nubbin/POU domain protein 1



pdm-1 is first expressed in the cellular blastoderm stage as two bands, each 8-10 cells wide, in the primordia of the abdominal segments, and in the head region in the anlage of the clypeolabium [Images]. The pattern of expression is the same as that of pdm-2. pdm-1 later exhibits a striped pattern at germ band extention where its pattern overlaps that of ftz. This expression is in the region of the neuroectoderm. During germ band retraction, pdm-1 transcripts are detected in the endoderm in the developing anterior and posterior midgut primordia (Affolter, 1993). pdm-1 is expressed later in a subset of CNS and PNS cells (Dick, 1991 and Lloyd, 1991). Thus, very specific neuroblasts express pdm-1. In the early part of the NB4-2 lineage, from which the RP2 motor neuron is derived, pdm-1 and pdm-2 are expressed. These genes are not required for the birth of the first ganglion mother cell (GMC4-2a) but both are involved in specifying its identity (Yeo, 1995).

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 neuroblast (NB) sublineages, Hb and Cas establish three pan-CNS compartments whose cellular constituents are marked by the expression of either Hb, Pdm, or Cas (see Lateral views of Drosophila CNS). 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. 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 (see Views of cephalic lobe neuroblasts) 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).

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).

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.

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).

The two closely related Drosophila POU-domain genes, pdm1 (nubbin) and pdm2, are co-expressed in the developing CNS (before stage 13) and have been shown (at least with respect to the specification of the first ganglion mother cell of the truncal NB4-2) to be functionally redundant. pdm1 is expressed in the trunk neuroectoderm during the first and second wave of NB segregation (stage 8/9), and transiently in most NBs at stage 10 and 11. In the procephalon the expression of the Pdm1 protein is highly dynamic. Until stage 10, Pdm1 is roughly restricted to the neuroectoderm of the antennal and ocular segments. Later, it is also found in the intercalary and labral ectoderm. At stage 9, NBs derived from Pdm1-positive neuroectoderm appear to be Pdm1 negative and initiate pdm1 expression at stage 10 or stage 11. At late stage 11, approximately one half of the brain NBs (about 52 NBs) express pdm1, including most deuto- and trito-cerebral NBs, as well as central ocular NBs and part of the labral 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).

Drosophila embryonic type II neuroblasts: origin, temporal patterning, and contribution to the adult central complex

Drosophila neuroblasts are an excellent model for investigating how neuronal diversity is generated. Most brain neuroblasts generate a series of ganglion mother cells (GMCs) that each make two neurons (type I lineage), but sixteen brain neuroblasts generate a series of intermediate neural progenitors (INPs) that each produce 4-6 GMCs and 8-12 neurons (type II lineage). Thus, type II lineages are similar to primate cortical lineages, and may serve as models for understanding cortical expansion. Yet the origin of type II neuroblasts remains mysterious: do they form in the embryo or larva? If they form in the embryo, do their progeny populate the adult central complex, as do the larval type II neuroblast progeny? This study presents molecular and clonal data showing that all type II neuroblasts form in the embryo, produce INPs, and express known temporal transcription factors. Embryonic type II neuroblasts and INPs undergo quiescence, and produce embryonic-born progeny that contribute to the adult central complex. These results provide a foundation for investigating the development of the central complex, and tools for characterizing early-born neurons in central complex function (Walsh, 2017).

It has been difficult to link embryonic neuroblasts to their larval counterparts in the brain and thoracic segments owing to the period of quiescence at the embryo-larval transition, and owing to dramatic morphological changes of the CNS that occur at late embryogenesis. Recent work has revealed the embryonic origin of some larval neuroblasts: the four mushroom body neuroblasts in the central brain and about 20 neuroblasts in thoracic segments. This study used molecular markers and clonal analysis to identify all eight known type II neuroblasts in each brain lobe and show they all form during embryogenesis, perhaps the last-born central brain neuroblasts. It was not possible to identify each neuroblast individually, however, owing to their tight clustering, movements of the brain lobes, and the lack of markers for specific type II neuroblasts (Walsh, 2017).

The single previously reported embryonic type II neuroblast formed from PntP1+ neuroectodermal cells with apical constrictions called a placode. This study did not investigate this neuroectodermal origin of type II neuroblasts in much detail, but multiple type II neuroblasts were seen developing from PntP1+ neuroectoderm. In the future, it would be interesting to determine whether all type II neuroblasts arise from PntP1+ neuroectoderm or from neuroectodermal placodes. Interestingly, one distinguishing molecular attribute of type II neuroblasts is PntP1, which is not detected in type I neuroblasts. Thus, a candidate for distinguishing type I/type II neuroblast identity is EGF signaling, which can be detected in the three head placodes and is required for PntP1 expression. Clearly, there are more PntP1+ neuroectodermal cells than there are type II neuroblasts, and expression of an EGF negative regulator such as Argos might be necessary to divert some of these neuroectodermal cells away from type II neuroblast specification. The earliest steps of type II neuroblast formation represent an interesting spatial patterning question for future studies (Walsh, 2017).

Now that the embryonic type II neuroblasts have been identified, it is worth considering whether there are differences between embryonic and larval type II neuroblasts or their INP progeny. To date, molecular markers do not reveal any differences between embryonic and larval type II neuroblasts, with the exception that embryonic neuroblasts transiently express the temporal transcription factor Pdm. Interestingly, type I embryonic neuroblasts require Cas to close the Pdm expression window, whereas this study found that cas mutants do not exhibit extension of the Pdm expression window in the earliest-born type II neuroblast or de novo expression of Pdm in the later-forming neuroblasts. Are there differences between embryonic and larval INPs? Larval INPs mature over a period of 6 h and then divide four to six times with a cell cycle of about 1 h. In contrast, embryonic INPs might have a more rapid maturation because Elav+ neurons were seen within 9D11+ INP lineages by stage 14, just 3 h after the first type II neuroblast forms. This study found that INPs undergo quiescence at the embryo-larval transition, as shown by the pools of INPs at stage 16 that do not stain for the mitotic marker pH3. The fate of these quiescent INPs -- whether they resume proliferation, differentiate or die -- remains to be determined (Walsh, 2017).

Neuroblasts in the embryonic ventral nerve cord use the temporal transcription factor cascade Hb>Krüppel>Pdm>Cas>Grh to generate neural diversity. This study shows that the type II neuroblasts are among the last neuroblasts to form in the embryonic brain, and that they sequentially express only the late temporal transcription factors Pdm (in the earliest-forming neuroblast) followed by Cas and grh (in all eight type II neuroblasts). It is unknown why most type II neuroblasts skip the early Hb>Krüppel>Pdm temporal transcription factors; perhaps it is due to their late time of formation, although several earlier-forming thoracic neuroblasts also skip Hb (NB3-3), Hb>Krüppel (NB5-5), or Hb>Krüppel>Pdm. This is another interesting spatial patterning question for the future. Furthermore, misexpression of the early factors (Hb and Krüppel) would be unlikely to affect the progeny produced by type II NBs during embryogenesis, as the competence window for Hb (i.e., the stage at which neuroblasts are responsive to Hb expression) closes with the loss of Dan/Danr expression in all neuroblasts at stage 12. Thus, most embryonic type II neuroblasts form after closing of the Hb competence window and would probably be unresponsive (Walsh, 2017).

Type I neuroblasts show persistent expression of the temporal transcription factors within neurons born during each window of expression (i.e. a Hb+ neuroblast divides to produce a Hb+ GMC which makes Hb+ neurons). In contrast, this study found that type II lineages do not show persistent Cas or grh expression in INPs born during each expression window, but do contain some Cas+ neurons. Both Cas and grh transcription factors can be seen in INPs immediately adjacent to the parental neuroblast, consistent with transient perdurance from the parental neuroblast, but they are typically lacking in INPs more distant. The function of Pdm, Cas and grh in embryonic type II neuroblasts awaits identification of specific markers for neural progeny born during each expression window (Walsh, 2017).

During larval neurogenesis, virtually all INPs sequentially express the temporal transcription factors Dichaete>Grh>Ey. In contrast, embryonic INPs express only Dichaete. These data, together with the short time frame of embryogenesis, suggest that INP quiescence occurs during the Dichaete window, preventing expression of the later Grh>Ey cascade. Interestingly, INPs in the posterior cluster (presumptive DL1 and DL2 type II neuroblast progeny) completely lack Dichaete; this is similar to the DL1 and DL2 larval lineages, which also do not express Dichaete. It is possible that DL1/DL2 neuroblasts make INPs that generate identical progeny (and thus do not require an INP temporal cascade), or perhaps these two neuroblasts use a novel temporal cascade in both embryonic and larval stages (Walsh, 2017).

