Zn finger homeodomain 1


REGULATION

Transcriptional Regulation

The mesodermal zfh-1 expression requires the products of the twist and snail genes. In twist mutants, all the early mesodermal staining with anti-ZFH-1 antibody posterior to the cephalic furrow is lost, although expression anterior to the furrow, as well as the later expression in the developing CNS is unaffected. A similar result is obtained in snail mutants (Lai, 1991).

During germ band elongation, widespread dpp expression in the dorsal ectoderm patterns the underlying mesoderm. These Dpp signals specify cardial and pericardial cell fates in the developing heart. At maximum germ band extension, dpp dorsal ectoderm expression becomes restricted to the dorsal-most or leading edge cells (LE). A second round of Dpp signaling then specifies cell shape changes in ectodermal cells leading to dorsal closure. A third round of dpp dorsal ectoderm expression initiates during germ band retraction. This round of dpp expression is also restricted to LE cells but Dpp signaling specifies the repression of the transcription factor Zfh-1 in a subset of pericardial cells in the underlying mesoderm. Surprisingly, cis-regulatory sequences that activate the third round of dpp dorsal ectoderm expression are found in the dpp disc region. The activation of this round of dpp expression is dependent upon prior Dpp signals, the signal transducer Medea, and possibly release from dTCF-mediated repression. These results demonstrate that a second round of Dpp signaling from the dorsal ectoderm to the mesoderm is required to pattern the developing heart and that this round of dpp expression may be activated by combinatorial interactions between Dpp and Wingless (Johnson, 2003).

If a second round of Dpp LE expression influences mesodermal cell fate during late stages of heart development, it would be necessary to be familiar with the wild-type expression patterns of cardial and pericardial cell markers such as Even-skipped (Eve), Seven-up, Zfh-1, and E7 3rd 63 (an enhancer trap in seven up. An unusual feature of Zfh-1 expression was noted that has not been previously described. Zfh-1 is a zinc finger and homeobox containing protein that is widely expressed in the dorsal mesoderm during early stages of heart development. Following germ band retraction, Zfh-1 becomes restricted to pericardial cells and is detected in these cells throughout the remainder of heart development. Analysis of zfh-1 loss of function mutations shows that at early stages of heart development zfh-1 is required to maintain Eve expression but that at late stages Zfh-1 and Eve are expressed in nonoverlapping sets of pericardial cells. Examination of late stage zfh-1 mutant embryos revealed morphological defects in the heart that reflect the effects of Zfh-1 on other, as yet unidentified, pericardial genes. It was noticed that the total number of pericardial cells expressing Zfh-1 decreases significantly from stage 13 to stage 17 in wild-type embryos (Johnson, 2003).

This study suggests that a second round of dpp LE expression leads to a second round of Dpp dorsal ectoderm to mesoderm signaling. The role of the second round of Dpp LE signaling appears to be the specification of a novel subset of pericardial cells by repressing the expression of the transcription factor Zfh-1. Sequences in the dpp disc region and the signal transducer Medea are required for the second round of dpp LE expression (Johnson, 2003).

Expression data reveal that a second round of dpp LE expression initiates during germ band retraction. Genetic analyses suggest that these Dpp signals influence pericardial cell fate specification by repressing Zfh-1 expression. These results provide an explanation for the observation that p-Mad is detectable in pericardial cells of germ band retracted embryos. Most likely, the second round of Dpp LE signaling stimulates p-Mad accumulation in pericardial cells during the repression of Zfh-1 expression (Johnson, 2003).

This study also extends previous studies of Zfh-1. To date, the only known function of Zfh-1 in the mesoderm is to maintain Eve expression in pericardial cells during early stages of heart development. However, late stage zfh-1 mutant embryos show heart defects not explainable by the failure to maintain Eve expression. These results led to the suggestion that Zfh-1 has additional roles during late stages of heart development. The presence of unexplained heart defects in zfh-1 mutant embryos supports the hypothesis that the second round of Dpp LE signaling functions, through repression of Zfh-1, to specify a single pericardial cell type during late stages of embryonic heart development. Perhaps the heart defects of zfh-1 mutants are due to an excess of non-Zfh-1-expressing pericardial cells (Johnson, 2003).

Two arguments are presented suggesting that the second round of Dpp dorsal ectoderm to mesoderm signaling is also conserved in vertebrate development. (1) BMP signaling represses endothelial cell fates in the cranial vasculature of zebrafish during late stages of embryonic development. Evidence for the repression of endothelial cell fate is an overabundance of endothelial cells in the cranial blood vessels of violet beauregarde (vbg) mutants. vbg encodes the BMP type I receptor similar to the Drosophila Dpp type I receptor Saxophone. The similarity of the mutant phenotypes (overabundance of Zfh-1 expressing pericardial cells in dppd6 mutants and overabundance of endothelial cells in vbg mutants) is intriguing. (2) SIP1 (Smad interacting protein) is most likely the vertebrate homolog of Zfh-1. Experiments in Xenopus show that SIP1 expression in the mesoderm is repressed by BMP overexpression and induced in the absence of BMP signaling. The similarity of the interactions and their tissue specificity (Dpp represses Zfh-1 in mesodermally derived pericardial cells and in BMP represses SIP1 in mesodermal cells) suggests evolutionary conservation (Johnson, 2003).

In summary, the role and regulation of a second round of dpp LE expression is described that corresponds to a second round of Dpp signaling from the dorsal ectoderm to the mesoderm during late stages of heart development. A second round of Dpp LE signaling represses the expression of the transcription factor Zfh-1 in a subset of pericardial cells. A subset of the enhancers driving this round of dpp LE expression are located in the dpp disc cis-regulatory region. Expression from these enhancers requires prior Dpp signaling and possibly release from dTCF-mediated repression by Wg. The continued analysis of the second round of Dpp LE signaling will provide new clues to understanding vertebrate cardiovascular development (Johnson, 2003).

Specification of Drosophila aCC motoneuron identity by a genetic cascade involving even-skipped, grain and zfh1

During nervous system development, combinatorial codes of regulators act to specify different neuronal subclasses. However, within any given subclass, there exists a further refinement, apparent in Drosophila and C. elegans at single-cell resolution. The mechanisms that act to specify final and unique neuronal cell fates are still unclear. In the Drosophila embryo, one well-studied motoneuron subclass, the intersegmental motor nerve (ISN), consists of seven unique motoneurons. Specification of the ISN subclass is dependent upon both even-skipped (eve) and the zfh1 zinc-finger homeobox gene. ISN motoneurons also express the GATA transcription factor Grain, and grn mutants display motor axon pathfinding defects. Although these three regulators are expressed by all ISN motoneurons, these genes act in an eve->grn->zfh1 genetic cascade unique to one of the ISN motoneurons, the aCC. The results demonstrate that the specification of a unique neuron, within a given subclass, can be governed by a unique regulatory cascade of subclass determinants (Garces, 2006).

Why do these three genes act in a unique fashion in aCC, and why is grn and zfh1 sensitive to Notch specifically in this ISN motoneuron? One explanation may be that the differential input from upstream regulators, such as Ftz, Pdm1, Hkb and Pros, acts to modify the genetic interactions between eve, grn and zfh1. Another possibility is that the relative level of each factor plays an important role in dictating different cellular fates. Studies of the related Isl1 and Isl2 LIM-homeobox genes suggest that their involvement in motoneuron subclass specification is not primarily the result of the unique activity of each gene, but rather by the combined 'generic', tightly temporally controlled, Isl1 and Isl2 levels. Similarly, the different expression levels of the transcription factor Cut have been shown to play instructive roles during the specification of neuronal cell identities within the PNS. Different levels of expression of Grn and Zfh1 have been observed; while Grn is strongly expressed in aCC and weakly in RP2, Zfh1 expression shows an opposite distribution. It is tempting to speculate that these levels may be instructive for ISN motoneuron specification (Garces, 2006).

In the VNC, mutually exclusive expression is observed between Grn and Hb9 (and Islet) in different subsets of interneurons and motoneurons. Cross-inhibitory interactions between eve and Hb9 has been shown to contribute to their mutually exclusive expression patterns, and functional studies demonstrate that eve and Hb9 regulate axonal trajectories of dorsally and ventrally projecting axons, respectively. These observations are reminiscent of the cross-repressive interactions between classes of regulators that act to determine, refine and maintain distinct progenitor domains along the dorsoventral axis of the vertebrate neural tube. eve is important for proper grn and zfh1 expression in aCC, but not in RP2. These results are consistent with previously reported observations that the requirement for eve in axonal guidance is somewhat more stringent in aCC than in RP2, leading the the proposal that there may be different target genes for Eve in these two motoneurons (Garces, 2006).

Zfh1 expression was previously shown to depend upon Notch signaling activity in the aCC/pCC sibling pair as mutations in spdo or mam, members of the Notch signaling pathway, lead to de-repression of Zfh1 in pCC. Using the same allelic combinations, de-repression of grn was also observed in the pCC. Whether or not grn is directly suppressed by the Notch pathway remains to be seen, but it is interesting to note that in vertebrates, gata2/3 have been identified as targets of Notch during the differentiation of specific hematopoietic lineages (Garces, 2006).

Within the ISN subclass, the aCC motoneuron pioneers the ISN to innervate the dorsal-most muscle, muscle 1. A number of genetic and cell-ablation studies have convincingly shown that aCC plays an instructive pioneer role and guides the follower U motoneurons along the ISN nerve. These results lend support for the proposed instructive role of aCC in ISN formation. However, these studies indicate that aCC may not be essential for ISN formation. First, using RN2-GAL4 to visualize aCC and RP2, aberrant innervation of muscle 8 were frequently found (35% of hemisegments) in grn mutants. However, an axonal projection was simultaneously observed at the vicinity of the dorsal muscles 2/10. In grn mutants, zfh1 expression is specifically lost in aCC but maintained in RP2. Given the role for zfh1 in motor axon pathfinding, it is proposed that aberrant innervation of muscle 8 in grn mutants, is caused by aCC and not by RP2, and that RP2 pathfinds normally to the muscles 2/10. If so, RP2 may function as a pioneer motoneuron for muscle 2 and project there without the aCC axon. Second, although the rescue of grn mutants using RN2-GAL4 is complete, it was found that using CQ2-GAL4 to specifically rescue U motoneurons does lead to a partial rescue (54% muscles 1/9 innervated compared with 15% in grn mutants). Thus, even in the absence of aCC pioneer function, the Us (presumably U1) can still project to the dorsal-most muscles. This is in line with previous studies showing that in eve aCC/RP2 mosaic mutants and in aCC/RP2 cell ablation experiments, there is still partial innervation of muscle 1/9 (Garces, 2006).

grn is part of an eve --> grn --> zfh1 transcriptional cascade crucial for specification of aCC motoneuron identity. However, the failure of grn to rescue eve, and of zfh1 to completely rescue grn, combined with the misexpression results, indicate additional roles for both eve and grn. These roles could be either in the regulation of other aCC determinants and/or in the regulation of genes directly involved in aCC axon pathfinding. Although there are no obvious candidates for additional aCC determinants, recent studies point to a candidate axon pathfinding gene. The Drosophila unc-5 gene encodes a netrin receptor and is expressed in subsets of neurons in the VNC. Misexpression of unc-5 is sufficient to trigger ectopic VNC exit in subsets of interneurons. Recent studies now show that unc-5 is specifically expressed in eve motoneurons, and that eve is necessary, but only partly sufficient for unc-5 expression. In line with these findings, it was found that whereas single misexpression of eve or grn in dMP2 neurons has very minor effects, co-misexpression of eve and grn can efficiently trigger dMP2 lateral axonal exit. This combinatorial effect of eve/grn occurs without apparent activation of zfh1. However, misexpression of zfh1 can also trigger dMP2 lateral exit. Thus, these genes appear to be able to act in an independent manner to trigger VNC exit, but in a highly context-dependent manner. A speculative explanation for not only the mutant and rescue results, but also these misexpression results, would be that all three regulators are needed for robust and context-independent activation of axon pathfinding genes such as, for example, unc-5 (Garces, 2006).

grn encodes a GATA Zn-finger transcription factor and is the ortholog of the closely related vertebrate gata2 and gata3 genes. In vertebrates, gata2/3 are expressed in overlapping domains in the nervous system, but relatively little is known about their function. Expression data and evidence from gene targeting suggest an involvement in neurogenesis, neuronal migration and axon projection. A role in specifying neuronal subtypes within the context of neural tube patterning is emerging and recently a role for gata2/3 during 5-HT neuron development has been reported. The role of gata3 in the development of the inner ear has been of particular interest, and in humans, mutations in this gene have been linked to HDR syndrome, which is characterized by hypoparathyroidism, deafness and renal defects. In the mouse, gata3 is expressed in auditory but not vestibular ganglion neurons during development. The mouse gata3 mutant shows auditory ganglion neuron loss and efferent nerve misrouting, revealing that gata3 regulates molecules associated with neural differentiation and guidance. These vertebrate studies, combined with the current results, suggest that gata2/3 genes, similar to other transcription factors specifying neuronal identities, such as islet1/2, evx1/2 or Hb9, and their respective orthologs in Drosophila, have maintained similar functions throughout evolution (Garces, 2006).

A Dpp signal from the dorsal ectoderm restricts the number of pericardial cells expressing the transcription factor Zfh1

During germ-band extension, Dpp signals from the dorsal ectoderm to maintain Tinman (Tin) expression in the underlying mesoderm. This signal specifies the cardiac field, and homologous genes (BMP2/4 and Nkx2.5) perform this function in mammals. A second Dpp signal from the dorsal ectoderm restricts the number of pericardial cells expressing the transcription factor Zfh1. Via Zfh1, the second Dpp signal restricts the number of Odd-skipped-expressing and the number of Tin-expressing pericardial cells. Dpp also represses Tin expression independently of Zfh1, implicating a feed-forward mechanism in the regulation of Tin pericardial cell number. In the adjacent dorsal muscles, Dpp has the opposite effect. Dpp maintains Krüppel and Even-skipped expression required for muscle development. The data show that Dpp refines the cardiac field by limiting the number of pericardial cells. This maintains the boundary between pericardial and dorsal muscle cells and defines the size of the heart. In the absence of the second Dpp signal, pericardial cells overgrow and this significantly reduces larval cardiac output. This study suggests the existence of a second round of BMP signaling in mammalian heart development and that perhaps defects in this signal play a role in congenital heart defects (Johnson, 2007).

A previous study suggested that a second round of Dpp dorsal ectoderm-to-mesoderm signaling, stimulated by enhancers located in the dpp disk region, initiates during germ-band retraction (stage 12; Johnson, 2003). This is referred to as the second round of signaling because a distinct set of enhancers located in the dpp Haplo-insufficiency (Hin) region activates Dpp dorsal ectoderm-to-mesoderm signaling during germ-band extension (stage 8). Further, the data revealed that dpp dorsal ectoderm expression driven by the Hin region enhancers persists long after germ-band retraction. These studies showed that Hin-region-driven dpp expression is sufficient for Dpp ectodermal functions such as dorsal closure and dorsal branch migration (Johnson, 2007).

Given these data, it appears that the dppd6 inversion prevents the augmentation of dpp expression in the dorsal ectoderm during germ-band retraction that is normally provided by disk region enhancers. The presence of numerous mesodermal phenotypes in dppd6 mutants (Johnson, 2003) suggests that the augmentation of dpp expression is necessary to boost Dpp dorsal ectoderm signals so that they can reach the underlying mesoderm. Perhaps there are barriers of distance or extracellular matrix density between these germ layers that must be overcome (Johnson, 2007).

The data are wholly consistent with the hypothesis that the dppd6 inversion prevents the augmentation of dpp expression provided by disk region enhancers during germ-band retraction. The data further suggest that the augmentation of dpp expression is necessary to boost Dpp dorsal ectoderm signals such that they can reach the underlying mesoderm. Finally, this study shown that during germ-band retraction Dpp signals maintain the boundary between pericardial cells and dorsal muscle cells via two distinct mechanisms: the regulation of gene expression and the restriction of cell proliferation. To regulate gene expression, Dpp signals directly to pericardial cells and restricts Odd and Tin expression in a zfh1-dependent manner. Dpp also limits Tin expression, independently of zfh1, by repressing the expression of mid, a stimulator of proliferation (Johnson, 2007).

With respect to zfh1-dependent regulation, the data support the hypothesis that Dpp restricts Zfh1 expression to regulate the number of pericardial cells derived solely from symmetrically dividing lineages. Lineage analyses have identified both symmetric and asymmetric cell divisions of myogenic and pericardial precursor cells. Pericardial cells are derived from four separate lineages that arise from four distinct precursor cells. Asymmetric precursor cell divisions initiating between stages 8 and 10 give rise to the Odd-positive/Seven up (Svp)-positive pericardial cells and the Eve-positive/Tin-positive pericardial cells (EPCs). In contrast, symmetric division, initiating at the same stage, establishes the Odd-positive/Svp-negative pericardial cells (OPCs) and the Tin-positive/Eve-negative pericardial cells (TPCs). dpp mutations do not affect the number of EPCs or the number of Odd-positive/Svp-positive cells. However, embryos bearing dpp mutations show an increase in the number of OPCs and TPCs. Therefore, the ectopic pericardial cells seen in dpp mutants derive from symmetrically dividing lineages (Johnson, 2007).

Previous reports have shown that regulation of asymmetric cell division is a key mechanism in establishing boundaries among the various cell types in the dorsal mesoderm. For instance, in the absence of Numb, a Notch pathway antagonist, asymmetric progenitor cell division is abrogated and the number of Odd-positive/Svp-positive cells and EPCs increases at the expense of the Svp-expressing cardial cells and Eve-expressing dorsal muscle cells, respectively. This study extends these observations by showing that pericardial cell types derived from symmetrically dividing lineages are also under strict regulatory control (Johnson, 2007).

With respect to zfh1-dependent regulation of pericardial cell number, Dpp restricts cell proliferation and, in turn, Tin expression by limiting mid expression. In wild-type embryos, cell division in the dorsal mesoderm is largely complete by the early stages of germ-band retraction (stage 11), whereas in dppd6 embryos cell proliferation in the dorsal mesoderm continues through stage 13. Interestingly, the number of cells expressing Zfh1 increases from stage 12 to stage 13 in wild-type embryos in the absence of cell division, demonstrating that patterning events subsequent to cell division regulate cell fate choices in the dorsal mesoderm. This hypothesis is supported by the fact that tracing pericardial cell lineages requires inducing mitotic clones by stage 8. Therefore, the ectopically dividing mesoderm cells observed in dppd6 embryos are derived from cells with the potential to become Tin-expressing cells (Johnson, 2007).

During stage 12, tin expression is reactivated in a subset of cardiac cells in a mid-dependent fashion, suggesting that tin expression in precursor cells alone is not sufficient for specifying the ultimate fate of their daughter cells. Moreover, misexpression of mid results in both ectopic cell division and expanded tin expression. Lineage studies support the necessity of reactivating Tin by showing that a single precursor cell gives rise to two Tin-positive/Eve-negative pericardial cells and two siblings that do not express Tin. Thus tin is not reactivated in all subpopulations of pericardial cells. The data suggest that, during stage 12, Dpp prevents tin reactivation in cells occupying lateral regions of the dorsal mesoderm by limiting mid expression (Johnson, 2007).

