The Interactive Fly
Genes involved in tissue and organ development
Gene expression during mesoderm development
Motoneurons regulate myoblast proliferation and patterning in Drosophila
Genetic control of the distinction between fat body and gonadal mesoderm
Role and regulation of starvation-induced autophagy in the Drosophila fat body
The two origins of hemocytes in Drosophila
The Drosophila lymph gland as a developmental model of hematopoiesis
Subdivision and developmental fate of the head mesoderm in Drosophila
Mononuclear muscle cells in Drosophila ovaries revealed by GFP protein traps
Please note: c, s, or v = expression in cardiac, somatic or visceral musculature; h = expression in head mesoderm, d = expression in dorsal medial cells, f = expression in fat body, h = expression in hemocytes
The transcription factor Twist initiates Drosophila mesoderm development, resulting in the formation of heart, somatic muscle, and other cell types. Using a Drosophila embryo sorter, enough homozygous twist mutant embryos were isolated to perform DNA microarray experiments. Transcription profiles of twist loss-of-function embryos, embryos with ubiquitous twist expression, and wild-type embryos were compared at different developmental stages. The results implicate hundreds of genes, many with vertebrate homologs, in stage-specific processes in mesoderm development. One such gene, gleeful/lame duck, related to the vertebrate Gli genes, is essential for somatic muscle development and is sufficient to cause neural cells to express a muscle marker (Furlong, 2001).
Formation of muscles during embryonic development is a complex process that requires coordinate actions of many genes. Somatic, visceral, and heart muscle are all derived from mesoderm progenitor cells. The Drosophila twist gene, which encodes a bHLH transcription factor, is essential for multiple steps of mesoderm development: invagination of mesoderm precursors during gastrulation, segmentation, and specification of muscle types. The role of twist in mesoderm development has been conserved during evolution, perhaps because it controls conserved regulatory mesoderm genes. For example, tinman and dMef2 are regulated by Twist in flies and are highly conserved in sequence and function in vertebrates (Furlong, 2001).
In Drosophila, somatic muscle forms from progenitor cells that divide to become muscle founder cells. Founder cells acquire unique identities controlled by transcription factors including Krüppel, S59, vestigial, and apterous. Each of the 30 body wall muscles in an abdominal hemisegment is initiated by a single founder cell and has unique attachments and innervations. To further clarify mechanisms underlying founder cell specification, myoblast fusion, and muscle patterning, Drosophila mutants together with microarrays of cDNA clones were used (Furlong, 2001).
twist mutant embryos develop no mesoderm. A population of mRNA species isolated from twist homozygous embryos was compared with that of stage-matched wild-type embryos. Drosophila lethal mutations are maintained as heterozygotes, in trans to balancer chromosomes. A twist mutation was established in trans to a balancer chromosome carrying a transgene encoding green fluorescent protein (GFP). Embryos were collected from wild-type and twist/GFP-balancer fly stocks at specific developmental stages. The twist/GFP-balancer collections contain a mixed population of embryos: one-quarter twist homozygotes lacking GFP, half heterozygotes with one copy of GFP, and one-quarter homozygous for the balancer chromosome with two copies of GFP. Homozygous twist mutant embryos were separated from their siblings using an embryo sorter. Putative homozygous twist embryos were assessed by immunostaining with an antibody to dMef2. More than 99% of the selected embryos had the twist phenotype (Furlong, 2001).
Three different periods of mesoderm development were analyzed: stages 9-10, 11, and 11-12 . During stage 9-10, the earliest time GFP is detectable in the balancer embryos, mesoderm cells migrate dorsally and become specified as somatic, visceral, cardiac, and fat body mesoderm. twist and its direct targets tinman and dMef2 are expressed throughout stage 9-10 mesoderm. The middle period contains stage 11 embryos and is a transition between the first period (stage 9-10) and the third period (late stage 11-12). During late stage 11 and stage 12, myoblast fusion begins and twist expression remains prominent in only a subset of the somatic muscle cells (Furlong, 2001).
For each developmental period, three independent embryo collections, embryo sortings, and microarray hybridizations were conducted. The microarrays used for the analysis contained over 8500 cDNAs corresponding to 5081 unique genes plus a variety of controls. Each embryonic RNA sample was compared with a reference sample, containing RNA made from all stages of the Drosophila life cycle to allow direct comparisons among all the experiments (Furlong, 2001).