Larval type II neuroblasts produce many intrinsic neurons of the adult central complex. This study shows that embryonic INPs also produce neurons that contribute to the adult central complex. The data show ~54 neurons (64 minus 10 due to 'leaky' expression) born from embryonic-born INPs survive to adulthood and innervate the central complex. It is likely that this is an underestimate, however, because (1) 9D11-gal4 expression is lacking from a few INPs in the embryonic brain and (2) the time to achieve sufficient FLP protein levels to achieve immortalization could miss the earliest born neurons. The variation in immortalization of the widefield ellipsoid body neuron might represent a neuron born early in the type II lineages, thus unlabeled in a subset of embryos. Additionally, some embryonic-born neurons might perform important functions in the larval/pupal stages but die prior to eclosion (Walsh, 2017).

Further studies will be required to understand the function of neurons born from embryonic type II lineages. It remains to be experimentally determined whether some or all embryonic progeny of type II neuroblasts (1) remain functionally immature in both the larval and adult brain, but serve as pioneer neurons to guide larval-born neurons to establish the central complex, (2) remain functionally immature in the larval brain, but differentiate and function in the adult central complex, or (3) differentiate and perform a function in both the larval and adult CNS. It will be informative to ablate embryonic-born neurons selectively and determine the effect on the assembly of the larval or adult central complex, and their role in generating larval and adult behavior (Walsh, 2017).

The selector gene cut represses a neural cell fate that is specified independently of the Achaete-Scute-Complex and atonal

The peripheral nervous system of Drosophila offers a powerful system to precisely identify individual cells and dissect their genetic pathways of development. The mode of specification of a subset of larval PNS cells, the multiple dendritic (md) neurons (or type II neurons), is complex and still poorly understood. A morphological categorization of md neurons reveals three subpopulations: md-da neurons are the most abundant subclass, which have extensive dendritic arborizations; md-bd neurons have bipolar dendrites; and md-td neurons extend their dendrites along tracheal branches. Within the dorsal thoracic and abdominal segments, two md neurons, dbd and dda1, apparently require the proneural gene amos but not atonal or Achaete-Scute-Complex (ASC) genes. ASC normally acts via the neural selector gene cut to specify appropriate sensory organ identities. Dbd- and dda1-type differentiation is suppressed by cut in dorsal ASC-dependent md neurons. Thus, cut is not only required to promote an ASC-dependent mode of differentiation, but also represses an ASC- and ato-independent fate that leads to dbd and dda1 differentiation (Brewster, 2001).

pdm1 and pdm2, two closely linked genes belonging to the POU family of transcription factors, are co-expressed in two PNS neurons that are potentially coincident with ASC/ato-independent neurons: dbd and a dorsal md-da neuron. In addition, the ligament cells of lch5 also weakly express these genes. To determine if the pdm-expressing cells are ASC-and ato-independent, the pdm1 expression pattern was examined in ASC and ato single and double mutant embryos. In all three mutant configurations pdm1 expression is present in dbd and a dorsal md-da neuron. The latter will be referred to henceforth as dda1 (dorsal da neuron #1). Thus, pdm specifically marks the ASC/ato-independent subclass of PNS neurons (Brewster, 2001).

In order to further characterize ASC/ato-independent md neurons, other markers expressed in these cells were sought. en and the lacZ reporter gene from the E7-3-49 enhancer trap line were identifed. In the PNS, en is expressed in one dorsal md neuron as early as stage 11. The E7-3-49 line confers lacZ expression to several PNS cells, including dbd and 2-4 dorsal da-md neurons. Co-incidence of expression of pdm1 with that of en and E7-3-49 was examined in the PNS. Double-labeling for expression of pdm1 and E7-3-49-lacZ reveals that they overlap in dda1 and dbd. Similarly, pdm and en are co-expressed in dda1 but not in dbd (Brewster, 2001).

The expression pattern of the homeobox neural selector gene cut encompasses all es organs and a large number of md neurons (the majority of which are related to es organs by lineage). cut is clearly not expressed in the readily identifiable dbd neuron. Since dbd and dda1 co-express the markers described above and perhaps are specified by the same proneural gene(s), whether dda1 is indeed negative for cut expression was examined. Embryos double-labeled for cut and E7-3-49-lacZ or pdm show that the pattern of cut expression in the dorsal PNS cluster is complementary to these markers. These results indicate that, unlike the majority of md neurons, dda1 and dbd (along with its sibling glial cell) are specified in a cut-independent fashion (Brewster, 2001).

The identity of the transformed md-da neurons in cut mutants was examined with markers for ASC/ato-independent neurons. Similar to E7-3-49, the expression of pdm1 is expanded to additional neurons in the dorsal and lateral PNS clusters. The cells expressing pdm1 ectopically are also positive for a marker, the E7-2-36 enhancer trap line, which is specific for all md neurons, suggesting that the extra Pdm1-positive neurons are indeed md neurons. The mechanism for restricting ectopic pdm1 but not E7-3-49- lacZ expression to the dorsal and lateral clusters is not known. Overall, these findings are consistent with the interpretation that in cut mutants many ASC-dependent md neurons are transformed towards an ASC/ato-independent rather than an ato-dependent fate (Brewster, 2001).

In contrast to these findings with E7-3-49-lacZ and pdm1 expression, when en expression was examined in cut mutants, the pattern of en-expressing PNS cells is unaltered, i.e. there is only one En-positive cell per dorsal cluster. Since en is not expressed in dbd but pdm1 is, the possibility that the invariance of en expression reflects the acquisition of a dbd rather than a dda1 cell fate in cut mutants cannot be ruled out. This possibility seems unlikely, however, since the morphology of supernumerary Pdm1-positive cells is unlike that of dbd, and a marker for the dbd-associated glial cell (repo) is not ectopically expressed in cut mutants (Brewster, 2001).

Taken together, it appears that in cut mutants the md neurons that normally depend on ASC in dorsal and lateral clusters are transformed towards an ASC/ato-independent fate, as determined by pdm and E7-3-49-lacZ expression. However, the postulated cell fate change may be incomplete due to the lack of ectopic en expression. This partial phenotype is not surprising, since cut (null) mutants also exhibit variability and incomplete phenotypic penetrance with respect to es organ transformation towards a ch fate. It is thus likely that gene functions other than cut also contribute to the restriction of en and pdm1, similar perhaps to the situation of ato-dependent md neurons, which do not express cut or pdm/en (Brewster, 2001).

Context-dependent utilization of Notch activity in Drosophila glial determination

During Drosophila neurogenesis, glial differentiation depends on the expression of glial cells missing. Understanding how glial fate is achieved thus requires knowledge of the temporal and spatial control mechanisms directing gcm expression. In the adult bristle lineage, gcm expression is negatively regulated by Notch signaling. The effect of Notch activation on gliogenesis is context-dependent. In the dorsal bipolar dendritic (dbd) sensory lineage in the embryonic peripheral nervous system (PNS), asymmetric cell division of the dbd precursor produces a neuron and a glial cell, where gcm expression is activated in the glial daughter. Within the dbd lineage, Notch is specifically activated in one of the daughter cells and is required for gcm expression and a glial fate. Thus Notch activity has opposite consequences on gcm expression in two PNS lineages. Ectopic Notch activation can direct gliogenesis in a subset of embryonic PNS lineages, suggesting that Notch-dependent gliogenesis is supported in certain developmental contexts. Evidence is presented that POU-domain protein Nubbin/PDM-1 is one of the factors that provides such context (Umesono, 2002).

Coexpression of constitutively active Notch with Nubbin generates ectopic glia outside dbd and dda lineages. This raises the possibility that Nubbin may be a part of the developmental context that allows Notch to promote gliogenesis. Within the embryonic PNS, dbd and dda neurons are the only two neurons that express Nubbin. In both lineages, Nubbin is present in both SOP daughter cells, at the time of glia versus neuron cell fate choice. Furthermore, temporal activation of Nubbin has been detected in presumptive glial cells derived from the NB1-1A lineage. Nubbin thus might create a permissive environment for the activation of gcm expression by the Notch signal. Since coexpression of Nubbin and constitutively active Notch does not cause glial transformation of all neurons, additional factors must exist that create a Notch-dependent gliogenic context (Umesono, 2002).

Nubbin is a POU-domain transcription factor with sequence-specific DNA-binding activity. The contextual role of Nubbin in Notch-dependent expression of gcm could employ a similar mechanism to the modulation of Notch activity in wing development, where Nubbin and Su(H) bind on the same enhancer element of Notch target genes. It will be interesting to further analyze the role of Nubbin in gliogenic lineages (Umesono, 2002).

