that the expression of certain terminal differentiation genes in neurons, but perhaps not subtype transcription factors, can be plastic throughout life, with changes commonly occurring in response to a developmental switch or physiological stimulus. Thus, terminal differentiation genes may retain complex transcriptional control in order to remain responsive to change. It is notable, however, that FMRFa, Nplp1 and PHM appear to be stably expressed at high levels in Tv1/4 neurons, and no conditions were found that alter their expression throughout life. Thus, these are considered to be stable terminal differentiation genes akin to serotonergic or dopaminergic markers in their respective neurons that define those cells' functional identity and, where tested, are actively maintained by their developmental inputs. Tv1/4 neurons undoubtedly express a battery of terminal differentiation genes, and sets of unknown transcription factors are likely required for their subtype-specific expression. Subtype transcription networks are considered to encompass all regulators required for differentiating the expression of all subtype-specific terminal differentiation genes. Further, differentiation of subtype identity is viewed as the completion of a multitude of distinct gene regulatory events in which each gene is regulated by a subset of the overall subtype transcription network. As highly restricted terminal differentiation genes expressed in Tv1 and Tv4 neurons, it is believed that Nplp1, FMRFa and PHM provide a suitable model for the maintenance of overall identity, with the understanding that other unknown terminal differentiation genes expressed in Tv1 and Tv4 may not be perturbed by knockdown of the transcription factors tested in this study. In the future, it will be important to incorporate a more comprehensive list of regulators and terminal differentiation genes for each neuronal subtype. However, it is believed that the principles uncovered in this study for FMRFa, Nplp1 and PHM maintenance will hold for other terminal differentiation genes (Eade, 2012).

Finally, it is proposed that the active mechanisms utilized for maintenance of subtype differentiation represent an Achilles heel that renders long-lived neurons susceptible to degenerative disorders. Nurr1 ablation in adult mDA neurons reduced dopaminergic markers and promoted cell death. Notably, Nurr1 mutation is associated with Parkinson's disease, and its downregulation is observed in Parkinson's disease mDA neurons. Adult mDA are also susceptible to degeneration in foxa2 heterozygotes, another regulator of mDA neuron differentiation that is maintained in adult mDA neurons. Studies in other long-lived cell types draw similar conclusions. Adult conditional knockout of Pdx1 reduced insulin and ß-cell mass and, importantly, heterozygosity for Pdx1 leads to a rare monogenic form of non-immune diabetes, MODY4. Similarly, NeuroD1 haploinsufficiency is linked to MODY6 and adult ablation of NeuroD in β-islet cells results in β-cell dysfunction and diabetes. These data, together with current results, underscore the need to further explore the transcriptional networks that actively maintain subtype identity, and hence the function, of adult and aging cells (Eade, 2012).


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Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 23 August 2014

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