Development of the dorsal musculature initiates when founder cells are specified in the mesoderm. These founder cells then fuse with neighboring cells to form syncitial myofibers. In the absence of Dpp, the pericardial cell domain expands into the dorsal muscle domain and reduces expression from the dorsal muscle genes Kr and Eve. Since the separation between pericardial and dorsal muscle cells is lost in dpp mutant embryos, it is concluded that Dpp maintains the pericardial-dorsal muscle cell boundary after it is established. Moreover, reducing pericardial cell number increases Kr expression after germ-band retraction, suggesting that cross-repressive interactions between pericardial and dorsal muscle cells contribute to patterning of the dorsal mesoderm. The presence of ectopic pericardial cells in the dorsal mesoderm reduces the number of myofibers comprising the dorsal muscles even though the dorsal muscle founder cells are, for the most part, correctly specified. pMad does not accumulate in Kr-expressing founder cells yet Kr expression is significantly reduced in dpp mutant embryos. Therefore, changes in Kr and Eve expression observed in embryos with altered dpp or zfh1 activity reflect alterations in the number of myoblast fusion events in the dorsal mesoderm (Johnson, 2007).

These data extend a previous study showing that misexpressing Zfh1 reduces dMef2 expression in somatic muscles. This study demonstrates that misexpression of Zfh1 induces ectopic pericardial cells and that the presence of pericardial cells in the dorsal muscle domain reduces myoblast fusion. Therefore, reduced dMef2 expression in embryos misexpressing Zfh1 is likely the result of reduced myoblast fusion and not of direct repression of dMef2 expression by Zfh1. Further, analysis of lmd mutants that have reduced numbers of myoblasts revealed that they also contain an excessive number of pericardial cells. Together, these results suggest that maintaining the pericardial-dorsal muscle cell boundary requires Dpp-mediated cross-repressive interactions between these cell types. Thus, in the absence of Dpp, the transformation of dorsal muscle cells into pericardial cells reduces the number of myoblasts available for fusion (Johnson, 2007).

Experiments in the larvae of Drosophila and other insects suggested that pericardial cells act as nephrocytes that filter the hemolymph. These studies also showed that pericardial cells secrete proteins into the hemolymph, suggesting that one pericardial cell function may be to provide short- or long-range signals. Consistent with this, reducing pericardial cell number reduces heart rate and increases the cardiac failure rate, suggesting that pericardial cells influence the development of cardiac cells (Johnson, 2007).

This study shows that pericardial cell hyperplasia reduces the luminal distance of the heart during systole as well as diastole, resulting in an overall decrease in average pulse distance of each contraction. However, pericardial overgrowth does not alter heart rate, indicating that cardiac cells develop appropriately in the presence of ectopic pericardial cells. Luminal measurements suggest a role for pericardial cells in the mechanics of heart function. One hypothesis for this is based on the fact that pericardial cell hyperplasia results in excess levels of extracellular matrix protein Pericardin (Prc) in the extracellular matrix (ECM) surrounding the heart. Prc is a collagen IV-like ECM protein secreted at high levels from pericardial cells. In dpp mutants, excess Prc is seen predominantly in the posterior of the heart where the pulse-distance reduction was observed. It is proposed that Prc secreted by pericardial cells limits the width of the dorsal vessel at diastole and thus modulates the pulse distance of each heart contraction. Pericardial cell overgrowth would increase Prc deposition, thereby reducing the size of the diastolic heart and the pulse distance. Consistent with this hypothesis, excessive expression of ECM proteins, including collagen IV, was correlated with heart failure in patients presenting with end-stage cardiomyopathy (Johnson, 2007).

It is well documented that many of the early events driving Drosophila embryonic heart development have been conserved in vertebrates. The data provide the first basis upon which to determine if Dpp regulation of Zfh1 or Tin late in heart development is also conserved (Johnson, 2007).

Two orthologs of zfh1, Sip1 and Kheper, have been identified in vertebrates. Zebrafish embryos injected with the Dpp homolog BMP4 show reduced Kheper expression while Xenopus embryos injected with the BMP antagonist Chordin display elevated Sip1 expression. These results suggest the possibility that Dpp repression of zfh1 expression may be conserved in vertebrates. In addition, mammalian Sip1 plays an essential role in heart development. In mice, Sip1 is expressed in neural crest cells (NCCs), paraxial mesoderm, and neuroectoderm. The subset of NCCs that express Sip1 give rise to the septum and large arteries of the heart. Sip1 knockout mice fail to form these NCCs and these mice die midway through gestation with numerous heart defects. Mice lacking the BMP receptors BMPRIA or ALK2 specifically in NCCs also display numerous cardiac phenotypes. In conditional knockout of ALK2 in NCCs, abnormalities are seen in the heart's outflow tract, and conditional knockout of BMPRIA in NCCs results in heart failure and early embryonic lethality similar to Sip1 knockout mice. Thus BMP signals are required for proper specification of NCCs, and loss of BMP signaling in NCCs phenocopies Sip1 knockout mice to an extent. It is tempting to speculate that, as in Drosophila, BMP signals regulate the Zfh1 ortholog Sip1 to correctly specify NCCs and, in turn, to properly pattern the mammalian heart (Johnson, 2007).

With regard to the conservation of late-stage Dpp regulation of Tin, a recent article describing a study of mice with a conditional knockout of Nkx2.5 where expression is missing only during late stages of heart development (post E14.5) is highly relevant. Utilizing rescue of Nkx2.5 mutant embryos with BMP-signaling-pathway components, the study identified a direct connection among BMP4 signaling, Nkx2.5 activity, and heart cell proliferation. Since Nkx2.5 is the Tin homolog, BMP4 is the Dpp homolog, and the mutant phenotype (heart cell hyperplasia) is the same in both species, this suggests that this aspect of Dpp signaling is conserved in mammals. Together with this study, these results suggest that defects in late-stage BMP signaling may play a role in congenital heart defects (Johnson, 2007).

Zfh-1 controls somatic stem cell self-renewal in the Drosophila testis and nonautonomously influences germline stem cell self-renewal

The ability of adult stem cells to maintain their undifferentiated state depends upon residence in their niche. While simple models of a single self-renewal signal are attractive, niche-stem cell interactions are likely to be more complex. Many niches have multiple cell types, and the Drosophila testis is one such complex niche with two stem cell types, germline stem cells (GSCs) and somatic cyst progenitor cells (CPCs). These stem cells require chemokine activation of Jak/STAT signaling for self-renewal. The transcriptional repressor Zfh-1 has been identified as a presumptive somatic target of Jak/STAT signaling, demonstrating that it is necessary and sufficient to maintain CPCs. Surprisingly, sustained zfh-1 expression or intrinsic STAT activation in somatic cells caused neighboring germ cells to self-renew outside their niche. In contrast, germline-intrinsic STAT activation was insufficient for GSC renewal. This data reveals unexpected complexity in cell interactions in the niche, implicating CPCs in GSC self-renewal (Letherman, 2008).

Thus Zfh-1 function is required to block differentiation of CPCs. zfh-1 may have conserved function as a stem cell factor, since it is expressed in somatic stem cell types in the Drosophila ovary, and the mammalian homologue zfhx1b is expressed in human ES cells (Boyer, 2005). As zfh-1 is also required to specify the CPC lineage, it joins a growing list of genes that act within a cell lineage to both to specify the lineage and later to maintain that cell type in the stem cell state (Letherman, 2008).

In the testis, the enrichment of Zfh-1 in CPCs and the fact that zfh-1 can be somatically induced in response to STAT activation suggested that zfh-1 is a target of the STAT self-renewal signal in CPCs. Consistent with this, Zfh-1 accumulation is reduced when STAT function is compromised. While it is not suspected that there will be other STAT targets in CPCs -- two candidates are the CPC-enriched genes SOCS36e and CG2264. zfh-1 is likely a key target, since sustained expression of zfh-1 elicits the same phenotype as somatic activation of STAT (the maintenance of stem cell fate in somatic daughter cells). However, it is also noted that Zfh-1 did not disappear upon stat inactivation, suggesting that another input(s) to CPCs remains to be identified (Letherman, 2008).

The data suggest strongly that the key activity of Zfh-1 is transcriptional repression. This is definitively the case from a gain-of-function test, and given the requirement found for the co-repressor CtBP in the somatic lineage, likely also so for Zfh-1 in CPCs in the niche. This is significant as recent embryonic stem cell studies suggest that a primary function for the self-renewal factors Nanog, Oct4, and Sox2 is to repress master regulators of differentiation pathways. Zfh-1 and its mammalian homologue zfhx1b repress various differentiation genes, including mef2 for muscle differentiation, brachyury for mesoderm formation, and E-cadherin for epithelial differentiation. In the testis niche, eya may be a target of Zfh-1 repression, since it is rapidly activated when zfh-1 is removed. Similarly, the Eya transcription factor is an essential differentiation factor in the somatic lineage. Thus, Zfh-1 shares functional similarity with the ES cell self-renewal factors in blocking differentiation (Letherman, 2008).

Among other potential targets for Zfh-1 in the CPCs is the repression of genes regulated by EGFR/raf signaling. Prior work showed that EGFR signaling is required in somatic cells, and the transcriptional branch of the EGFR pathway culminates in the activation of ETS transcription factors, regulating gene expression in the somatic cells. Interestingly, transcriptional repression by members of the Zfh-1 family can be overcome by ETS proteins acting in synergy with the transcription factor c-Myb. Perhaps in the testis niche Zfh-1 prevents differentiation by inhibition of ETS targets until an essential ETS synergizing factor is produced, or until Zfh-1 levels simply decay in CPC daughters (Letherman, 2008).

The ability of sustained zfh-1 expression to non-autonomously cause continual GSC self-renewal is a surprising result that questions the idea that germline Jak/STAT activation is instructive for GSC self-renewal. The data shows that Jak/STAT activation in the GSCs is not sufficient for GSC renewal outside the niche; rather, Jak/STAT activation in CPCs (with Zfh-1 activation) is necessary for niche independence. The gain-of-function experiment suggests several possibilities for normal GSC renewal. One is that Zfh-1 normally inhibits a differentiation signal sent from somatic to germ cells, and as a consequence of sustaining Zfh-1, GSCs stay in a 'default' stem cell state. This option is supported by the ability of GSCs to proliferate in the absence of EGFR activation in somatic cells. A second possibility is that both STAT activation and a zfh-1-dependent signal delivered by somatic cells are required for GSC renewal. For instance, the zfh-1-dependent signal could activate a transcription factor in germ cells that assisted phosphorylated STAT in GSC gene regulation. In this scenario, a requirement for STAT phosphorylation might be bypassed if enough of the second factor is produced (as when zfh-1 expression is sustained in CPC daughters). Interestingly, mouse ES cells require two signals for self-renewal: LIF activation of STAT3 and BMP activation of SMADs. A requirement for BMP signaling in testis GSCs has already been demonstrated, and it has been suggested that Jak/STAT signaling leads to production of the BMP ligand Dpp. However, a BMP ligand could only constitute part of the signal, as overexpression of BMP does not cause the same phenotype as does sustaining zfh1 expression (Letherman, 2008).

A requirement for a second signal assisting in GSC self-renewal has precedent in the Drosophila ovarian niche. There, a BMP was the first signaling pathway discovered to act in GSC self renewal. However, newly identified somatic escort stem cells (ESCs) surround GSCs, just as CPCs do in the testis. Like CPCs, ESCs intrinsically require STAT, as STAT loss non-autonomously causes GSC loss, demonstrating the requirement for an ESC-dependent signal in GSC renewal. In both the female and male niches, if somatic stem cell types are indeed required for GSC renewal, this would provide a means of balancing the two stem cell populations, since GSCs would not be able to survive without CPCs/ESCs, and an overabundance of CPCs/ESCs might increase the available GSC 'renewal' signal, causing additional germ cells to become GSCs via dedifferentiation or symmetric stem cell division. Proof of such a requirement for CPCs in the testis will require removing zfh-1 from many or most CPCs; since zfh-1 mutant CPCs are lost so rapidly, it has not been possible to follow the fate of neighboring GSCs. Attempts to knockdown zfh-1 in all CPCs by dsRNA transgenesis have thus far been unsuccessful in achieving significant reduction of Zfh-1 protein (Letherman, 2008).

This work suggests the existence of a hub to CPC to GSC self-renewal relay signal: Upd, secreted by hub cells, activates Zfh-1 in CPCs, which in turn causes a signal to be sent to the GSCs resulting in their self-renewal. Whether this relay is a required component of the GSC renewal, or simply an amplification of a hub renewal signal, it demonstrates a higher degree of complexity in cell-cell interactions than has been previously found in a stem cell niche. Recent work in other niches increasingly points toward the existence of (and a need for) such complex interactions. For example, in the Drosophila ovary, a feedback loop between stem cells and niche cells has recently been discovered. Similar to the Drosophila testis, the mammalian hair follicle niche supports two stem cell populations -- melanocyte and hair follicle stem cells. The potential for coordination between these two populations has not been explored, and could be relevant to the prevention of melanocyte stem cell loss, which results in hair graying. Neural stem cells were recently found to be much more diverse than expected, and their identity is dependent on their location, implying that niche signals for these stem cells must do more than just keep them in an undifferentiated state. Finally, hematopoietic stem cells reside in two distinct niches, associated with either osteoblasts or endothelial cells. Both niches require a second cell type, CXCL12-abundant reticular cells, for stem cell maintenance. How signals from these different cell types interact to coordinate self-renewal is completely unknown. Models based on the self-renewal relay described here will be a starting point for beginning to explore the complex cell interactions in these niches (Letherman, 2008).

chinmo is a functional effector of the JAK/STAT pathway that regulates eye development, tumor formation, and stem cell self-renewal in Drosophila: zfh1 acts downstream of Stat92E to maintain cyst stem cells

The Drosophila STAT transcription factor Stat92E regulates diverse functions, including organ development and stem cell self-renewal. However, the Stat92E functional effectors that mediate these processes are largely unknown. This study shows that chinmo is a cell-autonomous, downstream mediator of Stat92E that shares numerous functions with this protein. Loss of either gene results in malformed eyes and head capsules due to defects in eye progenitor cells. Hyperactivation of Stat92E or misexpression of Chinmo results in blood cell tumors. Both proteins are expressed in germline (GSCs) and cyst stem cells (CySCs) in the testis. While Stat92E is required for the self-renewal of both populations, chinmo is only required in CySCs, indicating that Stat92E regulates self-renewal in different stem cells through independent effectors. Like hyperactivated Stat92E, Chinmo misexpression in CySCs is sufficient to maintain GSCs nonautonomously. Chinmo is therefore a key effector of JAK/STAT signaling in a variety of developmental and pathological contexts (Flaherty, 2010).

This study has revealed important information about chinmo and its role in Stat92E-dependent biological processes, including eye development, hematopoeisis, and stem cell self-renewal. chinmo was identified as a cell-autonomously induced downstream mediator of JAK/STAT activity that shares loss- and gain-of-function phenotypes with Stat92E in several tissues. Although chinmo was originally identified in a screen for genes required for temporal identity of mushroom body neurons, no factors that regulate its expression had been identified. The fact that chinmo and Stat92E exhibit a high degree of functional overlap suggests that chinmo performs multiple Stat92E-dependent functions, including growth of the eye disc, formation of melanotic tumors, proliferation of mature hemocytes, self-renewal of adult stem cells, and repression of Ser. Furthermore, the results raise the interesting hypothesis that the JAK/STAT pathway is also required for the temporal identity of neurons in the mushroom body. It was also shown that Chinmo, like stabilized Stat92E, is expressed in GSCs and in CySCs in the testis. However, unlike Stat92E, Chinmo is required intrinsically only for the self-renewal of CySCs and not of GSCs. These data clearly indicate that Stat92E acts through distinct effector genes in these stem cells to promote cell-autonomous self-renewal. Finally misexpression of chinmo in CySCs results in the expansion of GSCs and CySCs, a phenotype also observed with misexpression of hopTum-l or of zfh1 in somatic cells. This provides additional evidence for the coordination of self-renewal and differentiation between adjacent GSCs and CySCs (Flaherty, 2010).

The BTB domain mediates protein-protein interactions, including dimerization, recruitment of transcriptional repressors to DNA, and protein degradation by acting as adaptors for Cul-3 E3 ubiquitin ligases. In the antenna, Ser was found to be cell-autonomously repressed by both Stat92E and Chinmo (this study; Flaherty, 2009). These data suggest that Chinmo might act as a transcriptional repressor, at least in the antennal disc, and that Ser might be one of its transcriptional targets. It should be stressed that these results do not rule out the possibility that Chinmo, through its BTB domain, can also act as an adaptor for Cul-3 and promote protein degradation. In fact, recent work has revealed that even BTB-ZF transcription factors like promyelocytic leukemia zinc finger (PLZF) can interact with Cul-3. Whereas the role of the BTB domain in PLZF-dependent transcriptional repression has been well documented, the physiological role of the PLZF-Cul-3 interaction and the proteins it modifies are as yet unknown. Future experiments will be needed to determine if Chinmo can act both as a transcriptional repressor per se and as an adaptor for E3 ligases. In either scenario, factors modified by Chinmo, in addition to Ser, will need to be identified and characterized (Flaherty, 2010).

This study shows that Chinmo, like Zfh1, is essential for the self-renewal of CySCs in the Drosophila testis. Furthermore, sustained expression of Chinmo in somatic cells, like that of Zfh1, is sufficient to induce the expansion of GSCs and CySCs. In addition, chinmo and zfh1 do not regulate each other's expression. chinmo and zfh1 both appear to act downstream of Stat92E to maintain CySCs, which raises the possibility that these factors function either in an epistatic or parallel manner in the somatic lineage (this study; Leatherman, 2008). chinmo does not act through zfh1, but it was not possible to determine if the reciprocal was true. However, it is hypothesized that if zfh1 is upstream of chinmo then CySCs lacking either of these factors should differentiate at the same time point. In fact, CySCs lacking zfh1 differentiate faster than those lacking chinmo, suggesting that zfh1 may not reside upstream of chinmo. Despite the unresolved genetic relationship between zfh1 and chinmo, the data are consistent with a model in which they function in a parallel pathway in the self-renewal of CySCs and the expansion of GSCs and CySCs (Flaherty, 2010).

Zfh1 has been shown to act as a transcriptional repressor (see Leatherman, 2008). The current data suggest that Chinmo may inhibit transcription directly or by post-translational modification of factors that silence genes. Zfh1 is expressed highly in CySCs and at low levels in early cyst cells. It is not expressed in late cyst cells. In contrast, Chinmo is expressed at high and comparable levels in CySCs and early cyst cells, but not in late cyst cells. Taking into account all of these results, two models are proposed to explain the function of Chinmo in the somatic lineage of the testis. The first hypothesizes that Zfh1 and Chinmo regulate distinct downstream effectors, all of which are required for the maintenance of CySCs. In this model, early cyst cells, which express high levels of Chinmo but low levels of Zfh1, can become late cyst cells only when Chinmo expression is sufficiently decreased there, allowing for full cyst cell differentiation and the complete development of mature germ cells. In the second model, Chinmo and Zfh1 regulate different genes critical for CySC self-renewal, but in contrast to the first, Chinmo only has a function in CySCs and not in early cyst cells. In this second model, the existence is invoked of cofactors expressed only in CySCs that act in concert with Chinmo, thereby restricting Chinmo function only to CySCs (Flaherty, 2010).