Proximodistal subdivision of Drosophila legs and wings: The elbow-no ocelli gene complex functions upstream of Nub in the formation of the wing primordium

Appendages are thought to have arisen during evolution as outgrowths from the body wall of primitive bilateria. In Drosophila, subsets of body wall cells are set aside as appendage precursors through the action of secreted signaling proteins that direct localized expression of transcription factors. The Drosophila homeodomain protein Distal-less is expressed in the leg primordia and required for formation of legs, but not wings. The homeodomain protein Nubbin is expressed in the wing primordia and required for formation of wings, but not legs. Given that insect legs and wings have a common developmental and evolutionary origin, attempts were made to identify genes that underlie the specification of all appendage primordia. Evidence is presented that the zinc-finger proteins encoded by the elbow and no ocelli genes act in leg and wing primordia to repress body wall-specifying genes and thereby direct appendage formation (Weihe, 2004).

Evidence suggests that the el and noc genes serve as mediators of the function of the Wg and Dpp signaling systems in specification of the appendage field within the imaginal discs. El and Noc are induced by Wg and Dpp and are required to repress the proximally expressed proteins Hth and Tsh. Previous work had identified Dll as a gene required for appendage formation in leg and antenna, and nub as a gene required for wing. This report identifies El and Noc as a pair of zinc-finger proteins that function in both ventral and dorsal appendages. However, there are interesting differences in the way that they do so, when examined in detail (Weihe, 2004).

Dll expression is required for the formation of all leg and antenna elements in the ventral (leg) discs, and until this work Dll was the earliest known marker for the distal region leg disc. Previous work has shown that repression of Hth and Tsh by Dpp and Wg was not required for expression of Dll in the leg, nor could Dll repress Hth and Tsh. Thus an essential mediator of the effects of Wg and Dpp was missing. The current results present evidence that El and Noc serve this function, since their removal leads to ectopic expression of Hth and Tsh. Removal of El and Noc does not cause loss of Dll expression, so it is concluded that Wg and Dpp act independently to induce El and Noc expression and Dll to define the distal region of the leg disc (Weihe, 2004).

The situation differs slightly in the wing. Repression of Tsh is the earliest marker for specification of the distal wing region, preceding the onset of Hth repression or of Nub induction. Loss of Tsh and Hth are required to allow Nub expression. Ectopic expression of Hth and Tsh and loss of Nub is observed in clones lacking El and Noc activity. Thus in the wing, expression of the distal marker Nub cannot be demonstrated to be independent of El and Noc (because ectopic Hth can repress Nub, but not Dll). The vestigial gene is also important for wing development and has been proposed to be a wing specifying gene. However, Vestigial is expressed all along the DV boundary of the wing, both in the wing primordium and in the body wall. This led to the suggestion that while Vestigial is essential for wing development, its expression cannot be taken as a molecular marker for wing identity per se, particularly at early stages. For this reason analysis of the relationship between El, Noc and Vestigial was not performed in this study (Weihe, 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 drawned.

  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).

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).


The pdm-1 gene is expressed in wing and leg discs. In the wing it appears to be required for the hinge, suggesting an involvement in proximal-distal growth control (Ng, 1995).

Flexible joints separate the rigid sections of the insect leg, allowing them to move. In Drosophila, the initial patterning of these joints is apparent in the larval imaginal discs from which the adult legs will develop. The later patterning and morphogenesis of the joints, which occurs after pupariation (AP), is described. In the tibial/tarsal joint, the apodeme insertion site provides a fixed marker for the boundary between proximal and distal joint territories (the P/D boundary). Cells on either side of this boundary behave differently during morphogenesis. Morphogenesis begins with the apical constriction of distal joint cells, about 24 h AP. Distal cells then become columnar, causing distal tissue nearest the P/D boundary to fold into the leg. In the last stage of joint morphogenesis, the proximal joint cells closest to the P/D boundary align and elongate to form a 'palisade' (a row of columnar cells) over the distal joint cells. The proximal and distal joint territories are characterized by the differential organization of cytoskeletal and extracellular matrix proteins, and by the differential expression of enhancer trap lines and other gene markers. These markers also define a number of more localised territories within the pupal joint (Mirth, 2002).

To identify distinct cell populations in the joints, the expression patterns of 10 joint markers were examined with respect to a posterior marker (engrailed lacZ) and a ventral marker (wingless lacZ). The leg discs of wandering larvae, and pupal legs at 24-28 and 34-38 h AP, were examined. Four of the joint markers were previously reported to be expressed in L3 and prepupal joints (Notch, disconnected lacZ, Nubbin, and odd-skipped lacZ). The rest were isolated for this study by screening Gal4 enhancer trap lines for those that drive expression of GFP in pupal leg joints (ckm78, ckm90, ckm239, ckm175, ok388, and ok483). Most of the joint markers do not change their expression domains between 24-28 and 34-38 h AP. Therefore, data is presented from wandering L3 discs only, and from legs at 34-38 h AP (Mirth, 2002).

In the L3 leg disc, joint markers fall into one of two categories, marking either the proximal joint territories (e.g., Nubbin) or the distal territories (e.g., Notch and odd-skipped lacZ). Of all the markers examined, only Nubbin (Nub), disconnected lacZ (disco lacZ), and odd-skipped lacZ (odd lacZ) are expressed in more than two joints in the L3 stage. Others mark one or two joints at this stage but are expressed in all joints during the pupal stage. Studies examining the expression of Notch and other elements of the Notch patterning cascade have also found that the joint seems to be divided into proximal and distal territories at this stage. Thus, proximal and distal joint domains have already been established by the late L3 (Mirth, 2002).

By 34-38 h AP, patterns of marker expression define three additional territories. First, a proximal-dorsal patch is high-lighted by two joint markers, ckm90 and ckm175, that drive GFP expression only in a patch above and includes the most proximal cells of the dorsal apodeme. The expression of GFP driven by ckm175 includes a greater number of cells than that driven by ckm90. The second domain identified was a mid-distal domain. Odd lacZ expression becomes largely restricted to a mid-distal group of cells in all but the tarsal joints. This corresponds to the region that does not accumulate collagen IV and marks the cells that push underneath the proximal joint cells. Odd lacZ is also expressed in the apodemes. Lastly, ok388 expresses GFP in the lateral anterior and posterior parts of the distal tibial/tarsal (but not tarsal) joint, but is excluded from the dorsal and ventral domains. This expression domain corresponds with the region of elongating cells seen in longitudinal sections of the leg (Mirth, 2002).

Two of the joint markers are expressed in both the proximal and distal portions in the developing adult joint: ckm239 and disco lacZ. Disco lacZ is expressed throughout the entire joint, and ckm239 is excluded from the ventralmost region (wingless lacZ-expressing region) (Mirth, 2002).

It seems likely that the domains of gene expression observed in the L3 leg disc correspond with those of the same genes in the developing adult joint, though this has not been verified directly. If so, proximal and distal joint domains are established before pupariation. These two joint territories separate cells that will invaginate [the cells in the odd lacZ domain, expressing the Notch target E(SPL)Mß] from those that will form the proximal palisade (the cells expressing Delta, Serrate, and Nubbin). During pupal development, the proximal and distal domains of the joint become further subdivided. Most of the enhancer trap markers identified are expressed in specific groups of cells within either the proximal or distal domain in the tibial/tarsal joint at 34-38 h AP. At the same time, the expression of some earlier markers becomes restricted to more specific territories. odd lacZ, which is expressed in some joints in the L3, is expressed most strongly in the mid-distal joint cells at 34-38 h AP. Ok388 expresses in the distalmost but not mid-distal joint cells, and is restricted to the lateral anterior and posterior sides. In the proximal joint, markers such as ckm90 and ckm175 express in only a small group of cells on the dorsal side. Thus, it seems that the tibial/tarsal joint may divided into three proximodistal domains based both on cell behavior and gene expression: proximal, mid-distal, and distalmost regions. Later during pupal development, the distalmost region subdivides into lateral anterior/posterior and dorsal/ventral domains and the proximal joint also subdivides into smaller territories. That further patterning and subdivision of the joint occurs after the prepupal stages is hardly surprising: the adult joint is too complex a structure to be derived simply from the proximodistal interactions that occur before pupal development (Mirth, 2002).

Nubbin and Teashirt mark barriers to clonal growth along the proximal-distal axis of the Drosophila wing

The division of the wing imaginal disc into anterior, posterior, dorsal, and ventral compartments is a critical step in Drosophila wing morphogenesis. This study investigated the existence of cell lineage restrictions along the proximal-distal (PD) axis of the wing disc. The existence of classical compartment boundaries in the hinge region was ruled out, but it was demonstrated that there are clonal restrictions corresponding to the expression domains of two transcription factors, Nubbin (Nub) and Teashirt (Tsh), present in distal and proximal cells, respectively. Unlike classical compartments, the Nub and Tsh domains do not define absolute lineage restrictions. Instead, due to regulation by Wingless signaling, the Nub and Tsh expression boundaries shift during development. Once established, the Nub and Tsh domains, and the intervening region in which neither factor is expressed, grow independently, because the progeny of cells present in one domain do not freely populate an adjacent domain. Despite shifting position, the Nub and Tsh domain boundaries, like compartment boundaries, impact the expression of secreted signaling molecules. Thus, like the vein/intervein divisions of the wing and mammalian rhombomeres, the Nub and Tsh domains share some of the attributes of classical compartments, but lack their stringent and immobile boundaries (Zirin, 2007).