To determine how transcription was affected by the twist mutation, SAM (significance analysis of microarrays) analysis was used. Genes that are normally highly expressed in mesoderm should have lower transcript levels in twist homozygotes. Genes in other tissues whose expression depends on signals from the mesoderm might also have reduced expression. Transcripts of 130 genes, the 'Twist-low' group, were significantly lower in twist mutants than in wild type. Conversely, cells that would have formed mesoderm may take on other fates in the absence of twist, such as neuroectoderm; therefore, many transcript levels could increase in twist mutants. Genes whose transcription is repressed by signals from the mesoderm would also be enriched in twist mutants. One hundred fifty genes, called the 'Twist-high' group, have increased levels of RNA in twist mutant embryos (Furlong, 2001).
In total, 280 of ~5000 genes had significant changes in transcript levels, with 10 false positives. The genes on the array include 15 previously characterized mesoderm-specific genes, all of which are significantly reduced in twist mutant embryos. The arrays also contain genes known to be transcribed in both mesoderm and other cell types. Significant changes in expression were detected for many of these genes (Furlong, 2001).
The 130 Twist-low genes were divided into three groups with similar trends of expression by a self-organizing map (SOM) clustering program. The 24 group A genes, which include tinman, dMef2, and bagpipe, have reduced transcript levels in twist mutants at all developmental stages assayed. Most of the Twist-low genes fall into the B and C groups. The 62 group B 'early genes' encode transcripts with reduced levels of expression in twist mutants only during stages 9-10, not later. One member of group B, stumps (dof/hbr) is essential for mesoderm cell migration. stumps RNA is abundant in the mesoderm at stages 9-10 and is strongly reduced by stage 11. At stage 11, stumps RNA accumulates in trachea, which is largely unaffected in twist mutants (Furlong, 2001).
The 44 group C genes have reduced transcript levels in twist mutant embryos only during late stage 11 and stage 12. These 'lategenes' include blown fuse, a gene essential for myoblast fusion; delilah, a gene required for somatic muscle attachment, and genes such as kettin, which is required to form contractile muscle. Given the predominantly early expression of twist, the early genes in groups A and B are the best candidates for direct transcription targets of Twist, though some indirectly activated genes may be present within these groups. Group C late genes are likely to be regulated by products of genes that are activated by Twist (Furlong, 2001).
In situ hybridizations were done using a previously uncharacterized representative of each Twist-low group. In each case, the hybridization pattern was consistent with the predicted time of transcription. A group A gene, CG15015 (GH16741), is transcribed in somatic muscle throughout stages 9-12. A group B gene, CG12177 (GH22706), is transcribed during early mesoderm development, but not later. CG14848 (GH21860), a group C gene, is expressed in the stomodeum but not the mesoderm during stages 9-10. Its mesoderm expression initiates during stage 11, the latest period of the twist experiment. Thus, combining loss-of-function mutant embryo analysis with staged embryo collections provides gene expression information for both tissue specificity and temporal expression (Furlong, 2001).
The mis-expression of twist in the ectoderm is sufficient to convert both neuronal and epidermal tissues to a myogenic cell fate. RNA from embryos with ubiquitous twist expression was used to evaluate the ability of Twist to initiate mesoderm-like gene expression in cells that would normally form other tissue types. Genes whose transcript levels decrease in twist loss-of-function embryos and increase when twist is ubiquitous are excellent candidates for regulators of mesoderm development or differentiation (Furlong, 2001).
To ectopically express twist, a dominant gain-of-function mutation of the maternal gene Toll (Toll10B) was used. Activated Toll induces the expression of twist and snail in early embryos and of immune response genes in older embryos. Thus, the effects of Toll10B on gene expression reflect the activities of Twist as well as Snail and Dorsal, or their combined actions. Toll10B embryos are essentially bags of mesoderm; epidermal structures are absent or greatly reduced, and they have been used successfully in subtractive hybridization screens to identify mesoderm genes. The gene transcription profile of Toll10B embryos was compared with that of wild-type embryos during four periods of development, using the reference sample to normalize experiments. The earliest period, stage 5, is when twist is initially expressed in presumptive mesoderm. The other three periods analyzed were those used in the twist mutant analysis: stages 9-10, 11, and 11-12 (Furlong, 2001).