Activation of the JAK/STAT signaling pathway in CySCs is sufficient to promote self-renewal of both CySCs and GSCs, indicating that CySCs can influence GSC maintenance (Leatherman, 2008). Although the mechanisms by which CySCs regulate GSC self-renewal have not yet been elucidated at the molecular level, two models have been proposed. Activated Stat92E, through its functional effectors, could (1) block CySC differentiation intrinsically, thus also inhibiting GSC differentiation at the same time, or (2) send one or more non-cell-autonomous signals from CySCs to GSCs, thus promoting GSC self-renewal. Either model would result in an increase in the number of CySCs and GSCs. In the first model, sustained activation of JAK/STAT signaling in CySCs allows these cells to continue proliferating. Therefore, they accumulate outside of the niche. Previous studies have shown that the mitoses of GSCs and CySCs must be linked in order to have the appropriate number of GSCs, and their progeny always encapsulated by two CySCs/cyst cells. In the second model, the self-renewal of GSCs depends on two independent signals: one is Upd sent from the niche, which activates Stat92E in adjacent GSCs, and the other is an unknown factor presumed to come from the neighboring CySCs. A similar situation occurs in female flies, where two signals are required for GSC maintenance. First, cap cells, which form the ovarian niche, produce Decapentaplegic (Dpp), which acts on GSCs by inhibiting the bam gene. Second, an Upd cytokine, produced by ovarian somatic support cells adjacent to the niche, acts on cap cells to increase Dpp production, thus influencing GSC self-renewal in a nonautonomous manner. It is currently unknown whether the nonautonomous signal from CySCs to GSCs in the testis is a secreted factor (Flaherty, 2010).

The results indicate that Chinmo has an important role in the self-renewal of CySCs, in the inhibition of CySC differentiation, and in the transduction of the nonautonomous signal from CySCs to GSCs. Furthermore, this expansion requires the BTB and ZF domains in Chinmo, suggesting that the molecular function of Chinmo in this process may be transcriptional repression and/or protein degradation. The fact that somatic misexpression of Chinmo, like that of Zfh1, can promote GSC self-renewal/expansion indicates that at least three factors play important roles in regulating stem cell self-renewal in a nonautonomous manner: activated Stat92E, Zfh1, and Chinmo. These observations raise many issues, such as the importance of JAK/STAT pathway activity in the soma, whether chinmo can bypass the requirement for Stat92E in CySCs, and the mechanism of nonautonomous self-renewal between adjacent stem cell populations. These issues need to be addressed at the molecular level in the future (Flaherty, 2010).

A protein BLAST search against the nonredundant mouse database identified mZFP509 as a potential Chinmo ortholog. mZFP509 is a 757 amino acid protein that has the same overall structure as Chinmo: an N-terminal BTB domain located between residues 20-120 separated from two C-terminal C2H2 zinc fingers by a stretch of ~400 amino acids. mZFP509 is 27% identical to Chinmo (with 36% identity in the BTB domain and 31% identity the ZF region) and is 83.7% identical to hZFP509. Microarray studies indicate that mzfp509 is enriched in normal and cancer stem cells. mzfp509 transcripts are present in mESCs but are substantially reduced during their differentiation. They are also significantly increased in PU.1-deficient preleukemic hematopoietic stem cells and normal mammary stem cells. These data suggest that ZFP509 and Chinmo are orthologs and that what is discovered about Chinmo in Drosophila may have a high probability of holding true for its mammalian counterpart. For example, blocking hZFP509 function may have therapeutic value in inhibiting cancer stem cells, thus offering better outcomes for human patients (Flaherty, 2010).

CSN maintains the germline cellular microenvironment and controls the level of stem cell genes via distinct CRLs in testes of Drosophila melanogaster

Stem cells and their daughters are often associated with and depend on cues from their cellular microenvironment. In Drosophila testes, each Germline Stem Cell (GSC) contacts apical hub cells and is enclosed by cytoplasmic extensions from two Cyst Stem Cells (CySCs). Each GSC daughter becomes enclosed by cytoplasmic extensions from two CySC daughters, called cyst cells. CySC fate depends on an Unpaired (Upd) signal from the hub cells, which activates the Janus Kinase and Signal Transducer and Activator of Transcription (Jak/STAT) pathway in the stem cells. Germline enclosure depends on Epidermal Growth Factor (EGF) signals from the germline to the somatic support cells. Expression of RNA-hairpins against subunits of the COnstitutively Photomorphogenic-9- (COP9-) signalosome (CSN; see CSN5) in somatic support cells disrupted germline enclosure. Furthermore, CSN-depleted somatic support cells in the CySC position next to the hub had reduced levels of the Jak/STAT effectors Zinc finger homeotic-1 (Zfh-1) and Chronologically inappropriate morphogenesis (Chinmo). Knockdown of CSN in the somatic support cells does not disrupt EGF and Upd signal transduction as downstream signal transducers, phosphorylated STAT (pSTAT) and phosphorylated Mitogen Activated Protein Kinase (pMAPK), were still localized to the somatic support cell nuclei. The CSN modifies fully formed Cullin RING ubiquitin ligase (CRL) complexes to regulate selective proteolysis. Reducing cullin2 (cul2) from the somatic support cells disrupted germline enclosure, while reducing cullin1 (cul1) from the somatic support cells led to a low level of Chinmo. It is proposed that different CRLs enable the responses of somatic support cells to Upd and EGF (Qian, 2014).

Targets of Activity

Drosophila zfh-1 is downregulated in embryos prior to myogenesis. Embryos with zfh-1 loss-of-function mutation show alterations in the number and position of embryonic somatic muscles, suggesting that zfh-1 could have a regulatory role in myogenesis. Zfh-1 is a transcription factor that binds E box sequences and acts as an active transcriptional repressor. When zfh-1 expression is maintained in the embryo beyond its normal temporal pattern of downregulation, the differentiation of somatic but not visceral muscle is blocked. One potential target of zfh-1 in somatic myogenesis could be the myogenic factor mef2. mef2 is known to be regulated by the transcription factor twist, and Zfh-1 is shown to bind to sites in the mef2 upstream regulatory region and inhibit twist transcriptional activation. Even though there is little sequence similarity in the repressor domains of vertebrate ZEB and zfh-1, evidence is presented that Zfh-1 is the functional homolog of ZEB and that the role of these proteins in myogenesis is conserved from Drosophila to mammals (Postigo, 1999c).

Among all zfh family members, Zfh-1 and ZEB share the most sequence similarity in the zinc fingers and homeodomain. The zinc fingers of ZEB bind to a subset of E boxes (and E box-like sequences), with highest affinity for the CACCTG site. The similarity in the zinc fingers of ZEB and Zfh-1 suggests that these motifs in Zfh-1 might also be DNA binding domains. Therefore, whether Zfh-1 can bind to the CACCTG site was tested. Both the N- and C-terminal zinc fingers of recombinant Zfh-1 bind to the site in gel retardation assays. This binding is abolished when the site is mutated. Furthermore, as observed for ZEB, Zfh-1 binds to only a subset of E box sequences; it fails to bind the CATTTG E box sequence. Interestingly, the Zfh-1 binding site also matches the high-affinity site recognized by the zinc finger protein Snail, and Zfh-1 binds quite efficiently to various Snail binding sites in the single-minded gene (Zfh-1 binds better than Snail to Sna5ab, the highest-affinity site). These results demonstrate for the first time that Zfh-1 is a DNA binding protein and that it shows DNA binding specificity similar to that of ZEB and Snail (Postigo, 1999c).

Both ZEB and snail are transcriptional repressors. To determine whether Zfh-1 has transcriptional activity, a reporter containing the CACCTG binding site 30 bp upstream of an enhancer was transfected in Drosophila Schneider L2 cells. These cells do not express endogenous Zfh-1 or Snail, and thus the presence of the E box site had no effect on promoter activity. However, cotransfection of a Zfh-1 or Snail expression vector results in repression. A similar level of repression by Zfh-1 was observed when the CACCTG sequence was moved 300 bp upstream of the enhancer, demonstrating that Zfh-1 (and ZEB) can repress at long range. In contrast, Snail failed to repress transcription at this long range. Expression of DNA-binding Zfh-1 (DB-Zfh-1), containing only the DNA binding domain of Zfh-1 but not the repression domain, did not repress, suggesting that the protein has separate DNA binding and repressor domains. These results demonstrate that Zfh-1, like Snail and ZEB, functions as an active transcriptional repressor when it binds to E box sequences (Postigo, 1999c).

Because of the overlap in DNA binding specificity, Zfh-1 could target the same genes as Snail. One such Snail-regulated gene is single-minded, which is normally restricted to midline cells and a subset of somatic muscle precursor cells. Snail functions to block ectopic expression of single-minded and other nonmesodermal genes in the mesoderm. Zfh-1 not only binds to the Snail sites on the single-minded promoter but also represses the activity of the single-minded promoter in transfection assays in Schneider cells even more efficiently than Snail, consistent with the finding that sites from the single-minded promoter bind to Zfh-1 more efficiently than Snail (Postigo, 1999c).

However, Snail also binds other sequences that are not shared with Zfh-1. In the rhomboid promoter, Snail sites are important to block the expression of rhomboid in the ventral regions during embryogenesis. Contrary to what was found for the Snail sites in the single-minded promoter, Zfh-1 showed little or no binding to the four Snail sites of the rhomboid promoter. And, accordingly, Zfh-1 fails to repress the transcriptional activity of the rhomboid promoter. These results demonstrate that Zfh-1 can interact with only a subset of Snail sites (Postigo, 1999c).

It is important to point out that Snail is required for zfh-1 expression and that Zfh-1 persists after Snail diminishes. Thus, the two proteins appear to be temporally distinguishable in the developing embryo. This suggests that the two proteins may regulate separate or perhaps partially overlapping sets of genes, albeit at distinct developmental stages or in distinct tissues (Postigo, 1999).

ZEB can also repress transcription in Drosophila cells. Whether Zfh-1 can repress transcription in mammalian cells was investigated. A reporter construct containing a CACCTG binding site upstream of an enhancer was used. Coexpression of DB-Zfh-1 (or DB-ZEB) does not repress the activity of the enhancer. However, transfection of an expression vector for either full-length Zfh-1 (or full-length ZEB) or DB-Zfh-1-RD-ZEB (RD-ZEB refers to the repressive domain of ZEB) does repress transcription through the binding site. Together, these results suggest that Zfh-1 also recognizes E box binding sites in mammalian cells and represses transcription when bound to these sites (Postigo, 1999c).

To determine whether Zfh-1 contains an independent repressor domain that can function when fused to a heterologous DNA binding domain, a construct was created where the region of Zfh-1 between the zinc finger domains (corresponding to the repressor domain in ZEB) was fused to the DNA binding domain of the yeast protein Gal4. Gal4-zfh-1 was tested in transfection assays with reporter plasmids containing Gal4 binding sites cloned upstream of various enhancers. Gal4-zfh-1 efficiently represses the SV40 enhancer and thymidine kinase (TK) promoter, indicating that Zfh-1 indeed contains an independent repressor domain located between the zinc finger regions (Postigo, 1999c).

The overall sequence similarity between Zfh-1 and ZEB in their repressor domains is very low. Nevertheless, when the ability of Zfh-1 to repress the activity of a number of transcription factors was tested, it was found that Zfh-1 and ZEB have similar transcription factor specificities in transfection assays. Zfh-1 is expressed in other tissues in addition to muscle (heart, gonadal cells, central nervous system), and the ability of Zfh-1 to repress various transcription factors may have a role in the regulation of gene expression in these tissues. These results indicate that Zfh-1 is an active transcriptional repressor and that the repressor domains in Zfh-1 and ZEB may be functionally similar (Postigo, 1999c).

Given the similarity between ZEB and Zfh-1 in DNA binding specificity and repressor activity, whether Zfh-1 could substitute for ZEB and block muscle differentiation in mammalian cells was tested. Transfection of myoD is sufficient to drive cells down a myogenic pathway by inducing a cascade of transcription factors including members of the myocyte enhancer family (e.g., mef2) that collaborate with myoD to amplify the muscle differentiation program. Overexpression of ZEB blocks this myogenic conversion. A construct encoding full-length Zfh-1 also efficiently blocks myogenic differentiation. As with ZEB, DB-Zfh-1 alone does not affect myogenic differentiation, even though DB-Zfh-1 binds DNA more efficiently than the full-length protein and efficiently displaces wild-type ZEB from the promoter. Therefore, Zfh-1 and ZEB do not block myogenic differentiation simply by displacing MRF proteins from the promoter; instead, their repressor domains are required for this activity. Accordingly, fusion proteins containing DB-Zfh-1 fused to RD-ZEB, DB-ZEB fused to RD-Zfh-1, or DB-Zfh-1 fused to RD-Zfh-1 also block myotube formation. These results indicate that the RD-Zfh-1 can block myogenesis in mammalian cells, suggesting that the function of zfh-1 and ZEB may be conserved from Drosophila to mammals (Postigo, 1999c).

The transfection assays in Drosophila and mammalian cells have suggested that Zfh-1 might play a negative role during muscle development in the Drosophila embryo. Loss of Zfh-1 function does not cause drastic alterations to muscle development. In zfh-1 mutant embryos, somatic and visceral muscles form and differentiate, but there are subtle defects such as loss, misplacement, and disorganization of some muscles. These results demonstrate that Zfh-1 is not required for muscle differentiation per se, but they are consistent with a regulatory role for Zfh-1 in the process. From these studies, there was no indication about its mechanism of action and whether Zfh-1 might act as a positive or negative regulator of myogenesis. Moreover, studies on the role of ZEB in muscle differentiation had been confined to in vitro assays. Therefore, the role of Zfh-1 during myogenesis in vivo was investigated (Postigo, 1999c).

Zfh-1 is downregulated prior to somatic muscle differentiation, raising the possibility that this downregulation is essential for the onset of myogenesis. While the loss-of-function phenotype appeared mild in muscle, it was of interest to see whether maintenance of Zfh-1 expression beyond the time that endogenous Zfh-1 diminishes might have a more drastic phenotype (e.g., a blocking of myogenesis as occurs in cultured cells (Postigo, 1999c).

Zfh-1 is initially expressed throughout the mesoderm, but after gastrulation it is downregulated in muscle precursors as well as most other mesodermal derivatives. Expression of Zfh-1 was maintained throughout embryogenesis by expressing the protein under control of the heat shock protein 70 promoter; muscle development was assayed by following MHC expression. First, the embryos were heat shocked at stage 9-10, which corresponds to the time that Zfh-1 is normally downregulated and is prior to MHC expression in muscle. Zfh-1 expression following heat shock was confirmed by immunohistochemistry. At stage 14, a loss of MHC expression in somatic muscles was observed; however, surprisingly MHC expression in visceral muscle appeared relatively normal. In embryos that completed embryogenesis, milder but still clear defects in MHC expression were observed in somatic muscles (Postigo, 1999c).

The embryos were also heat shocked to induce Zfh-1 expression after the onset of MHC expression (stage 12-13). In this case, little, if any, defect in MHC expression was observed in somatic muscles, indicating that once the muscles cells begin to express MHC, they are refractory to the negative effects of Zfh-1 expression. Taken together, these results are consistent with a model in which extinction of Zfh-1 expression in embryonic muscle precursors is necessary to allow muscle differentiation to proceed (Postigo, 1999c).

It was noticed that maintaining Zfh-1 expression results in a muscle differentiation phenotype similar to that seen with the loss of Mef2 (where there is a block in MHC expression in somatic muscle with less effect on visceral muscle). This phenotype is also similar to that observed when the transcriptional activator Twist is disrupted after gastrulation via a temperature-sensitive mutant. Twist is required for activation of the mef2 gene in somatic muscle, and these observations raised the possibility that Zfh-1 may act to inhibit somatic myogenesis by blocking the expression of mef2 (Postigo, 1999c).

The pattern of mef2 expression is complex and dynamic in the embryo, but mef2 expression increases in muscle precursors as they appear in the embryo. mef2 is first evident at the late cellular blastoderm stage in mesoderm primordia and continues to be expressed throughout the mesoderm during mesoderm invagination. At mid-germband extension, mef2 expression is reduced in the ventrolateral mesoderm but maintained in the dorsal region. During germband retraction, expression increases in visceral mesoderm and in somatic muscle precursors. This is around the time when Zfh-1 is downregulated. mef2 expression then increases dramatically in all somatic mesoderm, and throughout germband retraction expression continues to be high in somatic muscles (Postigo, 1999c).

Zfh-1 is also expressed in a dynamic fashion in the mesoderm, and it is downregulated in muscle precursors as they began to appear. When embryos were double immunostained for Mef2 and Zfh-1, it was found that the expression of Zfh-1 and that of Mef2 were mutually exclusive (Postigo, 1999c).

Taken together, the above results suggested that Zfh-1 might have some role in controlling the pattern of mef2 expression in muscle precursors. To test this possibility, the pattern of mef2 expression was analyzed in embryos where Zfh-1 expression is maintained by using the heat shock construct. Wild-type embryos exhibited a normal Mef2 pattern following heat shock at all stages examined. However, heat-shocked-expressing Zfh-1 embryos showed a range of defects. (1) In the most severe cases, mef2 was highly disrupted. These embryos failed to complete germband retraction and appear not to have developed far past this stage. (2) Other embryos showed a fairly normal morphology and completed embryogenesis. In these embryos, there was still clear disruption of mef2 in somatic muscle and a reduction in the number of Mef2-positive cells (stage 12). These results suggest that the downregulation of zfh-1, associated with the onset of somatic myogenesis, is required for expression of mef2 (Postigo, 1999c).

mef2 expression has been shown to depend on the existence of an enhancer element 2.3 kb upstream of the mef2 gene that is directly activated by Twist. Examination of the mef2 promoter sequence has revealed multiple Zfh-1 sites throughout the sequence that bind Zfh-1 in gel retardation assays. Do the Zfh-1 sites in the mef2 promoter block transcriptional activation by Twist? In transfection assays, it has been shown that Zfh-1 blocks transcriptional activation by Twist, and it is proposed that Zfh-1 blocks twist-mediated activation of the mef2 gene in muscle precursors until Zfh-1 expression diminishes (Postigo, 1999c).

The transcription factor Zfh1 is involved in the regulation of neuropeptide expression and growth of larval neuromuscular junctions in Drosophila melanogaster

Different aspects of neural development are tightly regulated and the underlying mechanisms have to be transcriptionally well controlled. This study presents evidence that the transcription factor Zfh1 is important for different steps of neuronal differentiation. First, it was shown that late larval expression of the neuropeptide FMRFamide is dependent on correct levels of Zfh1, and this regulation is presumably direct via a conserved zfh1 homeodomain binding site in the FMRFamide enhancer. Using MARCM analysis the requirement for Zfh1 was additionally examined during embryonic and larval stages of motoneuron development. Zfh1 was shown to cell autonomously regulate motoneuronal outgrowth and larval growth of neuromuscular junctions (NMJs). In addition, the growth of NMJs is dependent on the dosage of Zfh1, suggesting it to be a downstream effector of the known NMJ size regulating pathways (Vogler, 2008).

FMRFa encodes a prohormone which is cleaved to give rise to several biologically active neuropeptides. It is expressed in many cells of the Drosophila larval brain and, very prominently, in a single cell within each thoracic hemineuromere, the Tv neuron. These neurons innervate the neurohemal organs which lie dorsally on the midline of these neuromeres. From these organs, the FMRFa peptides are thought to be subsequently released into the hemolymph to modulate the contraction strength at neuromuscular junctions. It has been shown earlier that the onset of FMRFa expression in the Tv neuron is governed by the combination of the transcription factors encoded by apterous, collier, dachshund, dimmed and eyes-absent which act in concert with BMP signaling at the end of embryogenesis. However, only Collier and BMP signaling are absolutely required for the onset of FMRFa expression, since weak levels of FMRFa are found in null mutants in any of the other transcriptional activators mentioned above. Conversely, in cells which are sensitive to BMP signaling, FMRFa can be ectopically induced by combined expression of apterous, dimmed and squeeze or apterous, dimmed and dachshund. This study shows that lowered levels of Zfh1, as found in zfh1865 hypomorphs, lead to a strong reduction of FMRFa expression in late larvae. Surprisingly, neuronal overexpression of Zfh1 also represses the expression of this neuropeptide, in wild type as well as a hypomorphic background where the overexpression is expected to be less strong. An explanation would be that the similar loss of function and overexpression phenotype is due to a dosage-dependent activation or repression of different target genes (Vogler, 2008).