The experiments described here investigate whether the Drosophila wing disc is divided by lineage restrictions beyond the well-established anterior, posterior, dorsal, and ventral compartments. This question stemmed from a previous observation that, following the initiation of tsh repression by Wg signaling, tsh is maintained in a repressed state by the PcG genes. If tsh repression was a stably inherited state, as is typically the case for PcG regulation, these cells would be expected to define a distinct lineage. The results presented here are consistent with this view, but also reveal that the tsh repressed state does not define a compartment, using the strict definition of this term. Nevertheless, the experiments suggest that like the vein/intervein divisions in the wing disc, and the rhombomere divisions of the developing vertebrate brain, the Tsh, gap, and Nub domains have many of the properties of compartments (Zirin, 2007).

Three domains were defined whose boundaries restrict the pattern of cell divisions along the PD axis. These three regions are comprised of cells that are Nub+ and Tsh− (the Nub domain), Nub− and Tsh− (the gap domain), and Nub− and Tsh+ (the Tsh domain). In wild type discs, the two boundaries that separate these three domains cannot be considered strict lineage restrictions because examples were found of clones that cross these boundaries even when induced as late as the mid third instar. However, like compartment boundaries, the proximal Nub and distal Tsh expression boundaries clearly affect the patterns of cell division within the disc. Beginning in the 2nd instar, clones tend to grow along these boundaries for many cells. Importantly, this behavior is not typical of most gene expression boundaries in the wing disc. For example, neutral clones readily cross the rn, pnr and Iro-C expression boundaries. Also lineage tracing experiments demonstrate that by the late 2nd instar, these three domains grow largely as independent units, since the progeny of cells from one domain rarely move into the neighboring domain. Thus, it appears that the cells in the Nub, gap, and Tsh domains have a strong, but not absolute, tendency to maintain their gene expression status. Again, this behavior is in contrast to other gene expression domains in the wing disc such as rn, where the progeny of a rn expressing cell can readily turn off rn expression and contribute to a neighboring expression domain (Zirin, 2007).

The primary distinction between a compartment and the three PD domains described in this study is that the boundary of a compartment remains constant during development. The experiments demonstrate that both the Tsh and Nub boundaries, and their associated clonal restrictions, shift during development. The Tsh boundary shifts proximally, due to wg signaling in the second and third instar. Similarly, the experiments suggest that the Nub domain is gradually expanding over time. This conclusion stems from the observation that nub-Gal4 induced mitotic recombination frequently results in small clones at the edge of the Nub domain that sometimes straddle the Nub expression boundary. The small size of these clones suggests that they were generated late in development and contrasts with the typically much larger clones present in the middle of the Nub domain. Unlike tsh repression, which is dependent on Wg signaling, it is not known what signals induce the expansion of the Nub domain in the proximal direction. However, previous work proposed the existence of a diffusible molecule made in vg-expressing cells that is required to turn on nub (Zirin, 2007).

One consequence of the changes in the Nub and Tsh boundaries is that the gap domain expands in size during development. Initially, the gap domain comes from cells that turn off tsh in response to wg signaling. Following this initial tsh repression, the data suggest that the gap domain increases in size primarily due to proliferation of these early Tsh− Nub− cells. This conclusion is based primarily on tsh-Gal4 lineage data, which show that, by the end of the 2nd instar, tsh expressing cells contribute very little to the gap domain. Since this domain continues to grow during the 3rd instar, it is concluded that the domain must expand due to cell proliferation, consistent with earlier reports suggesting that Wg induces cell proliferation within this region of the disc (Zirin, 2007).

The initial motivation for analyzing lineage restrictions along the PD axis was the observation that tsh repression is maintained by PcG silencing. It was possible, therefore, that this initial tsh repression domain established a lineage restricted domain that is maintained for the remainder of development. The experiments suggest that this is not the case but that instead two imperfect clonal restrictions form along the PD axis that correspond to the Nub and Tsh boundaries. For example, in M+ clonal experiments, clear examples were observed of clones that extend across most of the gap domain, arguing that an invisible clonal restriction is not present in this region of the disc. Instead, clones made at the end of the 2nd instar or later have a strong tendency to respect the Nub and Tsh boundaries. These observations suggest that these gene expression boundaries create these clonal restrictions, a conclusion that is supported by the observation that nub− clones fail to respect the Nub boundary, and Tsh+ clones fail to respect the Tsh boundary. Thus, because the Tsh and Nub expression domains are changing during development, so are the barriers to clonal growth that are created by these boundaries. The changing position of these boundaries accounts for the behavior of the clones and lineages characterized in this study: they should largely respect these borders, but there should be exceptions to this rule since these domains change over time (Zirin, 2007).

What might the mechanism be for creating these clonal restrictions? One possibility, which is favored, is that once Nub is activated or once Tsh is repressed, these states of gene expression are heritably maintained even in the absence of the initiating signal. This is known to be true for the repression of tsh in the wing pouch, in which the initial repression by Wg is maintained by PcG silencing. This two-step repression of tsh also appears to hold true for the later phases of tsh repression in the hinge. Hinge clones that cannot transduce the Wg signal show tsh derepression when they are induced in the second instar, but not when they are induced in the third instar suggesting that, as in the pouch, tsh repression is maintained by a wg-independent mechanism. If nub activation is also maintained by a heritable mechanism, then it would be expected that clones generated in any of these three domains would remain in these domains. Thus, although a stable lineage restriction can be observed only when Wg signaling is blocked, it is suggested that the same phenomenon -- the epigenetic inheritance of gene expression states -- is occurring as new cells are recruited to the gap domain due to tsh repression or to the Nub domain due to nub activation (Zirin, 2007).

It is also noted that while cell affinity differences may also play a role in forming the Nub and Tsh domain boundaries, affinity differences appear not to be sufficient to create the clonal restrictions described in this sudy. Previous work has demonstrated, for example, that pnr expressing cells do not readily mix with non-pnr expressing cells, indicating a clear difference in cell affinities. Yet despite this affinity difference, the pnr expression boundary does not influence clonal growth. One possible distinction between these expression domains is that, unlike nub and tsh, the pnr gene expression status is not locked into place by an epigenetic (e.g. PcG-dependent) mechanism (Zirin, 2007).

Why have PD clonal restrictions? For both the D/V and A/P boundaries, it is known that compartmental interfaces create sources of secreted signals that are critical for the subsequent patterning and growth of the disc. Might the same phenomenon be occurring at the Nub and Tsh boundaries? wg expression in the IR is thought to be induced by a non-autonomous signal coming from vg-expressing cells and received by nub-expressing cells, and has been suggested to involve four jointed, fat, dachsous and dachs. At the time the IR is first induced, the Tsh and Nub domains abut each other, raising the possibility that this interface may also be important for IR induction. Although there is no definitive answer to this question, several results reported in this study are consistent with this view. First, in ap>TCFDN discs it was found, intriguingly, that Wg expression is observed in cells immediately adjacent and distal to the Tsh/Nub interface, in Nub-expressing cells. Second, in flip-out clones that ectopically express Tsh in the hinge, often a non-autonomous induction of wg expression is observed in cells adjacent to the clone. Third, in flip-out clones expressing a tsh RNAi hairpin construct, de-repression of wg is observed in the RNAi-expressing cells. However, an intriguing aspect to this experiment is that wg expression is not only observed at the Tsh+/- interface, but throughout the tsh RNAi clones. Thus, although the Tsh boundary may play a role in inducing wg, the RNAi experiment raises the more conventional possibility that tsh is a repressor of wg expression in this region of the wing disc (Zirin, 2007).

Another role for these domains may be to allow the orientation of cell divisions to differ along the PD axis. In the wing pouch, the predominant pattern of clonal growth is parallel to the PD axis. In contrast, it was found that, by the end of the 2nd instar, the predominant pattern of clonal growth in the gap domain is perpendicular to the PD axis. There is also a shift in clonal shape from relatively isometric in the mid second instar to long in the third instar. Interestingly, cells divide with a predominantly PD orientation in the wing pouch. Based on the shape of the clones observed, mitoses in the hinge may predominantly orient perpendicular to the PD axis. This orientation may help determine the shape of the adult hinge (Zirin, 2007).

Strikingly, analysis of the PD axis in the wing does not apparently apply to the ventral imaginal discs. Lineage tracing experiments performed in the leg disc indicate that cells in the Tsh domain readily lose tsh expression and contribute to the growth of the distal leg regions. Since there are no rings of Wg associated with the Tsh boundary in the leg disc, perhaps it is the unique relationship between Tsh and Wg in the wing disc that necessitates a more stringent clonal restriction (Zirin, 2007).