In Toll10B embryos, 447 genes had significant changes in RNA levels compared with stage-matched wild-type embryos, 16 of which are predicted to be false positives. Transcripts from 166 genes were reduced in Toll10B embryos compared with wild type. These genes may be involved in neuroectoderm events that are blocked when cells are turned into mesoderm. Transcripts of 281 'Toll-high' genes were increased in Toll10B embryos. Of the 21 previously characterized mesoderm-specific genes on the arrays, 18 have significantly increased transcript levels in Toll10B embryos. The remainder may require activators other than Toll, such as signals from the severely altered ectoderm (Furlong, 2001).
Genes with altered transcription in twist and Toll10B mutants were analyzed with a hierarchical clustering program to identify similar transcription profiles. The genes were divided into putative 'mesoderm' and 'non-mesoderm' groups. Non-mesoderm genes were defined as having increased transcript levels in mesoderm-deficient (twist) embryos and/or decreased expression in mesoderm-enriched (Toll10B) embryos. Mesoderm genes were defined as having decreased transcript levels in twist mutants and/or increased transcripts in Toll10B mutants. The mesoderm genes group would also contain genes expressed in other tissues in a mesoderm-dependent manner (Furlong, 2001).
Non-mesoderm genes in clusters B and C are repressed in Toll10B mutants. Cluster B genes have increased RNA levels in twist mutant embryos, whereas cluster C genes do not change significantly. The overexpression of twist in the presumptive ectoderm in Toll10B embryos results in a conversion of ectodermal cell fate into mesoderm. snail and dorsal are ectopically expressed in Toll10B embryos and transcriptionally repress the expression of neuroectoderm and ectoderm genes. The conversion of ectoderm to mesoderm due to twist misexpression, and the ability of Snail and Dorsal to repress ectoderm genes, suggests that the B and C clusters should contain primarily neuroectodermal genes. Indeed, the non-mesoderm genes include 31 previously characterized neuroectodermal genes. One previously unknown cluster B gene that encodes a putative cell adhesion protein is transcribed in the ventral nerve cord. Another previously unknown gene within cluster C is transcribed within the developing brain (Furlong, 2001).
The Twist-low and Toll-high genes have in common 51 genes that are highly likely to be involved mesoderm development. For example, transcription of the genes in cluster D is reduced in twist mutants and increased in Toll10B mutants during most or all time periods (Furlong, 2001).
A complete overlap between Twist-low and Toll-high gene sets is not expected for three reasons: (1) development of dorsal mesoderm, and of muscle founder cells marked by apterous and connectin, requires the Decapentaplegic signal and perhaps others. Changed characteristics of cells that form ectoderm in Toll10B embryos interfere with these signaling events. A significant reduction is observed in dpp RNA levels in Toll10B embryos. (2) During midgut development, endoderm cells migrate along the mesoderm. Midgut endoderm development is affected in twist mutants. Some Twist-low genes with unchanged expression in Toll10B embryos are transcribed in the midgut. (3) Ectopic Twist inhibits visceral mesoderm and heart development and promotes excess somatic muscle development. Toll10B embryos produce high levels of Twist throughout the embryo, so genes that have reduced RNA levels in both twist and Toll10B mutant embryos are likely to be visceral muscle and heart genes. Indeed, bagpipe and connectin, genes expressed in visceral mesoderm, are among the 79 Twist-low genes not induced by ectopic Twist (Furlong, 2001).
Of the 281 Toll-high genes, 230 are unaffected in twist mutants. Some of the 230 are normally expressed late in embryogenesis in wild-type embryos but are expressed prematurely in Toll10B embryos due to ectopic Twist. These include Myo61F, MSP-300, and Paramyosin, genes normally active in terminally differentiated muscle (stage 16). Ectopic Snail and Dorsal in Toll10B embryos may activate genes that are unaffected in twist mutants. Snail can repress neuroectodermal genes and may also activate mesoderm genes. Dorsal activates immune response genes later in development. relish, drosomycin, and metchnikowin genes -- all immuneresponse genes -- have higher transcript levels in Toll10B embryos (Furlong, 2001).
Data from loss- and gain-of-function experiments, combined with careful staging, yield a useful picture of genes that are likely to be required for mesoderm specification and muscle differentiation. Of 360 identified mesoderm genes, 273 have not been the focus of developmental studies. The predicted proteins encode transcription factors, signal transduction molecules, kinases, and pioneer proteins. The stage at which each gene is active is one criterion for assigning possible functions. Another key criterion will be finding a mutant phenotype. As a pilot, this additional step was undertaken for the gene CG4677 (LD47926). Changes in CG4677 transcript levels were also observed in a Toll10B subtractive hybridization screen (Furlong, 2001).