Unfortunately it was not possible to analyze FMRFa expression in zfh1 null mutant embryos, thus it is not known whether Zfh1 is also necessary for the early onset of the expression of this gene, thereby being a part of the above mentioned combinatorial code of transcription factors. However, since zfh1 is expressed at various levels in most Ap+ cells and in many other neurons, a requirement for Zfh1 to ectopically activate FMRFa after ectopic expression of apterous, dimmed and dachshund appears possible. In accordance with this possibility Zfh1 alone was not able to ectopically induce FMRFa expression, similar to what has been reported for each of the genes of the combinatorial code. Whether Zfh1 is acting in concert with those factors remains to be elucidated. A preliminary test for genetic interaction between zfh1 and Mad in this context showed that reduction of Mad activity was able to partially rescue the phenotype caused by Zfh1 reduction. However, this could also be due to differences in the genetic background because wishful-thinking did not genetically interact with zfh1 (Vogler, 2008).

With respect to later developmental stages it is not clear whether any of the factors necessary for the onset of FMRFa expression is also involved in the maintenance of its expression. For the transcription factor Apterous it could be shown that it plays a fundamental role during the early initiation of FMRFa but seems to be less important during larval stages. In contrast to that, Zfh1 is necessary for the maintenance of FMRFa expression, because residual FMRFa within neurohemal organs could often be detected although the contacting Tv neuron has already lost this expression. It is thought that this regulation occurs rather directly because an evolutionarily conserved putative Zfh1 homeodomain binding site was detected within the Tv neuron specific FMRFa-lacZ reporter construct PWFE17. Interestingly, exactly the same binding site has been shown to be one of three sites which can bind Apterous (binding site C) and which are necessary for Ap-dependent regulation of FMRFa in the Tv neurons. This study now found that the regulatory region of the reporter construct is also dependent on Zfh1 because it reacts similar to the endogenous FMRFa gene upon altered levels of Zfh1. It is possible that binding site C is in fact binding to Zfh1 and thereby necessary for the maintenance of FMRFa expression. This hypothesis is additionally supported by the earlier finding that by mutating site C the early onset of the reporter gene expression is normal but the expression is weaker during larval stages. This is quite similar to what was have found for the reporter gene expression in zfh1 hypomorphs (Vogler, 2008).

The possibility that the transcriptional repressor Zfh1 acts positively on FMRFa expression by binding with its homeodomain suggests that in Drosophila there could be a correlation between the mode of activity of Zfh1 and the binding domain used. So far different groups have provided experimental evidence for both: Zfh1 proteins have been shown to bind via the homeodomain to the sequence GCTAATTG but also by a two-handed binding mode of the zinc finger clusters to E box sequences (CACCT). It is provocative that in those cases where Zfh1 is thought to bind via its homeodomain it was found to act as an activator, while DNA binding with zinc fingers seems to correlate with a repressor function. It is therefore tempting to speculate that the activating or repressing effect of Drosophila Zfh1 could at least partially be governed by the type of target sequence (Vogler, 2008).

Earlier work on zfh1 indicated that it is expressed predominantly in motoneurons and necessary for proper axonal outgrowth of these cells. However, zfh1 is also expressed in developing embryonic muscles which makes it difficult to judge if a given phenotype is due to an autonomous requirement of this gene within the affected motoneurons. This study used the MARCM technique to generate zfh1 null mutant motoneurons which enabled the identification of individual mutant neurons within motoneuronal branches in an otherwise wild type environment. This allowed a cell autonomous mutational analysis at a very high resolution. By comparing the numbers of wild type and mutant motoneurons normalized to the number of larval preparations, it was found that zfh1 mutant motoneurons of the SNb and SNc are found at much lower frequency than those of the ISN. This is not due to mistargeting of motoneurons towards other muscles, since multiple muscle innervations different from wild type were not found. Instead the most likely explanation is that these motoneurons cannot extend their axons into the periphery and therefore are not detected in the analysis. This would be consistent with evidence evidence that motoneurons projecting through the SNb and SNc are selectively affected by loss of Zfh1 function. By the current analysis it was additionally showm that there are different requirements for Zfh1 even between motoneurons projecting within the same nerve. For example, muscles 3 and 19 have a similar dorsoventral position and both are innervated via the ISN. On muscle 3, equal numbers of clones are found, whereas for muscle 19 the number of mutant clones is much smaller. Muscle 3 is innervated by one U neuron derived from the early S1 neuroblast 7-1, whereas the motoneuron innervating muscle 19 is a progeny of the late S1 neuroblast 3-2. Such a differential requirement does not seem to be reflected by the different strength of Zfh1 expression. VUM motoneurons express highest levels of Zfh1 but no reduction is found in the frequency of labeled VUM motoneurons or any morphological changes on light microscopic level after removing Zfh1 function in these neurons (Vogler, 2008).

Taken together, these results support and extend earlier findings that Zfh1 is needed for certain motoneurons to be able to exit the CNS. and these results furthermore show that this function seems to be more important for ventrally than for dorsally projecting neurons. However, the data also reveal that some motoneurons are still able to exit the CNS and can innervate the correct target muscles even in the absence of Zfh1 (Vogler, 2008).

During larval growth a synaptic homeostasis between a given motoneuron and the innervated muscle is thought to be regulated by cell-cell-signaling at the synapse, retrograde signaling towards the neuron's cell body and proper neuronal response. The molecular architecture of the Drosophila NMJ is well studied, however there are only a few transcription factors described to be involved in these processes. Since this study found zfh1 is continuously expressed in motoneurons, its capability to regulate NMJ growth during larval stages was tested. The experiments revealed that reduced levels of Zfh1 limit the ability of NMJs to grow as they are significantly smaller and have fewer boutons. Likewise, motoneuronal overexpression of Zfh1 leads to an increase in NMJ size with significantly more boutons than in wild type. The only other transcription factors showing such effects are D-Jun, D-Fos, CREB and phosphorylated Mad. D-Jun and D-Fos act either as a heterodimeric immediate-early transcription factor called AP-1 (D-Jun+ D-Fos or D-Fos acts as a homo- or heterodimer independent of D-Jun. Their activity is regulated by JNK MAP kinase and this modulates synapse number in a Fasciclin2-dependent, and synaptic strength in a CREB-dependent manner. In addition, BMP signaling mediated by the type-II receptor wishful-thinking (wit) is required for synaptic growth and promotes the formation of a transcriptionally active Mad-Medea heterodimer. Since synaptic strength in zfh1 mutants was not evaluated, it is currently not known if and how zfh1 might contribute to these different pathways. Loss and gain of function experiments indicate that Zfh1 might act synergistically in at least one of these pathways and there are observations which support a connection especially between zfh1 and BMP signaling. Neurons which show highest levels of phospho-Mad in the embryo show Zfh1 immunoreactivity as well as nuclear localized Medea. Among these cells are Tv neurons and all motoneurons. Additionally, both Zfh1 activity and BMP signaling are involved in the regulation of FMRFa expression. Because vertebrate homologues of Zfh1, δEF-1 and SIP-1, have been shown to be able to bind to SMAD proteins one might speculate whether Zfh1 has this capacity as well. Therefore, whether zfh1 and members of the BMP signaling pathway genetically interact was tested. This does not seem to be the case: the size of larval NMJs of Mad, zfh1 transheterozygotes were not different from wild type or either of the single mutant alleles. This raises the alternative possibility that zfh1 could act via another pathway in this context, e.g., wingless or JNK. Recent findings on the role of zfh1 homologue SIP-1 during mouse hippocampus formation hint toward such an interaction (Vogler, 2008).

To understand the role of Zfh1 in NMJ growth regulation it would be important to know its targets in this context. There are several techniques which allow the identification of target genes of a transcription factor, e.g. DamID. For even-skipped which is involved in axon targeting and late differentiation processes of motoneurons, DamID identified genes encoding components of neuronal electrical properties. A similar approach for Zfh1 could be very promising, especially because one could correlate such data with the list of known genes necessary for motoneuronal development. The identification of such target genes should then allow examination how Zfh1 is involved in the integration of developmental signals to regulate the morphology and function of individual motoneurons (Vogler, 2008).


DEVELOPMENTAL BIOLOGY

Embryonic

Antisera against Zfh-1 and Zfh-2 were used to investigate their expression patterns during embryonic development. The zfh-1 gene is expressed in the mesoderm of early embryos. ZFH-1 protein is first detected in the presumptive procephalic mesoderm. By the early stages of gastrulation, ZFH-1 protein also begins to appear in a narrow ventral band of mesodermal anlagen extending the length of the embryo. At the start of germ band elongation, ZFH-1 protein is seen in the pole cells. In late embryos ZFH-1 protein is found in a number of mesodermally-derived structures of late embryos, including the dorsal vessel, support cells of the gonads, and segment-specific arrays of adult muscle precursors. In addition, zfh-1 is expressed in the majority of identified motor neurons of the developing CNS. Identified neurons positive for ZFH-1 include RP-13, muscle pioneers, aCC (but not pCC) ventral unpaired medial, ventral intersegmental and lateral ipsisegmental neurons (Lai, 1991).

In Drosophila as well as many vertebrate systems, germ cells form extraembryonically and migrate into the embryo before navigating toward gonadal mesodermal cells. Just how the gonadal mesoderm attracts migratory germ cells is not well understood in any system. A genetic approach has been taken to identify genes required for germ cell migration in Drosophila. The role of zfh-1 is described in germ cell migration to the gonadal mesoderm. Zfh-1 protein is initially expressed in all mesodermal cells, but by stage 10, Zfh-1 levels have declined in most mesodermal cells, although high levels are maintained in extreme anterior and posterior mesodermal cells. The cells within the anterior cluster are likely to be hemocytes. During stage 10, Zfh-1-expressing mesodermal cells located at the posterior end of the embryo migrate anteriorly in two bilaterally symmetric groups between the endoderm and the interior of the dorsal mesoderm. These cells have been termed the 'caudal visceral mesoderm' as they contribute to the midgut musculature at later stages. Crocodile was used as a marker for the caudal visceral mesoderm. Croc is not expressed in the caudal visceral mesoderm in zfh-1 mutant embryos. Caudal visceral mesoderm is in close proximity to migratory germ cells during late stage 10. In zfh-1 mutant embryos, the initial association of germ cells with their final destination, gonadal mesoderm made up of the somatic gonadal precursors (SGP), is blocked. Instead, some germ cells remain attached to the gut, leading to a cluster of germ cells in the middle of the embryo during later stages of development (Broihier, 1998).

The zfh-2 gene displays a more limited expression pattern, largely restricted to the CNS of late embryos. The expression patterns of zfh-1 and zfh-2 suggest that both genes may be involved in Drosophila neurogenesis and that zfh-1 may have additional functions in mesoderm development (Lai, 1991).

Subdivision and developmental fate of the head mesoderm in Drosophila

This paper defines temporal and spatial subdivisions of the embryonic head mesoderm and describes the fate of the main lineages derived from this tissue. During gastrulation, only a fraction of the head mesoderm (primary head mesoderm; PHM) invaginates as the anterior part of the ventral furrow. The PHM can be subdivided into four linearly arranged domains, based on the expression of different combinations of genetic markers (tinman, heartless, snail, serpent, mef-2, zfh-1). The anterior domain (PHMA) produces a variety of cell types, among them the neuroendocrine gland (corpus cardiacum). PHMB, forming much of the'T-bar' of the ventral furrow, migrates anteriorly and dorsally and gives rise to the dorsal pharyngeal musculature. PHMC is located behind the T-bar and forms part of the anterior endoderm, besides contributing to hemocytes. The most posterior domain, PHMD, belongs to the anterior gnathal segments and gives rise to a few somatic muscles, but also to hemocytes. The procephalic region flanking the ventral furrow also contributes to head mesoderm (secondary head mesoderm, SHM) that segregates from the surface after the ventral furrow has invaginated, indicating that gastrulation in the procephalon is much more protracted than in the trunk. This study distinguishes between an early SHM (eSHM) that is located on either side of the anterior endoderm and is the major source of hemocytes, including crystal cells. The eSHM is followed by the late SHM (lSHM), which consists of an anterior and posterior component (lSHMa, lSHMp). The lSHMa, flanking the stomodeum anteriorly and laterally, produces the visceral musculature of the esophagus, as well as a population of tinman-positive cells that is interpreted as a rudimentary cephalic aorta ('cephalic vascular rudiment'). The lSHM contributes hemocytes, as well as the nephrocytes forming the subesophageal body, also called garland cells (de Velasco, 2005).

The mesoderm is a morphologically distinct cell layer that can be recognized in early embryos of most bilaterian phyla and that gives rise to tissues interposed between ectodermal and endodermal epithelia, including muscle, connective, blood, vascular, and excretory tissue. Besides the differentiative fate of tissues derived from it, the mesoderm shares several common properties in regard to its formation during gastrulation. The anlage of the mesoderm is sandwiched in between the anlage of the endoderm and the neurectoderm. This has been documented in most detail in anamniote vertebrates, where signals from the vegetal blastomeres (the anlage of the endoderm) act on the adjacent marginal zone of the future ectoderm to induce mesoderm. Although gastrulation proceeds quite differently in arthropods from the way it does in chordates, the proximity of the mesodermal anlage to future endoderm and neurectoderm is conserved, and numerous signaling pathways and transcriptional regulators that share similar function and expression patterns in arthropods and chordates have been identified (de Velasco, 2005 and references therein).

Following gastrulation, the mesoderm is subdivided along the dorso-ventral axis into several subdivisions laid out in a distinct dorso-ventral order. In vertebrates, cells located in the dorsal part of the mesoderm anlage give rise to notochord and somites, which in turn produce muscular, skeletal, and connective tissue. Next to the somitic mesoderm is the intermediate mesoderm that will form the excretory and reproductive system. The ventral mesoderm (lateral plate) gives rise to blood, vascular system, visceral musculature, and coelomic cavity. In arthropods, fundamentally similar mesodermal subdivisions can be recognized, and similarities extend to the relative positions these domains obtain relative to each other and relative to the adjacent neurectoderm. For example, precursors of visceral muscles, vascular system, and blood are at the edge of the mesoderm facing away from the neural primordium (ventral in vertebrates, dorsal in arthropods (de Velasco, 2005 and references therein).

The subdivision of the vertebrate mesoderm into distinct longitudinal tissue columns with different fates is seen throughout the trunk and head of the embryo. However, several significant differences between the head and the trunk are immediately apparent. For example, cells derived from the anterior neurectoderm form the neural crest that migrates laterally and gives rise to many of the tissues that are produced by mesoderm in the trunk. As a result, the fates taken over by the head mesoderm are more limited than those of the trunk mesoderm. In contrast, the head mesoderm produces several unique lineages, such as the heart (cardiac mesoderm) and a population of early differentiating macrophages. Moreover, some of the signaling pathways responsible for inducing different mesodermal fates in the trunk appear to operate in a different manner in the head. A recently described example is the Wnt signal that induces somatic musculature in the trunk, but inhibits the same fate in the head (de Velasco, 2005 and references therein).

The head mesoderm of arthropods, like that of vertebrates, also appears to deviate in many ways from the trunk mesoderm. For example, specialized lineages like embryonic blood cells and nephrocytes forming the subesophageal body (also called garland cells) arise exclusively in the head. That being said, very little is known about how the arthropod head mesoderm arises and what types of tissues derive from it. The existing literature mainly uses histology, which severely limits the possibilities of following different cell types forward or backward in time. In this paper, several molecular markers have been used to initiate more detailed studies of the head mesoderm in Drosophila. The goal was to establish temporal and spatial subdivisions of the head mesoderm and, using molecular markers expressed from early stages onward, to follow the fate of the lineages derived from this embryonic tissue. Besides hemocytes and pharyngeal muscles described earlier, the head mesoderm also gives rise to several other lineages, including visceral muscle, putative vascular cells, nephrocytes, and neuroendocrine cells. The development of the head mesoderm is discussed in comparison with the trunk mesoderm and in the broader context of insect embryology (de Velasco, 2005).

The Drosophila head mesoderm, as traditionally defined, includes all mesoderm cells originating anterior to the cephalic furrow. The formation of the head mesoderm is complicated by the fact that (unlike the mesoderm of the trunk) only part of it invaginates with the ventral furrow; by far, the majority of head mesoderm cells, recognizable in a stage 10 or 11 embryo, segregate from the surface epithelium of the head after the ventral furrow has formed. Another complicating factor is that head mesoderm cells derived from different antero-posterior levels adopt very different fates, unlike the situation in the trunk where mesodermal fates within different segments along the AP axis are fairly homogenous, with obvious exceptions such as the gonadal mesoderm that is derived exclusively from a subset of abdominal segments. Using several different markers, this study has followed the origin, migration pathways, and later, fates of head mesoderm cells (de Velasco, 2005).

The anterior part of the ventral furrow, called primary head mesoderm (PHM) in the following, includes cells that will contribute to diverse tissues, including muscle, hemocytes, endoderm, and several ill-defined cell populations closely associated with the brain and neuroendocrine system. For clarification, the anterior ventral furrow will be divided into the following domains:

The anterior lip of the T-bar (PHMA) is the source of the corpus cardiacum, as well as other gt-positive cells that at least in part end up as nerve cells flanking the frontal connective and frontal ganglion. These cells continue the expression of giant throughout late embryonic development; they represent a hitherto unknown class of nonneuroblast-derived neurons (de Velasco, 2005).

The posterior lip of the T-bar (PHMB) can be followed towards later stages by its continued expression of htl. These cells, called the procephalic somatic mesoderm, form a bilateral cluster that moves dorso-anteriorly into the labrum and becomes the dorsal pharyngeal musculature. Htl expression almost disappears in these cells around late stage 11, but is reinitiated at stage 12 and stays strong until stage 14, when the dorsal pharyngeal muscles differentiate. Many of the genes expressed in the somatic musculature of the trunk and its precursors (Dmef2, beta-3-tubulin) are also expressed in the procephalic somatic mesoderm (de Velasco, 2005).

The part of the ventral furrow posteriorly adjacent to the T-bar (PHMC) expresses srp, forkhead (fkh), and other endoderm/hemocyte markers. After the ventral furrow closes in the ventral midline (stage 7/8), these cells form a compact median mass, most of which represents part of the anterior endoderm that gives rise to the midgut epithelium. Starting at around this stage, the lateral part of the hemocyte-forming 'secondary head mesoderm' ingresses in between the endoderm and the surface ectoderm. It is likely that some of the PHMC cells invaginating already with the ventral furrow, along with the cells that form the anterior endoderm, also give rise to hemocytes. Precursors of hemocytes and midgut are difficult to distinguish during and shortly after ventral furrow invagination since both express srp and other markers shared between hemocytes and midgut precursors. At around stage 9, the two populations of precursors disengage. The endoderm remains a compact mesenchyme attached to the invaginating stomodeum; hemocyte precursors move dorsally and take on the shape of expanding vertical plates interposed in between endoderm and ectoderm (de Velasco, 2005).

Domain PHMD, the short portion of the ventral furrow situated posterior to the endoderm, along with a considerable portion of the mesoderm behind the cephalic furrow, forms the mesoderm of the three gnathal segments (mandible, maxilla, labium). The gnathal mesoderm in many ways behaves like the mesoderm of thoracic and abdominal segments. It gives rise to somatic muscle (the lateral pharyngeal muscles), visceral muscle, and fat body. Unlike trunk mesoderm, gnathal mesoderm does not produce cardioblasts and pericardial cells. Instead, a large proportion of gnathal mesoderm cells, joining the anteriorly adjacent secondary procephalic mesoderm, adopt the fate of hemocytes (de Velasco, 2005).