The developmental domains defined in this study correspond to the body/hinge and hinge/wing blade anatomical boundaries of the adult fly. As insect wings may have evolved from a proximal outgrowth of a pre-existing multi-branched appendage, it may be that the formation of distinct domains was important for the independent growth and morphological modification of this outgrowth to become a wing (Zirin, 2007).

In closing, it is suggested that the phenomenon of imperfect lineage restrictions defined in this study may be more general in animal development than classical compartments. Although lineage restrictions clearly exist in the vertebrate hindbrain and forebrain, it has also been noted that these boundaries are not absolute. Krox-20, a zinc-finger transcription factor responsible for the specification and segmentation of even-numbered rhombomeres, is expressed in a gradually expanding domain. It is suggested that cell proliferation and changes in the expression patterns of such key genes, as is the case for the Nub and Tsh domains in the wing disc, underlie the leakiness of these boundaries (Zirin, 2007).

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).

Effects of Mutation, Deletion or Over-expression

The nubbin gene of Drosophila was originally identified as a viable spontaneous mutation which results in a dramatic reduction in the size of wings and halteres, but which does not otherwise affect adult morphology. Cloning of the DNA responsible for this phenotype, shows that the gene responsible is pdm-1 (Ng, 1995).

The role of pdm1 has been investigated during the elaboration of the GMC-1-->RP2/sib lineage. Also studied in this lineage was the functional relationship between pdm1 and pdm2. Deletion of pdm1 causes a partially penetrant GMC-1 defect, while deletion of both pdm2 and pdm1 results in a fully penetrant defect. This GMC-1 defect in pdm2 and pdm1 mutant embryos can be rescued by the pdm1 or pdm2 transgene. Rescue is observed only when these genes are expressed at the time of GMC-1 formation. Overexpression of pdm1 or pdm2 well after GMC-1 is formed results in the duplication of RP2 and/or sib cells. These results indicate that both genes are required for the normal development of this lineage and that the two collaborate during the specification of GMC-1 identity (Bhat, 1995).

The phenotype for mutations of the nubbin gene consists of a severe wing size reduction and pattern alterations, such as transformations of distal elements into proximal ones. nub expression is restricted to the wing pouch cells in wing discs from the early stages of larval development. These effects are also observed in genetic mosaics where cell proliferation is reduced in all wing blade regions autonomously, and transformation into proximal elements is observed in distal clones. Mutant clones are approximately 50% smaller than control clones or else they fail to grow in 50% of the cases. Clones located in the proximal region of the wing blade cause an additional nonautonomous reduction of the whole wing. Cell lineage experiments in a nub mutant background show that clones respect neither the anterior-posterior nor the dorsal-ventral boundary but that the selector genes decapentaplegic and engrailed are correctly expressed from early larval development. The phenotypes of nub elbow and nub dpp genetic combinations are synergistic and the overexpression of dpp in clones in nub wings does not result in overproliferation of the surrounding wild-type cells (Cifuentes, 1997).

Upregulation of Mitimere and Nubbin acts through Cyclin E to confer self-renewing asymmetric division potential to neural precursor cells

In the Drosophila CNS, neuroblasts undergo self-renewing asymmetric divisions, whereas their progeny, ganglion mother cells (GMCs), divide asymmetrically to generate terminal postmitotic neurons. It is not known whether GMCs have the potential to undergo self-renewing asymmetric divisions. It is also not known how precursor cells undergo self-renewing asymmetric divisions. Maintaining high levels of Mitimere or Nubbin, two POU proteins, in a GMC causes it to undergo self-renewing asymmetric divisions. These asymmetric divisions are due to upregulation of Cyclin E in late GMC and its unequal distribution between two daughter cells. GMCs in an embryo overexpressing Cyclin E, or in an embryo mutant for archipelago, also undergo self-renewing asymmetric divisions. Although the GMC self-renewal is independent of inscuteable and numb, the fate of the differentiating daughter is inscuteable and numb-dependent. These results reveal that regulation of Cyclin E levels, and asymmetric distribution of Cyclin E and other determinants, confer self-renewing asymmetric division potential to precursor cells, and thus define a pathway that regulates such divisions. These results add to understanding of maintenance and loss of pluripotential stem cell identity (Bhat, 2004).

Maintenance of a self-renewing fate can be viewed as a state where activities of certain genes maintain that state. Once the activity of such genes is switched off, the cells become committed to a differentiation pathway. The results reported in this study indeed support this type of mechanism. That POU genes might be a class of genes that maintain a self-renewing capacity is indicated by the fact that the Oct4 POU gene (Pou5f1 -- Mouse Genome Informatics), which is expressed in pluripotent stem cells of the mouse early embryo, is turned off when these cells begin to differentiate (Rosner, 1990). Similarly, SCIP is expressed in the progenitors of oligodendrocytes, but it is downregulated when these cells are induced to differentiate (Collarini, 1992). The current results provide direct evidence that these genes can induce a cell that is committed to a differentiation pathway to acquire a self-renewing capability in a lineage specific manner. Moreover, studies undertaken in the past several years using the Drosophila nervous system as a paradigm have revealed how asymmetry can be generated during cell division to produce two distinct postmitotic cells. However, there is very little information on how an asymmetric self-renewing division pattern is determined. In this paper, results are presented that provide insight into this particular process. (Bhat, 2004).

The strongest evidence that a GMC-1 undergoes a self-renewing type of asymmetric division in embryos overexpressing miti/nub or CycE, and in embryos mutant for ago, comes from the presence of hemisegments with two sibs and one RP2. There are two ways the second sib cell can be generated: (1) a self-renewed GMC-1 generates another sib when it divides, and (2) some other cell is transformed into a sib. The following set of evidence indicates the former scenario: (1) the second sib cell always appears later in development, i.e. at ~8.5 hours of age (as opposed to in wild type where the GMC-1 terminally divides by ~7.5 hours of age into an RP2 and a sib); (2) the dynamics of Eve expression itself in the sib -- expression of eve is switched off in a sib during the asymmetric division of GMC-1 and there is no de novo synthesis of Eve thereafter. If a postmitotic cell from an Eve-negative lineage transforms into a sib, it would be negative for Eve and would not be detected. The development of the other Eve-positive neuronal lineages is normal in these embryos, thus it is unlikely that a cell from those Eve-positive lineages is transformed into a sib. (3) The Eve and Spectrin staining of UAS-nub; ftz-GAL4 embryos provides more direct evidence for the self-renewal of GMC-1. In ~8. 5-hour-old UAS-nub; ftz GAL4 embryos, the larger GMC-1 (this Eve-positive cell is Zfh1 negative, indicating that it is indeed a GMC-1) can be observed undergoing asymmetric cytokinesis for the second time. From the heat-shock induction experiments of nub or miti mutant embryos, it can be argued that higher levels of these proteins in the parental NB4-2 cause later born GMCs to adopt a GMC-1 fate. However, the GMC-1 self-renewing phenotype observed following targeted expression of nub using the ftz-GAL driver makes this scenario unlikely. (4) The results obtained with the mitiP; insc and mitiP; nb double mutant embryos (P referring to prolonged expression), and the mis-localization of Insc in GMC-1 of these embryos, are also consistent with this conclusion. (Bhat, 2004).

These results indicate that the level, timing and duration of presence of Miti or Nub proteins determine the dynamics of the GMC-1 division pattern. For example, the asymmetric divisions (which generate the 3-cell phenotypes) and the symmetric divisions (which generate the 4-cell phenotype) were observed when the transgenes were induced for 20-25 minutes. However, the multiple cell-phenotype was observed only when the transgenes were induced for 90 minutes. Once the induction was stopped and the levels returned to normal, the two GMC-1s appeared to exit from the cell cycle to generate postmitotic cells. Similarly, when the transgene was induced with ftz-GAL4, only the 3-cell phenotypes, and not the 4-cell or multi-cell phenotypes were observed. Thus, the following picture emerges from these results. Although high levels of Miti and Nub proteins are required for the specification of GMC-1 identity, their level must be downregulated in order for the GMC-1 to divide asymmetrically into postmitotic RP2 and sib. Maintaining a high level of these proteins in GMC-1 commits that cell to adopt a self-renewing stem cell type of division pattern. The results described here also show that Miti and Nub prevent GMC-1 from exiting the cell cycle by upregulation of CycE (Bhat, 2004).

The results clearly show that upregulation of CycE in late GMC-1 is the cause for the adoption of a self-renewing asymmetric division pattern. In other words, presence of high levels of CycE in late GMC-1 and its unequal distribution to one of the two daughter cells prevents this cell from exiting the cell cycle. Since this daughter cell still maintains the GMC-1 identity and has sufficient CycE to divide again, a further asymmetric division(s) is ensured. The cell that has lower amounts of CycE becomes committed to a differentiation pathway (RP2 or sib) (Bhat, 2004).