CG4677 is transcribed in the visceral mesoderm at stages 10-13 and the somatic mesoderm during stages 11-13. This gene encodes a C2H2 zinc finger transcription factor with high sequence similarity to vertebrate Gli proteins: the gene has been named gleeful (gfl). Mammalian Gli proteins act downstream of Hedgehog signaling proteins to control target gene transcription (Furlong, 2001).
The role of gfl in mesoderm development was assessed by disrupting its function using RNA interference. Injection of a double-stranded RNA (dsRNA) control sequence had no effect on mesoderm development. In contrast, gfl dsRNA injection causes severe loss and disorganization of somatic muscle cells, whereas heart and visceral muscle are unaffected. A similar phenotype is seen in Df(3R)hh homozygous embryos: the deficiency removes gfl but not the nearby hedgehog gene (Furlong, 2001).
To determine whether gfl can induce muscle cell development, a UAS-gfl transgenic fly strain was generated. Ectopic expression of gfl using an en-GAL4 driver results in lethality and induction of ectopic dMEF2 expression in the ventral nerve cord. Remarkably, Gfl is sufficient to induce expression of a muscle gene in neuronal cells. Previous studies have shown an essential role for Sonic hedgehog signaling in the formation of slow muscle in avian and zebrafish embryos. gfl may be performing a similar role in Drosophila somatic muscle development (Furlong, 2001).
Drosophila muscles originate from the fusion of two types of myoblasts -- founder cells (FCs) and fusion-competent myoblasts (FCMs). To better understand muscle diversity and morphogenesis, a large-scale gene expression analysis was performed to identify genes differentially expressed in FCs and FCMs. Embryos derived from Toll10b mutants were employed to obtain primarily muscle-forming mesoderm, and activated forms of Ras or Notch were expressed to induce FC or FCM fate, respectively. The transcripts present in embryos of each genotype were compared by hybridization to cDNA microarrays. Among the 83 genes differentially expressed, genes known to be enriched in FCs or FCMs, such as heartless or hibris, previously characterized genes with unknown roles in muscle development, and predicted genes of unknown function, were found. These studies of newly identified genes revealed new patterns of gene expression restricted to one of the two types of myoblasts, and also striking muscle phenotypes. Whereas genes such as phyllopod play a crucial role during specification of particular muscles, others such as tartan are necessary for normal muscle morphogenesis (Artero, 2003).
The Toll10b mutation gives rise to embryos composed primarily of somatic mesoderm. In these embryos FCs and FCMs are readily detected, and they respond to the Ras and Notch signaling pathways in the same way as their wild-type counterparts. Advantage was taken of this fact to enrich Toll10b mutant embryos for FCs or FCMs, which allowed a concentration on the transcription in these two specific cell types within the context of the entire embryo. Genes known to be expressed and regulated in FCs or FCMs emerged from the screen in the proper categories. Not all known FC/FCM genes were detected in the screen for several reasons: the high stringency set for interpretation of the array data; the presence of only about one-third of the genome on the arrays; the loss of Dpp in the Toll10b background, and the specific window of myogenesis (5- to 9-hours) that was the focus of this investigation. However, a plethora of potential new muscle regulators were uncovered, including known genes with no previously recognized function in the mesoderm (such as phyl and asteroid), and genes predicted from the Drosophila genome sequence but not previously analyzed (Artero, 2003).
Various tests were applied to ascertain the validity of the results. Available databases were analyzed to find evidence that the known and predicted genes were expressed at the correct time and place. In addition, Northern analysis with eleven genes tested the reliability of the microarray detection and selection criteria; the results from all genes tested agreed with the array data (Artero, 2003).
A Toll10b sample on the Northern blots allowed ascertainment of why a gene is enriched in a particular condition. For example, in the case of FC enriched genes, the signal in the Ras and Notch lanes can be compared with Toll10b alone to determine whether the Ras/Notch ratio for a gene is due to activation by Ras or repression by Notch. Those genes that are 'enriched under Notch conditions', for example, could reflect a variety of transcription mechanisms that would result in a ratio of less than 0.6. By Northern analysis, many of the 'Notch-regulated' genes, and hence the predicted FCM genes, were found to be repressed by Ras signaling and slightly activated by Notch. As a case in point, hibris is induced by Notch (2-fold) and repressed by Ras (10-fold), both by Northern analysis and by in situ hybridization in embryos (Artero, 2003).