Besides the ventral furrow, other parts of the ventral procephalon produce head mesoderm in a complex succession of delamination and ingression events. The head mesoderm that forms from outside the ventral furrow will be called 'secondary mesoderm' (SHM) in the following. Based on the time of formation and the position relative to the stomodeum, the following phases and domains of secondary head mesoderm development can be distinguished.

Following the obliteration of the ventral furrow at stage 8, the eSHM delaminates from the ventral surface 'meso-ectoderm' (considering that this epithelium still contains mesodermal progenitors!) flanking the endodermal mass. The eSHM forms two monolayered sheets that gradually move dorsally and posteriorly; by stage 9, the eSHM cells line the basal surface of the emerging head neuroblasts. An undefined number of primary head mesoderm cells derived from domain PHMC of the ventral furrow are mingled together with the eSHM cells. The ultimate fate of the eSHM is that of hemocytes: they express srp, followed slightly later by other blood cell markers (e.g., peroxidasin and asrij). A subset of hemocytes, called crystal cells, derive from precursors that form a morphologically conspicuous cluster at the dorsal edge of the eSHM, identifiable from early stage 10 onward by the expression of lz. The mechanism by which at least part of the eSHM delaminates is unique. Thus, it is formed by the vertically oriented division of the surface epithelium, whereby the inner daughters will become eSHMe and the outer ones ectoderm. The focus of vertical mitosis has named the procephalic domain in which it occurs 'mitotic domain #9' (de Velasco, 2005).

From late stage 9 onward, the early SHMs are followed inside the embryo by the closely adjacent posterior late SHMs. One cluster of posterior late secondary head mesoderm (lSHMp) cells delaminates from the surface epithelium flanking the posterior lip of the stomodeum; a second lSHMp cluster appears at the same stage at a slightly more posterior level. The first cluster seems to contribute to the hemocyte population; the posterior cluster gives rise to the nephrocytes forming the subesophageal body (also called garland cells; labeled by CG32094). Garland cell precursors are initially arranged as a paired cluster latero-ventrally of the esophagus primordium; subsequently, the clusters fuse in the midline and form a crescent underneath the esophagus. Garland cells are distinguished from crystal cells by their size, location, and arrangement: crystal cells are large, round cells grouped in an oblong cloud dorso-anterior to the proventriculus. Garland cells are smaller, closely attached to each other, and lie ventral of the esophagus (de Velasco, 2005).

During stages 10 and 11, cells delaminate beside and anterior to the stomodeum, originating from the anlage of the esophagus and the epipharynx (labrum). These cells, called anterior late secondary head mesoderm cells (lSHMa), can be followed by their expression of tin. Two groups can be distinguished. The tin-positive cells delaminating from the esophageal anlage (es) give rise to the visceral musculature (vm) surrounding the esophagus. These cells lose tin expression soon after their segregation, but can be recognized by other visceral mesoderm markers such as anti-Connectin. More dorsally, in the anlage of the clypeolabrum (cl) delaminate, the dorsal subpopulation of the lSHMas, which rapidly migrates posteriorly on either side and slightly dorsal of the esophagus, can be found. These cells retain expression of tin into the late embryo. They assemble into two longitudinal rows stretching alongside the roof of the esophagus primordium. During late embryogenesis, they move posteriorly along with the esophagus towards a position behind the brain commissure. Many of the tin-positive SHMs apparently undergo apoptosis: initially counting approximately 25 on either side, they decrease to 12-15 at stage 14 to finally form a single, irregular row of about 15 cells total in the late embryo. These cells come into contact with the anterior tip of the dorsal vessel. This formation of previously undescribed cells, for which the term 'procephalic vascular cells', is proposed, is interpreted as a rudiment of the head aorta, which forms a prominent part of the dorsal vessel in many insect groups (de Velasco, 2005).

On the basis of additional molecular markers, the tin-positive procephalic vascular cells are further subdivided into two populations. The first subpopulation expresses the muscle and cardioblast-specific marker Dmef2; the second type is Dmef2-negative. In the dorsal vessel of the trunk, tin-positive cells also fall into a Dmef2-positive and a Dmef2-negative population. Dmef2-positive cells of the trunk represent the cardioblasts, myoendothelial cells lining the lumen of the dorsal vessel. Dmef2-negative/tin-positive cells form a somewhat irregular double row of cells attached to the ventral wall of the dorsal vessel. The ultimate fate of these cells has not been explored yet. However, preliminary data suggest that they develop into a muscle band that runs alongside the larval dorsal vessel. This would correspond to the situation in other insects in which such a ventral cardiac muscle band has been described (de Velasco, 2005).

The role of tinman in the formation of the procephalic vascular rudiment was investigated by assaying tin-mutant embryos for the expression of Dmef2. Similar to the cardioblasts of the trunk, the Dmef2-positive cells of the procephalic vascular rudiment are absent in tin mutants. It is quite likely that the (Dmef2-negative) remainder of the procephalic vascular rudiment is affected as well by loss of tin, but in the absence of appropriate markers (besides tin itself, which is not expressed in the mutant), it was not possible to substantiate this proposal (de Velasco, 2005).

At the time of appearance of the ventral furrow, segmental markers such as hh do not allow the distinction between distinct 'preoral' segments. Thus, hh is expressed in a wide procephalic stripe in front of the regularly sized mandibular stripe. During stage 7, the procephalic hh stripe splits into an anterior, antennal stripe and a posterior, short, intercalary stripe. The anterior lip of the ventral furrow (domain PHMA) coincides with the anterior boundary of the antenno-intercalary stripe. Thus, the primary head mesoderm and endoderm originating from within the anterior ventral furrow can be considered a derivative of the antennal and intercalary segments. This interpretation is supported by the expression of the homeobox gene labial (lab) found in the intercalary segment. The labial domain covers much of the anterior ventral furrow, including domains PHMB-C (de Velasco, 2005).

Morphogenetic movements in the ventral head, associated with the closure of the ventral furrow, the formation of the stomodeal placode, and the subsequent invagination of the stomodeum result in a shift of head segmental boundaries. The antennal segment tilts backward, as can be seen from the orientation of the antennal hh stripe that from stage 8 onward forms an almost horizontal line, connecting the cephalic furrow with the sides of the stomodeal invagination (which falls within the ventral realm of the antennal segment, in Drosophila as well as other insects). Since the expression of hh, like that of engrailed (en), coincides with the posterior boundary of a segment, the territory located ventral to the antennal hh stripe falls within the intercalary segment. This implies that most, if not all, of the posterior late SHM, is intercalary in origin. It is further plausible to consider that the anterior lSHM belongs to the intercalary and antennal segment. The vascular cells of the head, a conspicuous derivative of the anterior lSHM in Drosophila, are derived from the antennal mesoderm in other insects. The labrum, with which much of the anterior lSHM is associated, represents a structure that has always been difficult to integrate in the segmental organization of the head. Most likely the labrum represents part of the intercalary segment; this would help explain some of the unusual characteristics of the head mesoderm (de Velasco, 2005).

In conclusion, several fundamental similarities are found between the mesoderm of the head and that of the trunk regarding the tissues they give rise to, and possibly the signaling pathways deciding over these fates. After an initial phase of structural and molecular homogeneity, the trunk mesoderm becomes subdivided into a dorsal and a ventral domain by a Dpp-signaling event that emanates from the dorsal ectoderm. The dorsal domain, characterized by the Dpp-dependent continued expression of tinman, becomes the source of visceral and cardiogenic mesoderm, among other cell types. A role of Dpp/BMP signaling in cardiogenesis seems to be conserved among insects and vertebrates. Subsequent signaling steps, involving both Wingless and Notch/Delta, separate between these two fates and further subdivide the cardiogenic mesoderm into several distinct lineages, such as cardioblast, pericardial cells, and secondary hemocyte precursors (lymph gland). As a result of these signaling events, Tinman and several other fate-determining transcription factors become restricted to their respective lineages: tin to the cardioblasts, odd to pericardial cells and hemocyte precursors, zfh1 and srp to hemocyte precursors and fat body. Dmef2 and several other transcription factors become restricted to various combinations of muscle types (somatic, visceral, cardiac) (de Velasco, 2005).

In the head mesoderm, the above genes are associated with similar fates. Tin and Dmef2 appear widely in the procephalic ventral furrow and the anterior lSHM before getting restricted to the procephalic vascular rudiment and/or the pharyngeal musculature, respectively. In contrast with the initially ubiquitous expression of Tin and Dmef2 in the trunk mesoderm, those parts of the head mesoderm giving rise to hemocytes (PHMC, posterior lSHM) never express these mesodermal genes. Previous work has shown that the head gap gene buttonhead (btd) is responsible for the early repression of tin in the above mentioned domains of the head mesoderm. The early absence of Tin and Dmef2 in the head mesodermal hemocyte precursors is paralleled by the presence of Srp and Zfh1 in these cells. Interestingly, Srp/Zfh-positive cells of the head produce only hemocytes and no fat body, suggesting that an as-yet-uncharacterized signaling step prevents the formation of fat body in the head. It is tempting to speculate that there exists within the mesoderm a 'blood/fat body equivalence group'. Blood cells and fat body share not only the expression of fate-determining genes such as srp and zfh1, but also, later, functional properties that have to do with immunity. In the trunk, the blood/fat body equivalence group gives rise mostly to fat body, producing only a limited number of hemocyte precursors in the dorsal mesoderm of the thoracic segments. In the head, on the other hand, all cells of the equivalence group become hemocytes (de Velasco, 2005).

Attention is drawn to another mesodermal lineage that produces related, yet not identical, cell types in the trunk and the head: the nephrocytes. Nephrocytes are defined by their characteristic ultrastructure (membrane invaginations sealed off by junctions) that attests to their excretory function. In the trunk, nephrocytes are represented by the pericardial cells that settle beside the cardioblasts; a newly discovered nephrocyte population ('star cells') invading the Malpighian tubules is derived from the mesoderm of the tail segments. In the head, nephrocytes aggregate near the junction between esophagus and proventriculus as the subesophageal body, also called garland cells. The fact that from the early stages of development onward different transcription factors are expressed in garland cells and pericardial cells suggests that these cells perform similar, yet not fully overlapping, functions (de Velasco, 2005).

Antagonistic function of Lmd and Zfh1 fine tunes cell fate decisions in the Twi and Tin positive mesoderm of Drosophila melanogaster

This study showS that cell fate decisions in the dorsal and lateral mesoderm of Drosophila depend on the antagonistic action of the Gli-like transcription factor Lame duck (Lmd) and the zinc finger homeodomain factor Zfh1. Lmd expression leads to the reduction of Zfh1 positive cell types, thereby restricting the number of Odd-skipped (Odd) positive and Tinman (Tin) positive pericardial cells in the dorsal mesoderm. In more lateral regions, ectopic activation of Zfh1 or loss of Lmd leads to an excess of adult muscle precursor (AMP) like cells. It was also observed that Lmd is co-expressed with Tin in the early dorsal mesoderm and leads to a reduction of Tin expression in cells destined to become dorsal fusion competent myoblasts (FCMs). In the absence of Lmd function, these cells remain Tin positive and develop as Tin positive pericardial cells although they do not express Zfh1. Further, it was shown that Tin repression and pericardial restriction in the dorsal mesoderm facilitated by Lmd is instructed by a late Decapentaplegic (Dpp) signal that is abolished in embryos carrying the disk region mutation dppd6 (Sellin, 2009).

Loss of Lame duck (Lmd) leads to an increase of pericardial cells and adult muscle precursor like cells: In embryos lacking Lmd function, staining for zinc finger homeodomain factor 1 (Zfh1) expression reveals a pericardial hyperplasia phenotype and a general excess of Zfh1 positive mesodermal cells. In wild type embryos, three types of pericardial cells (PCs) have been described: Tin positive (TPCs), Odd positive (OPCs) and Eve positive (EPCs) pericardial cells, all of which express Zfh1 and the handC- GFP reporter. Closer inspection of the pericardial cells in lmd mutant embryos revealed that the number of TPCs and OPCs is dramatically increased, while the number of EPCs is normal. All OPCs co-express the handC- GFP reporter and Zfh1 in wild type and lmd mutant embryos. In contrast, a considerable number of ectopic Tin positive cells, though positive for handC- GFP, do not express Zfh1 in lmd mutant embryos. The absence of β3Tubulin expression in these cells is consistent with earlier reports in which a normal set of cardioblasts was described in lmd mutant embryos. To decide whether the Zfh1 negative/Tin positive cells are atypical pericardial cells or dorsal mesodermal cells that fail to differentiate, a triple staining waa conducted for Tin, Zfh1 and Pericardin (Prc), a collagen that is secreted by differentiated pericardial cells. Prc protein was observed surrounding all Tin positive/Zfh1 negative cells, suggesting that they are ectopic pericardial cells. However, due to the fact that Prc is a secreted protein the possibility cannot be ruled out that there might be occasional Tin positive/Zfh1 negative cells in lmd mutant embryos which do not express Prc themselves, but remain in an uncommitted, dorsal mesodermal state (Sellin, 2009).

For further analysis of the ectopic pericardial cells, the number of OPCs in stage 16-17 embryos was counted. An average of 206.3 OPCs was observed in lmd mutant embryos as compared to 97.8 in wild type embryos, thereby representing a ~2-fold increase. It has been reported that the Odd subgroup of pericardial cells (OPCs) originates from two different lineages: a symmetric lineage (two OPCs from one precursor) and an asymmetric lineage (two OPCs from two precursors), adding up to a total of four OPCs per hemisegment. Of note, the siblings of the asymmetrically derived OPCs, the Seven-up (Svp) positive cardioblasts, are normal in lmd mutants, thus suggesting that the asymmetrically derived OPCs do not contribute to the lmd phenotype. Since the two different types of OPCs can not be distinguished directly because the anti-Svp antibody stains the precursor cells and the cardioblast siblings, but not the final PCs at later stages, their abundance was measured in lmd mutants indirectly. The fact was utilized that in inscutable (insc) mutants, asymmetric cell division fails, and all siblings of the asymmetric OPC lineage become Svp positive cardioblasts. The difference in OPC number between insc; lmd and lmd mutant embryos therefore corresponds to the number of asymmetrically derived OPCs in lmd mutant embryos. A loss of ~45 OPCs was observed in insc; lmd double mutant embryos as compared to lmd mutant embryos. This number is reasonably close to the number of ~38 OPCs that are lost in insc mutant embryos when compared to wild type embryos. In addition, the number of Svp positive precursors, which give rise to the asymmetric Odd lineage, is normal in lmd mutant embryos at early stage 13. Altogether, these data strongly support the initial hypothesis that there is the normal amount of asymmetrically derived OPCs in lmd mutant embryos and the phenotype is not caused by a failure of asymmetric cell division (Sellin, 2009).

An excess of Zfh1 positive cells was also observed in the lateral mesoderm of lmd mutant embryos, where it is normally expressed in the adult muscle precursor cells (AMPs). These imaginal myogenic cells retain Twist (Twi) expression, but do not express any other myogenic genes in the embryo. Instead, they are maintained in a less differentiated state during embryogenesis and are dormant until metamorphosis, when they start to differentiate and give rise to the adult musculature of the fly. In the embryo, they are arranged as groups of cells in the thoracic segments, while six solitary cells (one dorsal, two dorsolateral, two lateral and one ventral) are present in the abdominal hemisegments. It was reported earlier that too many Twi positive cells persist in the lateral mesoderm of lmd mutant embryos. Together with the fact that both Zfh1 and Twi are present in AMPs in the wild type, it appeared likely that both factors are also co-localized in embryos mutant for lmd. Indeed, double staining for Zfh1 and Twi showed a complete overlap in the lateral mesoderm and confirmed that both populations of ectopic cells are identical. They also express the gene holes in muscles (him) which is another marker specific for AMPs. For further characterization, the expression patterns were analyzed of several myogenic markers in lmd mutant embryos. No expression was detected of the muscle specific genes myocyte enhancing factor 2 (Mef2), β3 Tubulin or the reporter rP298 (Duf-lacZ) in Twi/Zfh1/Him positive cells (Sellin, 2009).

Twist expression is normally present during early stages of somatic muscle development in myoblasts that are not yet differentiated. Zfh1, which has been implicated in the repression of mef2, might help in keeping AMPs in the undifferentiated state until metamorphosis. The gene him, which is also expressed in AMPs, was recently reported to be involved in maintaining cells in an undifferentiated state by inhibiting the myogenic signal provided by Mef2 function. Consequently, the ectopic Zfh1/Twist/Him positive cells in the lmd mutant embryos are likely to be cells with myogenic potential, as are the endogenous AMPs, and hence can be considered to be ectopic AMP like cells. To assess if enhanced proliferation is also involved in generating an increased amount of cells in lmd mutant embryos, staining was oerfirned for phosphorylated Histone 3 (pH3), which specifically marks dividing cells. Over-proliferation in the dorsal and lateral mesoderm was not observed in lmd mutant embryos when compared to wild type embryos. Although there is a considerable number of the additional, AMP like cells that persist until the end of embryogenesis, their number is reduced between stage 13 and 16/17. Staining with Nile Blue A revealed a general excess of dying cells during these stages in lmd mutant embryos as compared to wild type, suggesting that not all ectopic cells survive until the end of embryogenesis (Sellin, 2009).

The supernumerary PCs and AMPs originate from the population of fusion competent myoblasts: While no general myogenic genes are expressed in the ectopic AMP like cells, it was however possible to show co-localization of Zfh1 and lmd mRNA in the somatic mesoderm of lmd mutant embryos, which is not observed in wild type embryos. In the wild type, lmd is expressed in fusion competent myoblasts (FCMs), which fail to differentiate in the absence of Lmd function. lmd mRNA is transcribed in a normal pattern in lmd mutant embryos. In situ hybridization with a lmd specific riboprobe therefore allowed visualization of the population of cells destined to become FCMs, although they do not express any other FCM specific genes in lmd mutant embryos. Since the ectopic Zfh1 positive cells co-express lmd mRNA, it is concluded that the ectopic AMP like cells in lmd mutant embryos originate from the FCM population and adopt AMP like characteristics instead. They are therefore generated by cell fate conversions, which is consistent with the observation that there are no additional cell divisions in lmd mutant embryos (Sellin, 2009).

In the dorsal mesoderm of lmd mutant embryos, the additional Zfh1 positive cells express Tin or Odd and Prc, indicating differentiation as pericardial cells. Pericardial cells usually develop from the dorsal cardiac mesoderm specified by Tin expression, while the somatic musculature is situated more laterally and is characterized by prolonged Twi expression. To address the question of whether a conversion of FCMs into PCs could also account for the pericardial hyperplasia phenotype in lmd mutant embryos in an analogous fashion to the ectopic AMP like cells, staining was carried out for Tin and lmd mRNA. It was reasoned that the ectopic Tin cells should also express lmd mRNA if they originate from the pool of mis-specified FCMs. Indeed, there is co-expression of lmd mRNA and Tin in ectopic pericardial cells in stage 13 lmd mutant embryos, indicating that cell fate conversions from FCM to ectopic PC fate are responsible for the observed pericardial hyperplasia phenotype (Sellin, 2009).