What lines of evidence support this conclusion? (1) In contrast with wild type, there is a significant amount of CycE present in a late GMC-1 in embryos overexpressing miti or nub. This CycE preferentially segregates to one of the two daughters of that GMC-1, usually the larger cell. When miti or nub genes are overexpressed only briefly, the level of CycE is downregulated after just one additional round of division, whereas with prolonged induction, the level is maintained at high levels in one or two cells of the multi-cell cluster for a prolonged duration of time (Bhat, 2004).

(2) Upregulation of CycE in a late GMC-1 is also observed in embryos mutant for ago, which is known to regulate CycE levels. In ago mutants, the two daughter cells of such a GMC-1 have unequal CycE levels accompanied by a self-renewing asymmetric division phenotype. The CycE is always downregulated after one additional GMC-1 division, which is consistent with the finding that the self-renewal occurs only once in these embryos. Since penetrance in ago mutants is partial, and CycE is downregulated in this lineage after just one additional division, there must be additional factors that mediate the downregulation of CycE in this lineage (Bhat, 2004).

(3) Embryos expressing high levels of CycE from a CycE transgene exhibit the same GMC-1 phenotypes as embryos expressing high levels of Miti or Nub. Thus, these results indicate that upregulation of CycE alone is sufficient for the GMC-1 to adopt a self-renewing type of division pattern. Finally, mitiP phenotypes are found to be dependent on CycE. That is, no multi-cell clusters were observed in mitiP; CycE double mutant embryos (Bhat, 2004).

In wild type, the downregulation of CycE in GMCs appears to occur through switching off CycE transcription and degradation of the protein by factors such as Ago. At what level does Miti or Nub regulate CycE? Since POU genes are thought to be transcriptional activators, they can regulate transcription of CycE either directly or indirectly. However, this does not seem to be the case since expressing high levels of miti does not have a discernible effect on the levels of CycE mRNA in GMC-1, as assessed by whole-mount RNA in situ hybridization. In addition, the putative promoter/enhancer region of CycE gene does not contain any consensus POU protein-binding sites. Therefore, it seems likely that Miti and Nub regulate factors that are involved in the degradation of CycE in late GMC-1 (Bhat, 2004).

The question arises as to how only one cell has a high level of CycE. There are several ways this can happen. There might be an asymmetric degradation of CycE. This scenario seems unlikely since there is only one of two daughter cells with high levels of CycE in ago mutants. Given that Ago downregulates CycE via a protein degradation mechanism, if there was an asymmetric degradation, in those hemisegments where the levels of CycE was elevated in GMC-1, it would initially be expected that both the daughter cells would have high CycE levels. However, this was not the case. An asymmetric transcription of the CycE gene also seems unlikely since the transcription of CycE ceases prior to GMC-1 division, as judged by whole-mount RNA in situ hybridization. The most likely possibility is that CycE is unequally distributed between the two daughter cells of GMC-1. The unequal distribution of CycE could be a passive process due to the size difference between daughter cells, especially in the GMC-1-->RP2/sib lineage. Moreover, no cytoplasmic crescent of CycE was observed during mitosis. By contrast, it could also be an active process. For instance, the size difference between an aCC and a pCC (or between a GMC1-1a and an aCC) is very small, and the fact that GMC1-1a undergoes a self-renewing asymmetric division suggests that the segregation of CycE may not be entirely a passive process (Bhat, 2004).

Finally, the results indicate that while a GMC that does not normally express Miti or Nub is insensitive to its ectopic expression (e.g., GMC1-1a of NB1-1; this GMC produces an aCC/pCC pair of neurons), a brief induction of CycE in the same GMC causes it to undergo self-renewing asymmetric division. Therefore, CycE can confer a stem cell type of division potential to more than one GMC. Another important conclusion one can draw from this result is that the segregation of CycE may be an active process. In the case of GMC1-->RP2/sib lineage, the cytokinesis of GMC-1 is asymmetric, and the size difference between an RP2 and a sib is significant. Thus, CycE can be asymmetrically segregated because of this size difference. However, the size difference between an aCC and a pCC (or between a GMC1-1a and an aCC) is very small, and the fact that GMC1-1a undergoes a self-renewing asymmetric division suggests that the segregation of CycE may not be entirely a passive process. It is possible that the difference between the levels of CycE needed to retain a cell within the cell cycle and the levels that do not support maintaining the cell within the cell cycle are quite small. Thus, even a minor change in the amount that a cell receives during division might be sufficient to make a difference. Thus, the segregation of CycE can still be a passive process. Nonetheless, these results reveal how a cell can adopt a self-renewing asymmetric division potential through CycE. (Bhat, 2004).

Pros has been implicated in inhibiting the ability of GMCs to divide more than once by preventing continued expression of cell-cycle genes. The caveat of this study, however, is that none of the GMC lineage was examined using cell-specific markers to determine whether GMCs continue to divide in embryos mutant for pros. The conclusion that Pros inhibits GMC division was mainly based on the presence of additional BrdU-positive cells in late stage (post 15-hours-old) pros mutant embryos. Pros is expressed in GMC-1 of the RP2/sib lineage and, in null alleles, this GMC-1 identity is not specified. In pros17, a loss-of-function allele, ~5% of the hemisegments had an RP2/sib lineage specified. In these hemisegments, the GMC-1 divides only once to generate an RP2 and a sib cell as in wild type. Moreover, specification of U and CQ lineages was observed in ~20% and ~13% of the hemisegments, respectively, and no additional cell division appeared to occur in these lineages. A previous study found that the aCC/pCC neurons (from GMC1-1a) have an abnormal axon morphology, but it did not find any additional neurons in this lineage. Similarly, NB6-4 of the thoracic segment produced the normal number of progeny in pros mutant embryos. These results suggest that Pros does not regulate cell division in RP2/sib, U and CQ lineages, and possibly not in many other neuronal lineages, and therefore it is unlikely to function in the miti/nub pathway (Bhat, 2004).

How is the specification of identity of one of the two progeny, either as an RP2 or as a sib, from a self-renewing asymmetric division of GMC-1 regulated? (Specification of the other progeny as GMC-1 is by high levels of CycE.) The results indicate that specification of an RP2 versus a sib identity to this differentiating cell is through a combination of low levels of CycE and localization of Insc. This is indicated by the finding that overexpression of Miti and Nub causes localization of Insc to be non-asymmetric. Non-asymmetric Insc also causes non-asymmetric localization of Numb. The cell that has lower levels of CycE and also has Numb becomes an RP2. Whenever the cell with lower levels of CycE fails to inherit Numb (the effect of overexpression of Miti or Nub on the localization of Insc is partially penetrant) that cell will become a sib. That the generation of an RP2 during the asymmetric division of GMC-1 is tied to Numb is also indicated by the analysis of mitiP; numb embryos. Although the self-renewal of GMC-1 in mitiP embryos is numb-independent, the commitment of a progeny to become a sib is numb-dependent. Thus, in ~13-hour-old mitiP; numb embryos, multiple cells are observed adopting a sib fate. An often overlooked fact is that in insc mutants the GMC-1 division is normal in ~30% of the hemisegments despite having no insc. Similarly, the penetrance of the symmetrical division of GMC-1 in pins (where Insc localization is affected as in mitiP embryos) is also partial, indicating the presence of additional (partially redundant) pathways for Insc that mediate asymmetric fate specification. These very same additional pathways must also influence the choice between a sib and an RP2 when the GMC-1 in mitiP embryos undergoes a self-renewing type of asymmetric division (Bhat, 2004).

CycE and Ago are part of a mechanism that converts a normal cell into a cancer cell. In ago mutants, CycE protein is not degraded and a number of cancer cell lines carry a mutation in ago. The current results showing that these genes are also involved in a stem cell type of division suggest a commonality between stem cells and cancer cells. These results also provide a molecular mechanism of how self-renewing asymmetric divisions are possible (Bhat, 2004).

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).

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).