A combination of in situ hybridization, immunostaining and confocal microscopy was used to verify that the differential expression changes that were observed in these overexpression embryos reflected true differential expression in the wild-type situation. The expression of nine genes from different functional categories was analyzed. For seven of these, expression was detected in the predicted type of myoblast. For two, asteroid (ast) and cadmus, no specific staining in embryos was detected by in situ hybridization. For those genes that fell into the category of 'specific role in muscle development uncertain', in situ hybridization of several (28%) showed expression in tissues other than somatic mesoderm that are present in the Toll10b background. These genes changed their expression levels in response to Ras or Notch, and may be Ras and Notch targets in non-mesodermal tissues (Artero, 2003).
The most stringent test, mutational analysis, was applied to a set of genes for which mutations are available. Preliminary analyses of another four FCM-enriched genes was carried out: Elongation factor Tu mitochondrial (EfTuM), Glutamine synthetase 1, cadmus and parcas. All four mutants have muscle defects, including muscle losses and aberrant muscle morphologies. Thus all the genes tested show some muscle defect, supporting the usefulness of the genetic and genomic approach (Artero, 2003).
Taken together, these data suggest that the majority of genes detected play important roles in FCs or FCMs during muscle development. Some of these genes might not have been found in traditional forward genetic screens because of partial or complete genetic redundancy. The data complement traditional forward genetic approaches for finding genes crucial for muscle morphogenesis (Artero, 2003).
Each of the thirty FCs per abdominal hemisegment is hypothesized to produce its own unique combination of transcriptional regulators, though the evidence for this is limited. In turn the combination of regulators would control the morphology of the final muscle. Although several transcriptional regulators have been linked to FC identity, the molecular description is far from complete. This screen contributed two more FC-specific genes. Previously known markers, such as slouch or eve, once induced in the muscle FC, are maintained throughout the remainder of development. Ubx, which emerged from this screen, is a similarly simple case, as its transcripts are steadily present in most FCs. By contrast, more complex patterns of gene expression have been identified in FCs, such as the transient transcription of asense in a subset of FCs. The subsequent transcriptional inactivation of asense may underlie temporal changes in cell properties (Artero, 2003).
Even less is known about transcriptional regulators controlling FCM differentiation. Only one gene, lame duck, has been shown to have a role in FCMs. This screen has uncovered three more potential players: delilah, E(spl)mß and CG4136, confirming that FCMs follow their own, distinct, myogenic program. Discovering what aspects of FCM biology are controlled by these transcriptional regulators awaits analysis of the loss-of-function phenotypes (Artero, 2003).
Notch and Ras signaling pathways interact during muscle progenitor segregation. The results suggest that phyl and polychaetoid (pyd) may be additional links between the two signaling pathways in FCs. phyl and pyd both interact genetically with Notch and Delta. The transcription of phyl, which promotes neural differentiation, is negatively regulated by Notch signaling during specification of SOPs and their progeny. This study shows a similar regulation in muscle cells, where Notch signaling represses phyl expression and Ras signaling increases phyl expression. Likewise, in the nervous system, the segregation of SOPs requires pyd, a Ras target gene, to negatively regulate ac-sc complex expression. Similarly, Pyd may restrict the muscle progenitor fate to a single cell, perhaps by regulating lethal of scute transcription. Thus, Pyd would collaborate with Notch signaling to restrict muscle progenitor fate to one cell (Artero, 2003).
FCMs appear to integrate Ras and Notch signaling differently. Two genes whose transcripts were enriched under activated Notch conditions, parcas and asteroid (ast), have been implicated in Ras signaling in other tissues, directly (ast) or indirectly (parcas). These data are suggestive of a role for Ras signaling in the FCMs, in addition to its role in FC specification. In addition, Notch signaling to FCMs may prime cells for subsequent Ras signaling during muscle morphogenesis, much as occurs in FCs where Ras signaling primes the cell for subsequent Notch signaling during asymmetric division of the muscle progenitor (Artero, 2003).
Embryos that lack or ectopically express phyl have morphological defects in specific muscles, for example, in LL1 and DO4 in response to diminished phyl function, and in DT1 and LT4 in response to increased phyl function. The morphological defects in the loss-of-function embryos appear to be due to a failure to specify particular FCs, a conclusion that is based upon missing or abnormal production of the FC marker Kr. In eye development and SOP specification, Phyl directs degradation of the transcriptional repressor Tramtrack. In a subset of the primordial muscle cells, Phyl may work similarly, targeting Tramtrack for degradation. The presence of Tramtrack would contribute to the specific identity program of the muscle. Since Tramtrack is expressed in the mesoderm, this possibility is likely. Alternatively, Phyl may be required for targeted degradation of some other protein in a subset of FCs. The molecular partner for Phyl during muscle differentiation is unknown, although preliminary data suggest that sina is also expressed in somatic mesoderm and thus may be its partner. These studies have identified a new role for Phyl in muscle progenitor specification and suggest the importance of targeted ubiquitination for proper muscle patterning (Artero, 2003).