Of note, there is a distinct overlap of lmd mRNA and Tin expression in the dorsal mesoderm of stage 12 embryos, both in wild type and lmd mutant background. This observation is consistent with the observed cell fate switch from FCM to PC fate and indicates that in wild type embryos, dorsal FCMs are specified in the dorsal, Tin positive mesoderm rather than the Twi positive somatic mesoderm. Indeed, dorsal muscle phenotypes can be observed in embryos mutant for tin, consistent with the conclusion that dorsal muscle cell types (i.e., FCMs) develop from the early dorsal mesoderm specified by Tin expression: If this domain is not specified, it can not generate dorsal FCMs (or other dorsal mesodermal derivatives, like heart or visceral mesoderm) (Sellin, 2009).

Co-expression of Tin and lmd mRNA is no longer detectable after germ band retraction (stage 13) in wild type embryos, but persists in lmd mutant embryos until the lmd mRNA signal fades (at about stage 14-15). Thus, it seems that repression of Tin in the dorsal mesoderm depends on the presence of Lmd protein. To substantiate this observation, Lmd was overexpressed in the whole mesoderm with the twi-Gal4 driver to assess its influence on Tin expression. Indeed, a reduction was observed of Tin expression in stage 12 embryos overexpressing Lmd compared to wild type, further confirming a negative influence of Lmd on Tin expression in the dorsal mesoderm. At later stages, the number of TPCs (and OPCs) remains reduced, while the cardioblasts are not affected. A model is therefore proposed in which the initial dorsal mesoderm specified by Tin expression is subdivided by Lmd into cardiac mesoderm and dorsal musculature by repression of Tin in lateral regions and induction of a myogenic differentiation program instead. During this process, Tin expression is maintained only in the cells that are destined to become pericardial cells (or cardioblasts), while Tin is repressed by Lmd in the dorsally localized FCMs. Loss of Lmd function consistently leads to an increased amount of Tin positive cells in the dorsal mesoderm from stage 13 onwards, which then can differentiate as ectopic pericardial cells as indicated by the expression of Prc. Taken altogether, these data suggest that, in the absence of Lmd function, the pool of unspecified FCMs can develop as ectopic PCs in the Tin-positive dorsal mesoderm and as AMP-like cells in the lateral and ventral mesoderm. However, increased cell death, and the possibility that a small number of ectopic Tin positive cells might exist without Prc/Zfh1 expression as mentioned earlier, suggest the possibility that not all cells of the FCM population follow alternative cell fates. Instead, some cells might remain in an uncommitted mesodermal state in lmd mutant embryos (Sellin, 2009).

Normally, instructive Dpp signals from the ectoderm are responsible for the specification of cardiac cell types by maintaining Tin expression solely in the dorsal mesoderm, while Twist activity in the lateral and ventral mesoderm leads to the development of the somatic musculature. To test if reduced Dpp signaling has a similar effect on PC number as overexpression of Lmd, by reducing the size of the Tin domain, embryos carrying the mutation mad1-2 were examined. mad1-2 is a weak hypomorphic allele of the Dpp effector Mad and causes larval lethality, thereby allowing observation of late stages of embryogenesis. Indeed, a decreased number of OPCs and TPCs was observed in mad1-2 mutant embryos, without any effect on cardioblast number, as is the case when overexpressing Lmd. Of note, the number of OPCs is decreased to a similar extent in mad1-2; lmd double, as compared to lmd single mutant embryos. Therefore, it is concluded that in the presence of the hypomorphic mad1-2 mutation, the dorsal mesoderm that is specified by Dpp-dependent Tin expression is reduced, resulting in a reduction of PCs in a Lmd independent manner. However, Lmd further restricts the number of PCs in the mad1-2 mutant background, as revealed by an increased number of PCs and the presence of TPCs without Zfh1 expression in mad1-2; lmd double mutants when compared to mad1-2 single mutants (Sellin, 2009).

Pericardial cells share their developmental origin with the myogenic cardioblasts in a similar fashion as AMPs with founder cells in the somatic musculature. During lateral inhibition, Notch activation promotes myogenic FCM fate as opposed to the progenitors of founder cells in the lateral mesoderm or cardiogenic progenitors in the dorsal mesoderm. Subsequently, during the process of asymmetric cell division, Notch activation renders the daughter cell always non-myogenic (PC or AMP fate). Although the AMPs have the potential to develop into muscle cells during metamorphosis, they are considered non-myogenic in this context because they do not yet express any myogenic genes, such as mef2, lmd or muscle structural genes in the embryo. In the case of pericardial cells, there is surprisingly little data available about their physiological role. While it is known that the OPCs contribute to the population of nephrocytes in postembryonic stages, TPCs and EPCs are not correlated with any function at all, and their developmental fate after embryogenesis is still unknown. A recent study described the development of adult muscular structures, the so called wing hearts, from a specialized subset of EPCs. This is the first hint that some pericardial cells might be considered as imaginal myogenic cells in an analogous fashion to AMPs, and it highlights the necessity to further characterize pericardial cells (Sellin, 2009).

It is currently known that PCs and AMPs have in common a dependency on active Notch signaling although they stem from different cell lineages and mesodermal primordia (Tin vs. Twi domain). FCMs, which adopt AMP or PC like characteristics in lmd mutant embryos, also need active Notch signaling. In fact, Lmd is a downstream target of N signaling and induces the FCM differentiation program. The observed lmd phenotype could be explained if, in the absence of Lmd, Notch activity always promoted AMP or PC (non-myogenic) fate, but not FCM fate, independently of the original pathway that is involved (lateral inhibition or asymmetric cell division). To assess this hypothesis, double mutants for lmd and genes involved in the Notch pathway were established. For this analysis kuzbanian and mastermind alleles were chosen because loss of either gene causes lethality only late in embryogenesis due to a maternal component, thereby allowing the analysis of later events in heart and muscle development. Both genes have also been well studied with respect to their molecular function and developmental implications. Kuzbanian (Kuz) is an ADAM metalloprotease that is known to process the Notch receptor following ligand binding. Zygotic loss of function mutations lead to defects in both lateral inhibition and asymmetric cell division in heart and muscle development, although the phenotype is far weaker than in embryos carrying N loss of function alleles. mastermind (mam) is involved in transducing the Notch signal and displays a stronger heart phenotype than kuz and a mild Notch-like muscle phenotype. Staining was perfomed for expression of Krüppel (Kr) and him mRNA, which are specific for a subset of muscle founders and AMPs/ PCs, respectively, and an increase of Notch negative cell types, corresponding to founders, was observed in the somatic mesoderm of kuz mutant embryos. This is accompanied by a reduction of AMPs, confirming the expected function of Kuz in facilitating N function in muscle cell differentiation. Furthermore, the number of FCMs as marked by Lmd expression is strongly reduced in kuz mutants, although the effect is not as complete as in N loss of function alleles (Sellin, 2009).

In kuz; lmd double mutant embryos, the increase of AMPs is milder than in lmd mutant embryos, which is consistent with a failure in lateral inhibition and a concomitant reduction of FCMs that are available for conversion to AMPs. The number of Kr-positive founder cells is increased to comparable levels in kuz and kuz; lmd mutant embryos, suggesting that Notch inactive cell fates (muscle founders and cardioblasts) are not influenced by the absence of Lmd, and that Notch acts as a permissive signal to allow the cell fate switch in lmd mutant embryos. mam; lmd double mutant embryos display a similar phenotype. Altogether, these findings suggest that in the double mutants, a general reduction of cell types with Notch activity (i.e. FCMs) occurs, followed by the conversion of the remaining potential FCMs to AMP or PC fate under the influence of N signaling in the absence of Lmd. Lame duck is present in stages 12-14, which is later than the period during which Notch activity is involved in facilitating cell fate decisions within the musculature. Hence, it appears that Notch can promote AMP or PC fate at a relatively late time point in the absence of Lmd (Sellin, 2009).

It was of interest to know if the endogenous set of AMPs, which develop through asymmetric cell divisions of muscle progenitors, is specified correctly in lmd mutant embryos. For example, the lateral AMPs are the siblings of the segment border muscle founder (SBM), and share with the latter the expression of the identity factor Ladybird early (Lbe). To discern ectopic cells and endogenous AMPs in lmd mutant embryos, co-staining was performed for Lbe and Twi expression. Indeed, the normal number of lateral AMPs, as marked by Lbe expression, is present in lmd mutant embryos, while far too many Twi-positive cells was observed in general. The latter are the ectopic AMP like cells that are presumed to be recruited from the FCM population. This observation further confirms that individual mesodermal lineages, such as the asymmetrically derived OPCs or individual AMPs, are not influenced by the loss of Lmd function (Sellin, 2009).

The proposed model of cell fate switches from myogenic FCM fate to non-myogenic AMP like or PC fate, but not myogenic fates (cardioblasts or founder cells), is consistent with the observation that Notch signaling is often employed to delay or inhibit the differentiation of stem cells or progenitor cells, especially in myogenesis. In vertebrates, Notch signaling prevents satellite cells (muscle stem cells) from entering a myogenic differentiation program in cell culture as well as in vivo, and impaired upregulation of its ligand Delta-like 1 in satellite cells has been correlated with a decreased capacity of aging muscle tissue to regenerate. While the data are consistent with the general function of Notch in preventing cells to enter the myogenic differentiation program by promoting the AMP or PC fate, they also highlight the special and unusual properties of Lmd - as a target of Notch signaling - in Drosophila muscle development. Although it is activated by Notch, it has the ability to induce myogenic differentiation. The data strongly suggest that the AMP or PC fate is the default consequence of Notch signaling in Drosophila myogenesis and that Lmd function overrules this signal to induce the FCM differentiation program in lateral or dorsal competence domains. It was shown that N has a biphasic function in heart differentiation analogous to the situation in the somatic mesoderm. At an early phase, N activity restricts the number of the sum of CBs and PCs, reflecting a function in the definition of early cardiac progenitors, likely by lateral inhibition. Subsequently, N activity is needed to promote pericardial cell fates in asymmetric cell division of the early progenitors. Although the last division step is in many cases a symmetric division seem to indicate that the majority of cardiac cell types is generated by asymmetric cell divisions segregating cardiac and pericardial fates. This might occur in some cases at one of the earlier division steps of the progenitor(s). Since these data indicate the generation of FCMs from the dorsal mesoderm, as reflected by co-expression of Tin and Lmd in stage 12 embyos, it might be suggested that dorsal FCMs originate from dorsal competence domains which also give rise to the above mentioned cardiac progenitors. These progenitors divide asymmetrically to generate CBs and PCs analogous to FC/AMP sibling pairs from more lateral competence fields, while it ia proposes that all or some of the remaining cells of the competence domains begin to express Lmd and generate FCMs under instructive influence of N signaling. In the absence of Lmd function (either in wild type in the N active daughter cells of the progenitors, or in lmd mutant embryos in all N active cells of the competence domains), the N signal promotes non-myogenic cell fates according to the mesodermal context (i.e., dorsal vs. lateral mesoderm). This would then result in the differentiation of the non-segregating population (normally developing as FCMs) as PCs in the Tin domain and AMPs in the somatic mesoderm (Sellin, 2009).

Lame duck and Zfh1 act antagonistically in mesodermal cell fate decisions: While loss of Lmd function results in an increased number of Zfh1-positive cell types, overexpression of Lmd leads to the opposite phenotype. The pan-mesodermally active twi-Gal4 driver line was used to induce Lmd expression in the whole mesoderm, and a reduction of OPCs, TPCs and AMPs was observed. To assess whether pericardial cell reduction might be a secondary effect of the early Tin repression caused by ectopic Lmd activity, the later and cardiac specific handCA-Gal4 driver, which is active in the heart from stage 12 onwards, was used. At this time point, the OPC precursors are already specified and are no longer expressing Tin. Since hand>Lmd overexpression severely reduces the number of all pericardial cells, it is concluded that their reduction is not only a secondary effect of the narrower Tin domain in embryos overexpressing Lmd. To further confirm this conclusion, the phenotype of zfh1 mutant embryos was compared with that of embryos overexpressing Lmd. The number of OPCs and TPCs is also reduced in zfh1 mutant embryos quite similarly to embryos overexpressing Lmd, although the early Tin expression pattern is normal in the absence of Zfh1 function. It is therefore unlikely that Lmd acts negatively on Zfh1 expression only by reducing Tin expression, but rather also independently of Tin function (Sellin, 2009).

There are however important differences in the phenotypes of twi > Lmd and zfh1 mutant embryos. Zfh1 appears to be involved in maintaining, but not in specification of OPCs, because it has been observed that loss of Zfh1 does not affect the number of OPC precursors at stage 13, but rather leads to a decrease of OPCs at later stages. This is in contrast to a reduced number of OPC precursors in stage 13 embryos overexpressing Lmd. Therefore, Zfh1 repression alone can not account for the loss of PCs in embryos ectopically expressing Lmd. Instead, it might be that the reduction of the dorsal Tin domain by ectopic Lmd expression results in the specification of fewer OPC precursor cells, followed by further reduction of the remaining OPCs by the negative effect of ectopic Lmd on Zfh1 expression. Consistently, a much stronger reduction of OPCs was observed after ectopic expression of Lmd as compared to the loss of OPCs in zfh1 mutant embryos. The observation that loss of Lmd function leads to the appearance of TPCs that do not express Zfh1, but Prc as a marker of pericardial differentiation, is another hint that both effects occur independently of each other and that pericardial differentiation can be accomplished in the absence of Zfh1 in lmd mutant embryos (Sellin, 2009).

Taken altogether, it does not seem likely that Tin and Zfh1 act in an epistatic hierarchy in dorsal mesodermal cell fate decisions. Instead, the data support the conclusion that Lmd regulates OPC and TPC number by two independent mechanisms: (1) Initially, Lmd restricts the cardiac field in general through repression of Tin, which leads to the reduction of early OPC precursors and the elimination of Tin expression in cells that do not express Zfh1 (which can differentiate as TPCs, as indicated by Prc expression, in the absence of Lmd function). (2) Later, it represses Zfh1, thereby reducing further the number of OPCs and TPCs. This is consistent with previous findings which described Zfh1-dependent and Zfh1-independent mechanisms for the regulation of OPC and TPC number (Sellin, 2009).

Of note, it was previously shown that Zfh1 overexpression leads to an increase in pericardial cell number (both OPCs and TPCs) and a concomitant loss of dorsal somatic muscle cells, indicating that overexpression of Zfh1 phenocopies the pericardial hyperplasia in lmd mutant embryos. It was shown further that overexpression of Zfh1 with the twist-Gal4 or 24B-Gal4 driver leads to an increased number of AMP like cells in the dorsal mesoderm although the effect is rather weak when compared to lmd mutant embryos. Zfh1 overexpression does not however alter the pattern of Lmd expression, indicating that Zfh1 does not antagonize Lmd function at the transcriptional level. To verify whether Zfh1 has an influence on Lmd at the posttranscriptional level, the intracellular distribution of Lmd was analyzed in embryos overexpressing Zfh1, because Lmd function has been shown to be modulated by its subcellular localization in wild type embryos. In embryos overexpressing Zfh1, the subcellular localization of Lmd does not appear to be altered, suggesting that Zfh1 does not influence the subcellular distribution of the Lmd protein (Sellin, 2009).

Taken together, these data indicate that Lmd and Zfh1 have generally opposite effects on dorsal mesoderm differentiation: Lmd loss-of-function or Zfh1 gain-of-function leads to increased AMPs or PCs, whereas Lmd gain-of-function and Zfh1 loss-of-function reduce these cell types. Consequently, Lmd and Zfh1 can be considered to be functional antagonists, although their repression is not mutual. One possible explanation for the antagonistic effect of Zfh1 overexpression might be due to its direct negative influence on mef2 expression, thereby counteracting the mef2 activating function of Lmd. The vertebrate functional orthologue of Zfh1, ZEB2 (or Sip1), also inhibits myotube development in culture and represses a number of myogenic genes, and is able to rescue Zfh1 function in Drosophila (Sellin, 2009).

Lmd is instructed to restrict Tin expression by a late, pro-myogenic Dpp signal: While in wild type embryos Tin expression is repressed in cells destined to become dorsal FCMs between stages 12 and 13, there is a prolonged co-localization of Tin and lmd mRNA in cells of the dorsal mesoderm in lmd mutant embryos. As a consequence, dorsal FCMs adopt pericardial cell fates in the absence of Lmd function. Of note, this effect can also be observed in embryos carrying the dppd6 disk region mutation. These embryos lack a late Dpp signal (beginning at about stage 12) that is involved in pericardial restriction. Early Dpp signaling does not seem to be affected since the dorsal mesoderm (characterized by Dpp-dependent Tin expression) is normal in dppd6 mutant embryos. Quite contrary to embryos with otherwise decreased Dpp signaling and a reduced pericardial field, such as mad1-2 embryos, the dppd6 mutant embryos display a pericardial hyperplasia phenotype that resembles in many aspects the phenotype observed in lmd mutant embryos. Too many OPCs, TPCs and atypical TPCs without Zfh1 expression are also detected, although the dppd6 mutant phenotype is milder than the lmd mutant phenotype. This resemblance in phenotypes suggested an epistatic relationship of Lmd and the late Dpp signal. In addition, the accumulation of phosphorylated Mad (pMad) has been traced in PCs and cells within the dorsal musculature that are not positive for founder specific Kr or Eve expression, and hence are likely to be FCMs. Altogether, these findings lead to the hypothesis that Lmd might be a target of the late Dpp signal in FCMs. However, Lmd is expressed in a normal pattern (both at the mRNA and protein levels) in dppd6 mutant embryos, indicating that Lmd expression is independent of Dpp signaling. Nevertheless, co-staining with anti-Tin antibody revealed a prolonged co-localization of Tin and lmd mRNA in dppd6 mutant embryos until stage 14/15, as observed in lmd mutant embryos, suggesting a requirement for late Dpp signaling in the process of pericardial restriction by Lmd. To assess if the restrictive influence of late Dpp signaling on Tin expression is indeed relayed by Lmd in the dorsal mesoderm, or if both negative effects are independent of each other, the late Dpp signal was enhanced in the lmd mutant background. For this purpose, the leading edge driver LE-Gal4 was used to overexpress Dpp, which was shown to reduce the number of OPCs and TPCs in the wild type background. It was reasoned that this effect would be lost in lmd mutant embryos if Lmd is responsible for the restricting effect on PC number. The number of OPCs was counted in LE > Dpp; lmd embryos in comparison to lmd mutant embryos. While overexpression of Dpp with the LE-Gal4 driver in the wild type background led to a reduction of OPCs by ~1.2-fold, no reduction of OPCs was observed in the lmd mutant background, indicating that Lmd is indeed necessary to interpret the late Dpp signal as pro-myogenic. Altogether, these data suggest that the pro-myogenic effect of the late Dpp signal is Lmd dependent, although not by inducing Lmd expression. Instead, the presence of Dpp activity seems to be a prerequisite for the negative influence of Lmd on Tin expression and might act as an instructive signal to modify Lmd activity to allow repression of Tin. If the late Dpp signal is lost -- as is the case in embryos carrying the hypomorphic allele dppd6 - repression of Tin fails even in the presence of Lmd protein, indicating that repressive activity of Lmd is dependent on Dpp signaling (Sellin, 2009).