Neuralized mediates asymmetric division of neural precursors by two distinct and sequential events: promoting asymmetric localization of Numb and enhancing activation of Notch-signaling

In the CNS, the evolutionarily conserved Notch pathway regulates asymmetric cell fate specification to daughters of ganglion mother cells (GMCs). The E3 Ubiquitin ligase protein Neuralized (Neur) is thought to activate Notch-signaling by the endocytosis of Delta and the Delta-bound extracellular domain of Notch. The intracellular Notch then initiates Notch-signaling. Numb blocks N-signaling in one of the two daughters of a GMC, allowing that cell to adopt a different identity. Numb is asymmetrically localized in a GMC and is segregated to only one of the two daughter cells. In the typical GMC-1 --> RP2/sib lineage, it was found that loss of Neur activity causes symmetric division of GMC-1 into two RP2s. It was further found that Neur asymmetrically localizes in a late GMC-1 to the Numb domain and Neur mediates asymmetric division via two distinct, sequential mechanisms: by promoting the asymmetric localization of Numb in a GMC-1 via down-regulation of the transcription factor Pdm1, followed by enhancing the Notch-signaling via trans-potentiation of Notch in a cell committed to become a sib. In neur mutants the GMC-1 identity is not altered but Numb is non-asymmetrically localized due to an up-regulation of Pdm1. Thus, both its daughters inherit Numb, which prevents Notch from specifying a sib identity. Neur also enhances Notch since in neur; numb double mutants, both sibling cells often adopt a mixed fate as opposed to an RP2 fate observed in Notch; numb double mutants. Furthermore, over-expression of Neur can induce both cells to adopt a sib fate similar to gain of function Notch. These results tie Numb and Notch-signaling through a single player, Neur, thus giving a more complete picture of the events surrounding asymmetric division of precursor cells. It was also shown that Neur and Numb are interdependent for their asymmetric-localizations (Bhat, 2011).

The results in this paper tie the localization of Numb and the signaling-processing of Notch through a single upstream player, Neur. This gives a more complete picture of the events that surround asymmetric division of neural precursor cells. The E3 Ubiquitin ligase protein Neur regulates asymmetric division of Numb and Notch-sensitive neural precursor cells in the CNS via two distinct, sequential mechanisms: first, by promoting the asymmetric localization of Insc and Numb in GMCs and second, via non-cell autonomously potentiating or enhancing the activation of Notch signaling in the Numb-negative daughter cell. While Neur is known to activate Notch-signaling by the endocytosis of Delta and the Delta-bound extracellular domain of Notch, an earlier role for it in asymmetric division via Insc and Numb localization has not been discovered. In fact, these results show that this is the primary role for Neur in generating asymmetry in the CNS. That Neur plays a secondary role or a role which is not absolute in the potentiation or enhancement of Notch signaling is indicated by the finding that in neur; numb double mutants, both sibling cells often but not always adopt a mixed fate as opposed to an RP2 fate seen in Notch; numb double mutants. If the role of Neur in Notch potentiation in this lineage is an absolute one, the same result would have been seen in neur; numb as N; numb mutants. Furthermore, over-expression of Neur can induce both cells to adopt a sib fate similar to gain of function Notch, however, the penetrance of this effect is weak (Bhat, 2011).

Previous studies had shown that the RP2-sib binary fate decision is regulated by unequal segregation of the Notch regulator Numb. The simplest interpretation of the current results would suggest that Neur is required for sib fate specification via Notch. However, the results indicate that the requirement of Neur for sib-specification to a daughter cell of a GMC-1 via regulating Notch is preceded by its requirement in GMC-1 for Numb localization, where Neur itself is expressed and becomes asymmetrically localized to the basal Numb-domain. Thus, the loss of sib identity in neur mutants appears to be mainly due to the non-asymmetric localization of Insc and Numb in GMC-1. Moreover, the levels of Pdm1 are responsive to both loss of function neur (Pdm1 level is up-regulated) and gain of function neur (the Pdm1 level is down-regulated), which are more likely a consequence of Neur function within GMC-1. This regulation of Insc and Numb localization appears to be via regulation of Pdm1 levels inside GMC-1, whereas regulating Notch processing is later and the source of Neur is from outside. By regulating asymmetric localization of Numb, Neur ensures that one of the two daughters is free of Numb, thus, later on the activation of Notch-signaling in that cell can occur. The source of Neur for this Notch processing appears to be from outside of the lineage since a division-arrested GMC-1 in numb; cyc A double mutant can still adopt a sib fate. Thus, the two roles of Neur in this lineage are distinct and separable. But then is it possible Notch has a role in the asymmetric localization of Numb and this activity of Notch is regulated by Neur? It certainly is possible but then one would have to disregard the presence of asymmetrically localized Neur in a GMC-1 as anything but of no consequence to the asymmetric division of GMC-1. It should also be pointed out that the identity of GMC-1 per se in neur is not altered, if it did, two neurons of some other identities would have been seen, not RP2s (or sibs) (Bhat, 2011).

A previous study in the sensory system of the PNS indicated that Neur protein localizes asymmetrically in the pI cell of SOP. It then segregates to pIIb, where it is thought to enhance the endocytosis of Dl to promote N activation in the pIIa cell. This represents a trans-differentiation mechanism to specify different cell fates. The results confirm the findings in SOP lineage but at the same time extends the data on SOP lineage in that this trans-determination process is a potentiation step to mediate a more efficient Notch-signaling-processing, but it is not necessarily a deterministic one. What is new and different from the SOP lineage is that Neur controls not only the asymmetric localization of Numb during mitosis, but also controls the localization of Insc, an apical cue that controls spindle orientation and participates in Numb basal localization. In neur mutant cells, Insc is no longer asymmetric indicating that Neur is somehow needed to localize Insc. The fact that Neur is somehow needed for Insc localization is also consistent with the finding that genetically insc is epistatic to neur, therefore that it is downstream of neur (Bhat, 2011).

Finally, while insc is epistatic to neur in the RP2 lineage defect in insc; neur double mutants, as for the neurogenic phenotype, neur is epistatic. This is not surprising since epistasis relationships are lineage/cell-type/tissue specific, depending upon whether or not the two genes in question are expressed in the same lineage and if the two single mutants give the same (or opposing) phenotype. Insc has no role during the neural versus ectodermal fate decisions and loss of function for insc does not cause a neurogenic phenotype, hence, the neurogenic phenotype of neur mutants is not expected to be present (epistatic) in the double mutant (Bhat, 2011).

It is clear from the results that Neur regulates asymmetric division of GMCs in the CNS. This was examined in at least two different GMCs, the GMC of the RP2/sib lineage (GMC-1 or GMC4-2a of NB4-2) and the GMC of the aCC/pCC lineage (GMC-1 or GMC1-1a of NB1-1). In neur, these GMCs symmetrically divide to generate two of the same cells, RP2 neurons in the case of GMC-1 and aCC neurons in the case of GMC1-1a. It is thought that many more GMC lineages are affected by loss of function for neur. Being a neurogenic protein, Neur is also involved in selecting neural versus ectodermal fates for the neuroectodermal cells. Due to its neurogenic property, the mutant will generate extra copies of many of the NBs in the nerve cord, which in turn, will generate more of the GMCs and neurons. Several lines of evidence indicate that symmetric division of a GMC indeed occurs at a high frequency in the CNS in neur mutants. For example, GMC-1 normally generates an RP2 and a sib, RP2 is larger than the sib and the two have distinct gene expression profiles and patterns. This is also the case for aCC/pCC pairs—they also have distinct gene expression profiles. These specific criteria were used to separate the ones that are generated by the symmetric division from those generated due to a neurogenic effect of neur mutation (Bhat, 2011).

Several additional pieces of evidence indicate a role for Neur in generating asymmetry. These include the asymmetric localization of Neur in GMCs, non-asymmetric localization of Numb in GMC-1 in neur mutants, non-asymmetric localization of Neur in numb mutants, genetic interaction results and effect on downstream players such as Pdm and Numb. All these results point to a specific role for Neur in regulating asymmetric mitosis of precursor cells (Bhat, 2011).

The results show that Neur itself is asymmetrically localized in GMC-1 to the Numb-domain and opposite to that of the Insc-domain (Neur is also localized to the basal end of several NBs, the significance of which is not known). In neur mutants, both Insc and Numb are not localized but found uniformly distributed along the cell cortex. This suggests that Neur is upstream of Insc and Numb localization but not their expression per se. The levels of Numb or Insc are also not affected in neur mutants indicating that Neur does not participate in Numb degradation (via ubiquitination, or otherwise). There is no evidence that Neur has a direct role in the localization of Numb. Do these results therefore mean Neur basically regulates the identity or the fate (i.e. gene expression program) of the GMC-1 prior to its division and therefore that Neur has only one function, which is potentiating Notch signaling? The GMC-1 was examined in neur mutants with several different GMC-1 markers (Eve, Pdm1, Zfh-1, Spectrin, etc.) and with the exception of a higher than normal Pdm1 in a late GMC-1, none of these markers were affected. A higher than normal levels of Pdm1 does not change the identity of a GMC-1. Indeed, several studies have shown that high levels of Pdm1 or its sister protein Pdm2 will induce a GMC-1 to undergo symmetric division to produce two GMC-1s and then two RP2s and two sibs. In order for a GMC-1 to change its identity, many of its genes should be turned off and a new set of genes has to be initiated. Such a drastic change does not occur in GMC-1 of neur mutants. Similarly, an identity change should result in this GMC-1 in neur mutants to produce different sets of neurons, which it does not. Instead, it produces two RP2s. Given these results and that Neur is necessary for the normal localization of Numb, whether this is directly mediated or indirectly mediated, the conclusion that Neur regulates asymmetric division at two different levels during the lineage development is based on firm grounds (Bhat, 2011).