A model is proposed in which the subdivision of the early Tin positive primordium into pericardial and dorsal muscle tissues is mediated via the antagonistic action of Lmd and Zfh1 under the instructive influence of late Dpp signals. While the early function of Dpp restricts Tin expression to the dorsal mesoderm, subsequent Dpp signaling provides pro-myogenic input to modulate the pericardial field in favor of the dorsal musculature. The present data show that the function of this late Dpp signal requires Lmd activity, strongly suggesting that Lmd is a target of Dpp for establishing the boundary between the dorsal musculature and pericardial field. Repression of Tin also appears to be dependent on Dpp signaling. The previous observation that pMad accumulation occurs in PCs and dorsal muscle cells, which are likely to be FCMs, is consistent with the finding that Lmd is needed to relay the pro-myogenic function of late Dpp signaling. These cells originate from the Tin-expressing dorsal mesoderm, and co-expression of Tin and lmd mRNA in wild type embryos at stage 12 can be observed. In the presence of Lmd protein, this co-expression is not maintained after stage 12 due to a repressive function of Lmd on Tin. Of note, it was previously shown that Lmd function depends on posttranscriptional mechanisms that modulate its specific subcellular localization and activity, and it might be speculated that Dpp signaling is involved in changing Lmd function into a repressive form. However, there is no evidence that the negative influence of Lmd on Tin expression is of a direct nature, or if there are other factors that are involved in the process. In this context, the following explanation for the antagonistic effect of Zfh1 overexpression without repression of Lmd could also be considered. Since the vertebrate homologue ZEB2 was shown to inhibit activation of target genes by Smads, an excess of Zfh1 might antagonize the late Dpp signal by repressing pMad-dependent interaction partners of Lmd, thereby preventing the repression of Tin (and/or other targets) in the dorsal mesoderm. Lmd expression and function would not be affected elsewhere which would be consistent with the observation that Zfh1 is not a general repressor of Lmd (Sellin, 2009).

Hh signalling is essential for somatic stem cell maintenance in the Drosophila testis niche

In the Drosophila testis, germline stem cells (GSCs) and somatic cyst stem cells (CySCs) are arranged around a group of postmitotic somatic cells, termed the hub, which produce a variety of growth factors contributing to the niche microenvironment that regulates both stem cell pools. This study shows that CySC but not GSC maintenance requires Hedgehog (Hh) signalling in addition to Jak/Stat pathway activation. CySC clones unable to transduce the Hh signal are lost by differentiation, whereas pathway overactivation leads to an increase in proliferation. However, unlike cells ectopically overexpressing Jak/Stat targets, the additional cells generated by excessive Hh signalling remain confined to the testis tip and retain the ability to differentiate. Interestingly, Hh signalling also controls somatic cell populations in the fly ovary and the mammalian testis. These observations might therefore point towards a higher degree of organisational homology between the somatic components of gonads across the sexes and phyla than previously appreciated (Michel, 2012).

Hh thus provides a niche signal for the maintenance and proliferation of the somatic stem cells of the testis. CySCs that are unable to transduce the Hh signal are lost through differentiation, whereas pathway overactivation causes overproliferation. Hh signalling thereby resembles Jak/Stat signalling via Upd. Partial redundancy between these pathways might explain why neither depletion of Stat activity nor loss of Hh signalling causes complete CySC loss (Michel, 2012).

This study has shown that loss of Hh signalling in smo mutant cells blocks expression of the Jak/Stat target Zfh1, whereas mutation of ptc expands the Zfh1-positive pool. Overexpression of Zfh1 or another Jak/Stat target, Chinmo, is sufficient to induce CySC-like behaviour in somatic cells irrespective of their distance. By contrast, Hh overexpression in the hub using the hh::Gal4 driver only caused a moderate increase in the number of Zfh1-positive cells relative to a GFP control. Ectopic Hh overexpression in somatic cells under c587::Gal4 control increased this number further. However, unlike in somatic cells with constitutively active Jak/Stat signalling, the additional Zfh1-positive cells remained largely confined to the testis tip, although their average range was increased threefold. Thus, Hh appears to promote stem cell proliferation, in part, also independently of competition (Michel, 2012).

It is tempting to speculate that further stem cell expansion is limited by Upd range. Consistently, cells with an ectopically activated Jak/Stat pathway remain undifferentiated, whereas ptc cells can still differentiate. Future experiments will need to formally address the epistasis between these pathways. However, the observations already show that Hh signalling influences expression of the bona fide Upd target gene zfh1, and therefore presumably acts upstream, or in parallel to, Upd in maintaining CySC fate (Michel, 2012).

In addition, the reduction in GSC number following somatic stem cell loss implies cross-regulation between the different stem cell populations that presumably involves additional signalling cascades, such as the EGF pathway (Michel, 2012).

In recent years, research has focused on the differences between the male and female gonadal niches. This paper instead emphasizes the similarities: in both cases, Jak/Stat signalling is responsible for the maintenance and activity of cells that contribute to the GSC niche, and Hh signalling promotes the proliferation of stem cells that provide somatic cells ensheathing germline cysts. In the testis, both functions are fulfilled by the CySCs, whereas in the ovary the former task is fulfilled by the postmitotic escort stem cells/escort cells and the latter by the FSCs. Finally, male desert hedgehog (Dhh) knockout mice are sterile. Dhh is expressed in the Sertoli cells and is thought to primarily act on the somatic Leydig cells. However, the signalling microenvironment of the vertebrate spermatogonial niche is, as yet, not fully defined. Future experiments will need to clarify whether these similarities reflect convergence or an ancestral Hh function in the metazoan gonad (Michel, 2012).

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

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

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

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

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

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

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

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

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

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

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

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

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

Robust intestinal homeostasis relies on cellular plasticity in enteroblasts mediated by miR-8-Escargot switch

This study used tracing methods that allow simultaneously capturing the dynamics of intestinal stem and committed progenitor cells (called enteroblasts) and intestinal cell turnover with spatiotemporal resolution. Intestinal stem cells (ISCs) divide 'ahead' of demand during Drosophila midgut homeostasis. Their newborn enteroblasts, on the other hand, take on a highly polarized shape, acquire invasive properties and motility. They extend long membrane protrusions that make cell-cell contact with mature cells, while exercising a capacity to delay their final differentiation until a local demand materializes. This cellular plasticity is mechanistically linked to the epithelial-mesenchymal transition (EMT) programme mediated by escargot, a snail family gene. Activation of the conserved microRNA miR-8/miR-200 in 'pausing' enteroblasts in response to a local cell loss promotes timely terminal differentiation via a reverse EMT by antagonizing escargot. These findings unveil that robust intestinal renewal relies on hitherto unrecognized plasticity in enteroblasts and reveal their active role in sensing and/or responding to local demand (Antonello, 2015).

The robustness of intestinal cell renewal relies on cellular plasticity in committed progenitor cells and a rather loose regulation of ISCs proliferation. One key finding is that stem cells divide continually and generate a 'stock' of committed progenitor cells that do not terminally differentiate right away but postpone their final differentiation for long time intervals in the absence of a local epithelial cell loss. Accordingly, one noticeable change in newborn progenitor cells after their (enterocyte) fate commitment is their transformation from rounded cells to spindle-shaped cells that appear to actively monitor their surroundings by extending long membrane actin-rich protrusions that make cell-cell contact with mature epithelial cells and their mother ISCs. Timely terminal differentiation with epithelial cell loss is orchestrated by activation of a conserved pro-epithelial microRNA, in turn, directly repressing the repressors of differentiation. A microRNA-induced repression of the repressors of differentiation provides a faster mechanism than one involving a transcriptional regulator since synthesizing a miRNA likely requires less time than synthesizing a protein. Importantly, mutual antagonism between the microRNA (MiR-8/miR-200) and its targets (Escargot/Snail2 and Zfh1/ZEB) may serve to slow down the mesenchymal-to-epithelial process inside individual mesenchymal/progenitor cells until they are successfully integrated in the epithelium. Consistently, abrupt transition as in mir-8 overexpressing midguts results in erroneous tissue repair (Antonello, 2015).

Supply and demand in business production involves frequently two alternative solutions called 'make-to-stock' and 'make-to-order'. In 'make-to-stock' or MTS, production is continuous so that response to customers can be supplied immediately. However, as production is not based on actual demand, the MTS solution is not robust against fluctuations in demand and errors in forecasting can result in shortages (if there is insufficient residual stock) or overproduction. In 'make-to-order', or MTO, production only starts upon receiving a customer's order, thereby precisely matching production to demand. However, the MTO generates a delay in the response and can be less efficient and competitive than the MTS paradigm. The dynamics of stem cells and committed progenitor cells in the midgut suggests a hybrid solution between MTS and MTO -- reminiscent to the business solution known as delayed differentiation. Thus, in basal homeostasis, production of new cells to replace cell loss occurs in two stages: (1) a 'make-to-stock' stage where committed progenitor cells are continually generated and 'stocked' in an 'undifferentiated' state; and (2) a 'make-to-order' stage where terminal differentiation takes place only in response to a local demand. In mice and humans, the rapid turnover that occurs in the small intestinal epithelium is thought to be the result of continual shedding of superficial cells balanced by the continual stem cell production. The mechanism described in this study may be more general than expected and could account for how murine cells after fate commitment like the secretory-committed cells defer for long periods their terminal differentiation (Buczacki et al, 2013; Antonello, 2015).

Escargot/Snail2 sustains the undifferentiated state and self-renewing divisions of midgut intestinal stem cells. However, the committed progenitor cells also express escargot and apparently at higher levels than the stem cells. It is hypothesized that below a certain threshold level, Escargot maintains stemness and a partial EMT that may facilitate regular cell division and a topologically confined position at the base of the intestinal epithelium. Conversely, when Escargot surpasses a certain threshold level, it promotes a full EMT that confers invasive properties and motility for the successful response and integration of the newly differentiated cells in the preexisting epithelium. Intriguingly, the enteroendocrine cells appear to escape from this block in terminal differentiation and differentiate at the normal rate in the absence of escargot. There is as yet no explanation for the behaviour of these progenitor cells (Antonello, 2015).

Mechanistically, the different levels of escargot could be achieved via Notch signalling pathway, which is prominently activated in enterocyte-committed progenitors. Notch signalling activates directly zfh1 gene and Zfh1, a homolog of the mammalian stemness and EMT-determinant Zeb1,2, and binds to the escargot promoter region, and this study shows that Zfh1 acts genetically upstream of escargot. Thus, progenitor cells receiving Notch signalling might enhance escargot transcriptional levels via Notch-induced zfh1 transcription. Such regulatory mechanism would explain, for example, that loss of Notch results in stem-like/round cells (Antonello, 2015).

In mammalian cell culture, the EMT process has been linked to the acquisition of stem-like nature via an interplay between the ZEB1,2 and Snail transcription factors and the microRNAs of the miR-200 family. Moreover, EMT determinants often regulate each other to promote EMT. Thus, the interactions between Escargot/Snail2, zfh1/Zeb and miR-8/miR-200 that were identified in this study exemplify the conservation of the regulatory mechanisms involved in EMT/MET and stemness in an in vivo context and a normal physiology of an adult organism. However, this study shows that escargot-zfh1 promotes stemness and full EMT/invasive properties in distinct cell populations and likely at different concentration levels, highlighting the utility of Drosophila midgut as a model to dissect out mechanisms linking physiological EMT to cellular plasticity and stemness as well as provide novel insights linking polyploidy and EMT towards stemness (Antonello, 2015).

Although midgut mesenchymal/progenitor cells have motility, most of them maintain their own local area as clearly defined by Flybow clonal analysis. This situation is similar to the leading edge mesenchymal cells during collective cell migration. Midgut enteroblasts retain contact via E-cadherin with their mother ISC, a process that might be regulated by escargot as in tracheal cells. Cell-cell contact is crucial to sustain Notch signalling in committed progenitor cells and likely to help to stabilize polarity of enteroblasts and their membrane protrusions that contact mature cells. Through these protrusions, mesenchymal/enteroblasts might actively monitor their surroundings. When a protrusion detects changes in tension and mechanical forces generated during the elimination of a dying cells, a positive input might be created that triggers the activation of expression of the microRNA mir-8 in the particular progenitor cell which, in turn, promotes the epithelial state and integration of the newly differentiated cell in the epithelium. Adhesion via E-cadherin could facilitate communication between an epithelial cells and a mesenchymal/progenitor cell in its vicinity so that a single, newly differentiated cell fills the gap left by the cleared cell (Antonello, 2015).

Dynamic pseudopodia in migrating cells have been proposed as a mechanism for temporal and spatial sensing during cell migration. Direction sensing is also consistent with time-lapse data showing individual progenitor cells re-adjusting position in the homeostatic midguts. Transduction of mechanical cues via YAP and TAZ (called Yorkie in flies) is functionally involved in differentiation of mesenchymal stem cells. Hence, Drosophila Hippo/Yorkie-YAP in mature enterocytes is a primary candidate pathway for a potential transduction of mechanical cues activating mir-8 in response to cell death (Antonello, 2015).

In summary, the miR-8-escargot-zfh1 axis and the EMT/MET programme provides a conceptual shift of the current stem cell-centred view of tissue renewal and offers a starting point for investigating how mature cells speak with neighbouring committed progenitor cells to ensure that epithelial cell loss and cell addition are kept in balance (Antonello, 2015).

Effects of Mutation or Deletion

Ectopic expression of the zfh-1 gene produces defects in the CNS. Adult survivors of both larval and pupal overexpression often have missing, misplaced, and duplicated thoracic macrochaetae. Roughened eyes are produced by ectopic expression during late larval development. The eye roughening is characterized by disordered, irregularly sized facets and is most pronounced in posterior eye regions (Lai, 1991).

Phenotypic analysis of zfh-1 mutant embryos reveals that the gene is not required for the initial segregation of the mesoderm or for the differentiation of mesodermally derived tissues. Rather, loss of zfh-1 function results in various degrees of local errors in cell fate or positioning. The ventral-oblique and the dorsal muscles are usually the most severely affected. The ventral-longitudinal and pleural muscles appear fairly normal in number and attachment but lack the taut straight appearance of the wild type. There are a variety of errors in segregation of muscle precursors. Mutants have missing muscles, misplaced muscles, and nuclei within a muscle are disorganized and spread further from the cluster than normal. The foregut and hindgut appear normal, but the midgut is abnormal. Constrictions start to form but rarely complete the subdivision of the yolk, and the elongation and narrowing of the gut occurs in a partial and very uneven fashion. In some mutants there is a prononunced kink in the heart. In others, there are breaks in anterior parts of the heart: the two sides fail to come together for part of the length of the heart or cells fail to join to make a continuous tube lengthwise. Gonads are abnormal: the normally compact structure of the gonads appears to be more dispersed. A range of defects are seen in pole cells. Adult muscle precursors are missing or displaced (Lai, 1993).

In many animal groups, an interaction between germ and somatic lines is required for germ-line development. In Drosophila, the germ-line precursors (pole cells), which form at the posterior tip of the embryo migrate toward the mesodermal layer where they adhere to the dorsolateral mesoderm, which ensheaths the pole cells to form the embryonic gonads. These mesodermal cells may control the expression of genes that function in the development of germ cells from pole cells. However, such downstream genes have not been isolated. In this study, a novel transcript, indora(idr), is identified that is expressed only in pole cells within the gonads. The nucleotide sequence of the 1.5 kb cDNA predicts a protein of 131 amino acids. The amino acid sequence shows no significant homology to any known proteins. The putative Idr protein is highly basic (calculated isoelectric pH is 10.1). During normal development, the expression of idr transcripts become discernible in pole cells at the embryonic stage 14, when pole cells are incorporated into the gonads. Expression persisted in pole cells until the completion of embryonic development. idr expression is undetectable in the adult germ line. However, the possibility that a trace amount of IDR mRNAs is expressed in somatic cells as well as in the germ line throughout most of the life cycle cannot be excluded, because Northern blot analysis reveals that idr transcripts are detectable from late embryogenesis to adulthood (Mukai, 1998).

Reduction of idr transcripts by an antisense idr expression causes the failure of pole cells to produce functional germ cells in females. Furthermore, idr expression depends on the presence of the dorsolateral mesoderm, but it does not necessarily require its specification as the gonadal mesoderm. In order to determine the source of the mesodermal cue, idr expression was analyzed in the absence of the mesodermal cells that make up the gonads. The origin and development of the somatic components of the gonads are described. The somatic gonad precursors (SGPs) are specified from the dorsolateral mesoderm within PS 10-12 at stage 11. In tin;zfh-1 double-mutants, no dorsolateral mesoderm is formed, which results in loss of SGPs. In these embryos, pole cells pass through the midgut epithelium, but subsequently they are dispersed around the midgut. idr expression is drastically reduced in tin;zfh-1 double-mutants. This result shows the requirement of the dorsolateral mesoderm for idr expression in pole cells. It was next asked whether the specification of the dorsolateral mesoderm as SGPs is needed to induce idr expression in pole cells. To examine this, abd-A and iab-4 mutations were used. abd-A function is required in the mesodermal cells for the specification of SGPs. In abd-A mutant embryos, pole cells pass through the midgut wall and are normally associated with the dorsolateral mesoderm. However, they do not coalesce with the pole cells to form the gonads due to their failure to be specified as SGPs. Consequently, pole cells are released from the mesoderm and scattered throughout the embryo. In these embryos, the dispersed pole cells express idr during stages 14-16. Furthermore, a regulatory mutation in the abd-A locus, iab-4, also has no deleterious effect on idr expression. Thus, the specification of the dorsolateral mesoderm as SGPs is dispensable for idr expression. These findings suggest that the induction of idr in pole cells by the mesodermal cells is required for germ-line development (Mukai, 1998).

The Drosophila Brachyury homolog brachyenteron (byn) is essential for the development of hindgut, anal pads and Malpighian tubules. byn is activated by the terminal gap gene tailless (tll) in a region of 0%-20% egg length of the syncytium (0% = posterior tip). With completion of cellularization, the byn expression becomes downregulated in the posteriormost cap of the embryo, which will later form the posterior midgut, by the terminal gap gene huckebein (hkb). Thus, the expression of byn is confined to a ring of cells from about 10%-20% egg length. The dorsal and the lateral aspects of that ring correspond to the proctodeum, from which the hindgut, the anal pads and the Malpighian tubules later develop. Intriguingly, hkb also determines the posterior extent of the ventral mesoderm primordium by repressing the mesodermal determinant snail (sna). This suggests that the ventralmost aspect of byn expression might comprise the posterior tip of the mesoderm primordium (Kusch, 1999).

The visceral musculature of the larval midgut of Drosophila has a lattice-type structure and consists of an inner stratum of circular fibers and an outer stratum of longitudinal fibers. The longitudinal fibers originate from the posterior tip of the mesoderm anlage, which has been termed the caudal visceral mesoderm (CVM). The CVM migrates in an orderly movement anteriorly and eventually forms an outer layer of longitudinal muscle fibers surrounding the midgut. The progenitors of a second tissue, the inner sheet of circular muscles of the midgut, are recruited from 11 parasegmentally arranged clusters of dorsal mesoderm in the trunk region and are therefore referred to as trunk visceral mesoderm (TVM) (Kusch, 1999).

In this study, the specification of the CVM has been investigated and particularly the role of the Drosophila Brachyury-homolog brachyenteron. Supported by fork head, brachyenteron mediates the early specification of the CVM along with zinc-finger homeodomain protein-1. This is the first function described for brachyenteron or fork head in the mesoderm of Drosophila. The mode of cooperation resembles the interaction of the Xenopus homologs Xbra and Pintallavis. Another function of brachyenteron is to establish the surface properties of the CVM cells, which are essential for their orderly migration along the trunk-derived visceral mesoderm. During this movement, the CVM cells, under the control of brachyenteron, induce the formation of one muscle/pericardial precursor cell in each parasegment. It is here proposed that the functions of brachyenteron in mesodermal development of Drosophila are comparable to the roles of the vertebrate Brachyury genes during gastrulation (Kusch, 1999).

During germband retraction and midgut closure, the progenitors of the the outer, longitudinally oriented fibers of the visceral mesoderm, the CVM, perform an ordered movement that can be subdivided into three phases. The first migratory phase starts at early germband retraction when the cells begin to move anteriorly from their position at the posterior tip of the mesodermal germ layer and split into two tightly packed, bilaterally symmetrical clusters on each side of the posterior midgut primordium. When these clusters have reached the anterior tip of the posterior midgut primordium, the cells detach from each other and disperse anteriorly as two rows along the germband, the second phase of the migration. During this movement, the cells are arranged along the dorsal and ventral edge of the midgut primordia and are in close contact with the band of progenitors of the circular muscle fibers. The band seems to serve as a migration substratum. During the last phase of the migration, which takes place as the midgut encloses the yolk, the progenitors of the longitudinal muscle fibers spread regularly over the underlying circular muscle fibers. The cells acquire a spindle shape, then stretch in an anteroposterior direction and form about 16-20 regularly spaced longitudinal muscle fibers. These fibers reach from the proventriculus to the midgut-hindgut transition where the ureters of the Malpighian tubules insert. The foregut and the hindgut lack any longitudinal muscles and are solely covered by the inner layer of circular muscles (Kusch, 1999).

The specification of the CVM and its fate were monitored by the detection of Byn protein or the expression of CVM-specific markers like croc-lacZ and cpo-lacZ. The initial byn expression at the posterior pole is regulated by tll and hkb. Thus it is likely that the CVM cells are specified under the control of the same genes. In fact, in hkb embryos, the size of the CVM primordium is enlarged and comprises more cells than normal. This corroborates the notion that the CVM primordium constitutes the most posteriorly located mesoderm primordium. tll expression reaches more anteriorly than the hkb domain and encompasses the primordia of the proctodeum and of the CVM. One would therefore expect that the formation of the CVM is entirely dependent on tll. Indeed, this is the case: the CVM is missing in tll mutant embryos. Part of the function of tll seems to be mediated by byn. In byn mutants, a significantly reduced number of CVM cells is seen, and these few cells form clusters that are less compact and migrate significantly slower than in wild type. Later, they fail to contact the TVM and do not distribute along the germband. During stage 11, most of the cells acquire a condensed appearance resembling apoptotic bodies. A high level of apoptosis is detected in the proctodeum of byn embryos as well as in the posteriormost mesoderm. By stage 13, cells with the properties of the CVM are not detectable any longer in the mutants and, as expected from this, the dissected midguts of byn embryos lack the outer, longitudinal muscle fibers (Kusch, 1999).

Only the anterior and the posterior mesoderm are competent to be specified by byn as CVM, in conjunction with fkh. Therefore, at least one other gene must exist that confines the competence to form CVM to these two regions. A good candidate for this gene is zinc finger homeodomain protein-1 (zfh-1). At the blastoderm stage, zfh-1 is expressed in high levels in the terminal regions of the mesoderm including the primordium of the CVM. zfh-1 is essential for the migration of the CVM: in zfh-1 mutant embryos, CVM-specific gene expression such as croc-lacZ is deleted. From the restricted effects of ectopic byn /fkh, it has been proposed that the two genes are capable of specifying CVM development only in the region of high zfh-1 expression. zfh-1, byn and fkh act in parallel downstream of tll. High levels of caudal zfh-1, as with byn and fkh, are dependent on tll, and there is no crossregulation between zfh-1, byn and fkh (Kusch, 1999).

The Drosophila tracheal system is a model for the study of the mechanisms that guide cell migration. The general conclusion from many studies is that migration of tracheal cells relies on directional cues provided by nearby cells. However, very little is known about which paths are followed by the migrating tracheal cells and what kind of interactions they establish to move in the appropriate direction. An analysis has been carried out of how tracheal cells migrate relative to their surroundings and which tissues participate in tracheal cell migration. Cells in different branches are found exploit different strategies for their migration; while some migrate through preexisting grooves, others make their way through homogeneous cell populations. Alternative migratory pathways of tracheal cells are associated with distinct subsets of mesodermal cells and a model is proposed for the allocation of groups of tracheal cells to different branches. These results show how adjacent tissues influence morphogenesis of the tracheal system and offer a model for understanding how organ formation is determined by its genetic program and by the surrounding topological constraints (Franch-Marro, 2000).

Tracheal cells are first specified as clusters of ectodermal cells at the embryonic surface. Since tracheal cells invaginate and form the tracheal pits they occupy the grooves between the muscle precursors of adjacent metameres. The formation of this groove is independent of tracheal invagination because it also forms between metameres that do not have tracheal placodes and it also develops in trh mutant embryos, which do not undergo tracheal invagination. A subset of the tracheal cells moves anteriorly, whereas another subset moves posteriorly until they reach the cells from the adjacent placodes. These cells will form the dorsal trunk, the most prominent tracheal branch that spans the embryo longitudinally. Those cells migrate across the adjacent precursors of somatic muscles and separate the precursors of the most dorsal muscles from the precursors of more ventral muscles. Other cells, those from the dorsal side of the tracheal pit, move dorsally along the longitudinal groove to form the dorsal branches that will end up fusing with the dorsal branches coming from the contralateral hemisegments. In the ventral side, the tracheal cells follow two different paths along the two clusters of lateral muscle precursors at each side of the groove. Anterior ventral cells will form the anterior lateral trunk while the posterior ventral cells will form the posterior lateral trunk. Finally, another group of cells from a midposition in the tracheal pit will migrate inward and will form the visceral branch (Franch-Marro, 2000).

The migrating cells of the dorsal trunk do not recognize any preexisting gap between the muscle precursor cells. Thus, the role of the lateral mesoderm in the migration of tracheal cells was studied. The lateral mesoderm, which comprises the fat body and the somatic gonadal precursors, is determined by the early functions of tinman (tin) and zinc-finger homeodomain protein-1 (zfh1). In embryos mutant for either zfh-1 or tin, the number of somatic gonadal and fat body precursors is reduced. Consistent with a role for the lateral mesoderm in the migration of the dorsal trunk cells, it was observed that the development of the dorsal trunk is impaired in these mutants. This is a nonautonomous effect since tin and zfh-1 are not expressed in the tracheal cells. The effect is quite mild, however. Since tin and zfh-1 have overlapping and partially redundant functions, mutant embryos for both genes were examined. In those embryos, the somatic gonadal mesoderm and the fat body precursors are virtually absent and the tracheal dorsal trunk is almost completely absent, but formation of the dorsal and ventral branches is not impaired. This defect is not due to a failure of bnl expression since it appears at the right position between the tracheal pits. (Franch-Marro, 2000).

Zfh1, a somatic motor neuron transcription factor, regulates axon exit from the CNS

Motor neurons are defined by their axon projections, which exit the CNS to innervate somatic or visceral musculature, yet remarkably little is known about how motor axons are programmed to exit the CNS. This study describes the role of the Drosophila Zfh1 transcription factor in promoting axon exit from the CNS. Zfh1 is detected in all embryonic somatic motor neurons, glia associated with the CNS surface and motor axons, and one identified interneuron. In zfh1 mutants, ventral projecting motor axons often stall at the edge of the CNS, failing to enter the muscle field, despite having normal motor neuron identity. Conversely, ectopic Zfh1 induces a subset of interneurons -- all normally expressing two or more 'ventral motor neuron transcription factors' (e.g., Islet, Hb9, Nkx6, Lim3) -- to project laterally and exit the CNS. It is concluded that Zfh1 is required for ventral motor axon exit from the CNS (Layden, 2006).

Zfh1 is a transcriptional repressor required for mesoderm development and cell migration. This study shows that Zfh1 regulates lateral axon growth and CNS exit in ventral-projection motor neurons, and that misexpression of Zfh1 can induce lateral axon projections and an interneuron to motor neuron fate transformation in cells competent to respond to ectopic Zfh1. In vertebrates, there are typically two Zfh1 homologs: ZFHX1A and ZFHX1B in humans, deltaEF1 and Sip1 (also called ZEB1 and ZEB2, respectively) in mouse, and Kheper in fish. These Zfh1 orthologs are required for proper muscle and CNS development. The worm homolog zag-1 is expressed in muscle, interneurons, and motor neurons where it regulates axon outgrowth. Thus, the Zfh1 family members appear to have a conserved role regulating CNS and mesoderm development (Layden, 2006).

Drosophila Zfh1 is detected in all embryonic somatic motor neurons, but not in the dMP2 visceral motor neuron. Thus, its expression correlates better with lateral axon projection or innervation of body wall muscles rather than simply motor neuron identity. Zfh1 is detected in at least one interneuron that does not project laterally out of the CNS, the Apterous AD interneuron, and thus Zfh1 is not sufficient to induce lateral axon projections (except in certain contexts as described below). Zfh1 is also expressed in about 20 neurons that are negative for the motor neuron marker pMad and the glial marker Repo. These cells could be interneurons or late-differentiating motor neurons, but due to the lack of axon projection markers, it has not been possible to determine the identity of these cells. Zfh1 is expressed in a subset of glia, including the exit glia that enwrap motor nerves and the surface glia. The association of Zfh1+ exit glia with Zfh1+ motor neurons is intriguing; no phenotype is available that suggests a role for Zfh1 glial function (Layden, 2006).

Initially Zfh1 was investigated as a candidate for regulating the difference between motor neurons and interneurons. Analysis of the zfh1 mutant phenotype clearly shows that it is not required to promote motor neuron identity or suppress interneuron identity. All known motor neuron markers (Eve, Islet, Hb9, Lim3, Nkx6, Late bloomer, and pMad) and interneuron markers (Apterous, Engrailed, and Dachshund) are normal in zfh1 mutant and/or misexpression embryos (Layden, 2006).

Instead, it was found that zfh1 mutants show a severe defect in lateral axon projections of the ventral nerves ISNb/SNa; axons typically extend laterally to the edge of the CNS before arresting. This explains why there are missing nerves (the axons do not leave the CNS) but a normal pattern of nuclear pMad in motor neurons (they are at the edge of the CNS where they may be exposed to BMP signals from ventral muscles). In contrast, the dorsal projecting ISN appears to exit the CNS normally (100%), but is often truncated and has pathfinding defects. Although mesodermal defects cannot be rule out as the origin of these phenotypes, there are several reasons to think that the ISN defects are due to loss of neuronal Zfh1. (1) grain mutant embryos show completely normal muscle development but fail to express Zfh1 specifically in the aCC motor neuron, and the aCC shows truncation and pathfinding defects that are qualitatively and quantitatively similar to the zfh1 mutant ISN phenotype. (2) The grain mutant phenotype can be partially rescued by zfh1 expression specifically in the aCC motor neuron. Thus, Zfh1 does not appear to be required for ISN motor axon exit from the CNS, but rather for later aspects of motor axon outgrowth and target recognition (Layden, 2006).

The fact that ISNb and SNa motor axons stall at the edge of the CNS raises the possibility that the phenotype is due to loss of Zfh1 in the exit glia. Exit glia express Zfh1, are intimately associated with motor axons as they exit the CNS, and regulate the site of motor axon exit from the CNS. However, no glial positioning or migration phenotypes were observed, and the motor axon exit phenotype can be rescued with neuronal Zfh1 expression but not glial Zfh1 expression. Moreover, the lateral exit glia are not required for motor axons to exit the CNS; the only motor neuron known to require a non-neuronal guidance cue to exit the CNS is the transverse nerve motor neuron, which requires a specialized pair of midline mesodermal cells to exit the CNS. It is concluded that the zfh1 mutant motor neuron exit phenotype is due to loss of zfh1 in motor neurons (Layden, 2006).

In addition to Zfh1, two other Drosophila transcription factors regulate motor axon exit from the CNS. nkx6 mutants show a 90% loss of ISNb projection out of the CNS, despite normal Zfh1 levels in the affected neurons. Thus, both Nkx6 and Zfh1 are required for motor axon outgrowth via the ISNb nerve. Similarly, eve mutants show 98% and 80% loss of aCC and RP2 motor axon projections out of the ISN nerve, respectively, but in these mutants, aCC/RP2 neurons have little or no Zfh1 expression. Thus, it is unclear if Eve alone is required for ISN axon outgrowth, or if Eve and Zfh1 function redundantly to promote ISN axon outgrowth (Layden, 2006).

It has been reported that the expression of the Drosophila Eph transmembrane receptor is restricted to interneuronal axons, and that RNAi knockdown of Eph levels leads to redirection of some interneuron axons laterally out of the CNS. It is tempting to speculate that Zfh1 keeps Eph levels low in motor neurons to allow them to exit the CNS. However, the situation is complicated by the recent finding that the Eph null mutant does not show abnormal interneuron projections out of the CNS, and thus the role of Eph in restricting interneuron axons to the CNS remains unclear (Layden, 2006).

Zfh1 is likely to have additional later functions in motor neurons that have not been addressed. Vertebrate Zfh1 homologs ZEB-1 (Smad interacting protein 1/Sip1) and ZEB-2 (deltaEF1) are known to bind pSmad, the nuclear effecter of the BMP signaling pathway. In flies, BMP signaling from the muscle field induces nuclear pMad in motor neurons. This raises the possibility that Zfh1/pMad may collaborate to regulate later aspects of motor neuron differentiation. Analysis of later aspects of zfh1 mutant phenotype in motor neurons (such as target recognition, synaptic branching, or formation of a functional synapse) is complicated by the fact that zfh1 mutants have reduced muscle numbers and size. Analysis of potential later functions of Zfh1 awaits methods for removing zfh1 expression from individual motor neurons, similar to recent elegant studies of Eve motor neuron function (Layden, 2006).

Ectopic Zfh1 does not induce all neurons to project axons laterally out of the CNS, but rather a modest thickening of the ISNb nerve and a slight increase in the number of pMad+ neurons, due in part to the aberrant projection of dMP2 and the EW1-EW3 interneurons. Why do just these interneurons respond to ectopic Zfh1? The EW1-EW3 and dMP2 neurons are the only neurons that express two or more 'motor neuron transcription factors' (Hb9, Islet, Nkx6, or Lim3) but do not project to the lateral body wall muscles. However, the fact that dual misexpression of Zfh1/Hb9, Zfh1/Nkx6, or Hb9/Nkx6 does not induce a detectable increase in pMad staining or lateral projections above that observed in any of the single misexpression experiments suggests that the combined activity of these motor neuron factors is not sufficient to drive axon exit from the CNS. There may be an additional, unknown positive factor present in the EW1–EW3 and dMP2 neurons that gives them competence to exit the CNS in response to Zfh1 misexpression; alternatively, most interneurons could express a negative factor preventing lateral axon projection that cannot be overcome by Zfh1 expression (Layden, 2006).

How does Zfh1 induce nuclear pMad expression and lateral axon outgrowth in the EW1-EW3 and dMP2 neurons? In the EW1-EW3 neurons, expression of a constitutively-activated Tkv type I BMP receptor does not induce nuclear pMad; thus, misexpression of Zfh1 in EW1-EW3 must induce competence to respond to the BMP ligand as well as promote lateral axon outgrowth. In contrast, the dMP2 visceral motor neuron is competent to respond to BMP signaling even without endogenous Zfh1 expression. Thus, misexpression of Zfh1 may only promote lateral axon outgrowth in dMP2 (Layden, 2006).

The results support a role for Zfh1 in regulating lateral exit of the ISNb motor neurons from the CNS. Both zfh1 and nkx6 mutants show a loss of ISNb motor axons in the muscle field, suggesting that both genes are required to promote proper lateral CNS exit of ISNb motor neurons. Because Zfh1 and Nkx6 do not require each other for expression in ventral projecting motor neurons (e.g. RP1, 3, 4, 5), these two genes must be independently activated but are likely to have common downstream genes to induce lateral axon outgrowth. It will be interesting to determine if common targets of Zfh1 and Nkx6 are involved in promoting lateral CNS exit and if they are required for lateral CNS exit in other motor neuron subtypes. One might predict that a gene that regulates lateral CNS exit in all motor neurons would be a target of Zfh1, Nkx6, and Eve. One potential candidate is unc5, which encodes a repulsive Netrin receptor that promotes cell and growth cone movement away from Netrin on the midline. Misexpression of Unc5 within the Drosophila CNS results in a subset of interneurons redirecting their axon projections laterally out of the CNS. However, misexpression of Unc-5, but not Zfh1, results in lateral displacement of the EW1–EW3 cell bodies, so it is unlikely that the Zfh1 EW1–EW3 phenotype is solely due to upregulation of Unc-5 expression. In addition, zfh1 mutant ISNb motor neurons initially project away from the midline, only stalling at the edge of the CNS, which is inconsistent with the phenotype being due to failure to express the Unc-5 receptor. In the future, it will be important to test directly whether Zfh1 regulates unc-5 directly or indirectly, as well as to identify novel direct targets regulated by Zfh1. Because eve and nkx6 mutants also show motor neuron axon exit defects, it will be especially interesting to compare direct targets of Eve, Nkx6, and Zfh1; common targets may help identify genes important for promoting motor neuron guidance out of the CNS in both flies and vertebrates (Layden, 2006).

Functional conservation of zinc-finger homeodomain gene zfh1/SIP1 in Drosophila heart development

Comparative genetic studies of diverse animal model systems have revealed that similar developmental mechanisms operate across the Metazoa. In many cases, the genes from one organism can functionally replace homologues in other phyla, a result consistent with a high degree of evolutionarily conserved gene function. Functional conservation was investigated between the Drosophila Zinc-finger homeodomain protein 1 (Zfh1) and its mouse functional homologue Smad-interacting protein 1 (SIP1). Northern blot analyses of SIP1 expression patterns detected three novel variants (8.3, 2.7, and 1.9 kb) in addition to the previously described 5.3 kb SIP1 transcript. The two shorter novel SIP1 transcripts were encountered only in developing embryos and both lacked zinc-finger clusters or homeodomain regions. The SIP1 transcripts showed complex embryonic expression patterns consistent with that observed for Drosophila Zfh1. They were highly expressed in the developing nervous systems and in a number of mesoderm-derived tissues including lungs, heart, developing myotomes, skeletal muscle, and visceral smooth muscle. The expression of the mammalian 5.3 kb SIP1 transcript in Drosophila zfh1 null mutant embryos completely restored normal heart development in the fly, demonstrating their functional equivalence in cardiogenic pathways. These data, together with the previously described heart defects associated with both SIP1 and Drosophila zfh1 mutations, solidify the conclusion that the Zfh1 family members participate in an evolutionary conserved program of metazoan cardiogenesis (Liu, 2006).

Without children is required for Stat-mediated zfh1 transcription and for germline stem cell differentiation

Tissue homeostasis is maintained by balancing stem cell self-renewal and differentiation. How surrounding cells support this process has not been entirely resolved. This study shows that the chromatin and telomere-binding factor Without children (Woc) is required for maintaining the association of escort cells (ECs) with germ cells in adult ovaries. This tight association is essential for germline stem cell (GSC) differentiation into cysts. Woc is also required in larval ovaries for the association of intermingled cells (ICs) with primordial germ cells. Reduction in the levels of two other proteins, Stat92E and its target Zfh1, produce phenotypes similar to woc in both larval and adult ovaries, suggesting a molecular connection between these three proteins. Antibody staining and RT-qPCR demonstrate that Zfh1 levels are increased in somatic cells that contact germ cells, and that Woc is required for a Stat92E-mediated upregulation of zfh1 transcription. These results further demonstrate that overexpression of Zfh1 in ECs can rescue GSC differentiation in woc-deficient ovaries. Thus, Zfh1 is a major Woc target in ECs. Stat signalling in niche cells has been previously shown to maintain GSCs non-autonomously. This study now shows that Stat92E also promotes GSC differentiation. The results highlight the Woc-Stat-Zfh1 module as promoting somatic encapsulation of germ cells throughout their development. Each somatic cell type can then provide the germline with the support it requires at that particular stage. Stat is thus a permissive factor, which explains its apparently opposite roles in GSC maintenance and differentiation (Maimon, 2014).


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Zn finger homeodomain 1: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 15 July 2015

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