The main question is how might Neur regulate Insc and Numb localization. A clue to this question comes from previous studies. It was shown that over-expression of Pdm POU transcription factors (Pdm1 or Pdm2) in GMC-1 causes non-localization of Insc and Numb and their segregation to both daughter cells of GMC-1; these cells then adopt an RP2 fate, with Numb blocking the N-signaling from specifying a sib fate. Pdm1 was up-regulated in GMC-1 in neur mutants and down-regulated with over-expression of Neur. This shows that the localization of Insc and Numb is altered in neur mutants indirectly via the up-regulation of Pdm protein. At the moment, it is not clear how an up-regulation of Pdm alters Insc or Numb localization. A most likely possibility is that Pdm proteins, being transcription factors, their over-expression may cause changes in the expression of genes that are needed for the proper localization of Insc and Numb but without altering the cell-identity itself (since this cell still produces RP2 neurons and not some other neurons). These conclusions are all consistent with the overall expression pattern and mutant effects of pdm genes: Pdm proteins are down-regulated in GMC-1 prior to its division, loss of function for Pdm causes loss of GMC-1 identity (Bhat, 2011).

The gain of function for these pdm genes indicates that the GMC-1 division is quite sensitive to varying levels and timings of expression of these POU proteins. For example, a high level of pdm gene expression in a GMC-1 from pdm transgenes causes a symmetric division of GMC-1 into two GMC-1s and then each of these GMC-1s generates an RP2 and a sib. In contrast, a symmetric division of GMC-1 into two RP2s can also be observed in these embryos. In this case, the cells from the GMC-1 express Zfh1; a GMC-1 does not continually express (a late GMC-1 about to divide does express Zfh1 at a very low level), a sib transiently expresses Zhf-1, and an RP2 stably expresses Zfh-1. Moreover, both these cells inherit Insc and Numb. No more cells are produced from these two cells, and each of these cells generates a projection as that of an RP2. When these genes are over-expressed for a prolonged period of time, a GMC-1 divides multiple times producing a GMC-1 and a differentiated progeny: First two unequal sized cells are observed. Only one of the two (the smaller cell) expresses markers such as Zfh1. Later on, three cells, and then five cells, etc., are sequentially seen, all in a tight cluster; from these clusters, as many as 5 RP2s are formed. Indeed, with this prolonged over-expression of pdm genes for 90 min from a heat shock promoter causes hemisegments with all the above types of divisions depending upon the time of over-expression. In contrast, it is not clear what the sensitivity range of GMC-1 is to varying concentrations in terms of the kind of division pattern generated. One clue to this comes from an earlier study, that GMC-1 in embryos carrying a duplication chromosome for the chromosomal region containing the two POU genes undergo a single self-renewing asymmetric division of GMC-1. This suggests that when the copy numbers for these genes are doubled, this presumably results in producing twice the amount of these proteins (from their own promoters), and causes a single self-renewing division. Having said that, it was also found that in neur mutants a GMC-1 rarely divides symmetrically into two GMC-1s and then each produces a sib and an RP2, or a GMC-1 dividing more than once with self-renewing asymmetric division as in pdm-GOF situations (Bhat, 2011).

Based on these results with gain of function for pdm genes, a loss of function for pdm genes should suppress the neur defects. However, this experiment is not possible to do since loss of function for the pdm genes causes loss of GMC-1 identity (GMC-1 becomes some other GMC) and therefore GMC-1 is undetectable with GMC-1 markers (Bhat, 2011).

While the exact mechanism as to how the level of Pdm1 is up-regulated in GMC-1 of neur mutants or down-regulated when Neur is over-expressed in GMC-1, is not known, one possibility is that Neur is involved in the degradation of Pdm1 in GMC-1. This scenario is most likely since Neur has the RING domain, one of the signature domains for E3 Ubiquitin-ligase proteins involved in protein degradation. Neur has also been shown to ubiquitinate proteins in vitro. One indication that Neur might be involved in the degradation of Pdm1 is the result that while ectopic or over-expression of full length neur from a transgene down-regulated Pdm1 and resulted in the same phenotype as loss of function for pdm genes, a similar ectopic or over-expression of a neur transgene missing the RING domain (Hs-neurΔRF) did not result in a down-regulation of Pdm1 or resulted in any phenotypes. Pdm1 appears to be specifically affected in GMC-1 of the RP2/sib lineage and not in other cells where Pdm proteins are present. Even if the up-regulation of Pdm proteins in neur mutants is via an indirect mechanism, say via factor X or Y, the results define a major role for Neur in regulating asymmetric division prior to the Notch-potentiation role of Neur: regulating Numb localization via down-regulating (directly or indirectly) Pdm proteins (Bhat, 2011).

Results from the analysis of neur, neur; numb double mutant embryos and neur gain-of-function embryos show that Neur functions to increase the efficiency of Notch-signaling but not essential for it. None of the previous studies have made this important distinction. Previous results have indicated that Neur activates Notch-signaling via endocytosis of Delta and the Delta-bound extracellular domain of Notch. However, in neur null mutants (embryos homozygous for a deficiency that removes neur completely), sib specification still occurs in ~ 10% of the hemisegments. While this may arguably be due to a partial redundancy for neur, there is another line of evidence that suggests a role for Neur in enhancing the efficiency of Notch signaling. That is, while in Notch; numb double mutants both daughter cells of a GMC-1 adopt an RP2 fate (note that for the specification of an RP2 fate Numb is needed only when there is an intact Notch-signaling), in neur; numb double mutants the daughters often adopt a mixed identity. This result indicates that Notch is still able to specify some features of a sib identity (i.e., reduced levels of Eve expression) in the absence of Neur activity. If Neur is absolutely needed for Notch signaling, the double mutant results would have been exactly the same as Notch; numb double mutants where both daughters adopt an unambiguous RP2 fate (Bhat, 2011).

In contrast, the results from Neur over-expression experiments indicate that when present at high levels Neur is able to overcome the Numb block and induce both the progeny of GMC-1 to adopt a sib fate. This phenotype is strikingly similar to the phenotype observed with the over-expression of the intracellular domain of Notch or the phenotype in numb mutants. These results suggest that over-expression of Neur leads to processing of Notch in the cell that has Numb. It is also pointed out that the source of Neur for the trans-effect on Notch-signaling need not be only from the “RP2” cell, but may also be from the neighboring cells. This is indicated by the previous result that while the GMC-1 in embryos mutant for cyclin A adopts an RP2 fate, the same GMC-1 in cyclin A; numb double mutants adopts a sib fate (Bhat, 2011).

These results show that the asymmetric basal localization of Numb in neur mutants and Neur in numb mutants is affected. This shows the interdependence of localization of these two proteins. Whether there is any initial localization of Numb or Neur in the two mutants was examined to determine if the localization of the one protein falls apart in the absence of localization of the other. However, no such initial localization was observed for either of the two proteins. It is possible that both Neur and Numb control the same pathway(s) that directly or indirectly mediates localization of the other. Perhaps Neur and Numb interact physically with each other in the cytoplasm prior to any localization and it is this Neur-Numb complex that gets localized to the basal pole; in the absence of either of the two proteins, no such complex is formed, and no localization occurs. This model has not been tested due to lack of appropriate reagents. In contrast, loss of Numb-localization in neur could be due to loss of Insc localization; loss of Neur localization in numb mutants could be more direct where Neur is downstream of Numb and Numb mediates directly or indirectly the localization of Neur. The function of Neur in GMC-1, however, appears to be required for the down-regulation of Pdm and allow localization of such proteins as Insc. Thus, Neur is both upstream and downstream of Numb in GMC-1. Another important distinction between Neur and Numb is that while non-asymmetric localization of Numb in GMC-1 will lead to both daughters of GMC-1 inheriting Numb and adopting RP2 fates, a non-asymmetric localization of Neur and inheritance of Neur by both daughters will not make them adopt an RP2 fate, but a sib fate (Bhat, 2011).

In numb mutants, the localization of Neur is affected in such a way that both daughters inherit Neur. Does this have a consequence? The results argue that unlike Numb there is no consequence to the non-asymmetric localization and segregation of Neur to both daughters. For instance, in wild type the sib cell does not inherit Neur, thus, the potentiation of Notch in this cell by Neur occurs in a cell non-autonomous mechanism (removing the extracellular domain of Notch bound by Delta) and there is no role for Neur in the sib itself. Thus, in numb mutants although both daughters inherit Neur, they still adopt a sib fate (Bhat, 2011).


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POU domain protein 1: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 20 December 2020

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