A role for ubiquitination in muscle differentiation is further reinforced by the identification of the RING finger-containing protein Goliath (Gol), induced by activated Notch conditions, and CG17492, induced by activated Ras conditions. Several RING-containing proteins function as E3 ubiquitin ligases, with the ligase activity mapping to the RING motif itself. Ligase function has been experimentally confirmed for the Gol ortholog GREUL1 in Xenopus. Thus, targeted protein degradation during muscle morphogenesis could serve a host of crucial functions, such as protein turnover, vesicle sorting, transcription factor activation and signal degradation (Artero, 2003).
The simplest view of the 'founder cell' hypothesis is that each FC contains all the information for the development of a particular muscle. By contrast, FCMs have been seen as a naïve group of myoblasts, entrained to a particular muscle program upon fusion to the FC. This work indicates that these two groups of myoblasts have distinct transcriptional profiles. These data raise the possibility of a greater role for FCMs in determining the final morphology of the muscle and emphasize a need to characterize fully those FCM genes. For example, this screen identified a protein kinase of the SR splice site selector factors (SRPK) whose transcripts are enriched in FCMs, suggesting that regulation of the splicing machinery is important for muscle morphogenesis. The Mhc gene undergoes spatially and temporally regulated alternative splicing in body wall muscles conferring different physiological properties on these muscles. This FCM-specific expression of SRPK may indicate that the production of a particular Mhc isoform is regulated by the FCMs that contribute to that muscle, rather than by the particular FC that seeds the muscle. In addition, a number of observations suggest that FCMs may be a diverse population of myoblasts, with different subsets having different potential to contribute to the final muscle pattern. For example, hbs expression suggests that only a subset of FCMs express the gene, and twist expression in lame duck mutant embryos persists in a subset of FCMs. This study provides additional genes for exploring whether FCMs are a heterogeneous population of myoblasts as well as determining the nature of FCM contribution to the final muscle (Artero, 2003).
The molecular events underlying complex morphological changes, such as migration, cell fusion, cell shape changes or changes in the physiology of a cell, require a rich and dynamic program of transcription changes. This study has described approximately one-third of this transcriptional profile. The FC- or FCM-specific transcription of seven genes, and the mutant phenotype of four selected genes, allowed the definition of new muscle mutations that specifically affect the morphological traits of a subset of muscles (Artero, 2003).
Motoneurons directly influence the differentiation of muscle fibers, regulating features such as muscle fiber type and receptor development. Less well understood is whether motoneurons direct earlier events, such as the patterning of the musculature. In Drosophila, the denervation of indirect flight muscles results in a diminished myoblast population and smaller or missing muscle fibers. Whether the neuron-dependent control of myoblast number is due to regulation of cell division, motoneuron-dependent apoptosis, or nerve-dependent localization and migration of myoblasts, was examined. Denervation results in a reduced rate of cell division, as revealed by BrDU incorporation. There is no change in the frequency of apoptotic myoblasts following denervation. Using time lapse imaging of GFP-expressing myoblasts in vivo in pupae, it was observed that despite denervation, the migration and localization of myoblasts remains unchanged. In addition to reducing myoblast proliferation, denervation also alters the segregation of myoblasts into the de novo arising dorso-ventral muscles (DVMs). To address this effect on muscle patterning, the expression of the founder-cell marker Dumbfounded/Kirre (Duf) in imaginal pioneer cells was examined. There is a strong correspondence between cells that express Dumbfounded/Kirre and the number of DVM fibers, consistent with a role for these cells in establishing adult muscles. In the absence of innervation the Duf-positive cells are no longer detected, and muscle patterning is severely disrupted. These results support a model where specialized founder cells prefigure the adult muscle fibers under the control of the nervous system (Fernandes, 2005).
The motoneuron exerts a mitogenic influence on IFM myoblasts. Following unilateral denervation, the BrDU birthdating experiments revealed a significant decline in the rate of proliferation. This decline is likely sufficient to account for the reduced myoblast population observed in denervated hemisegments (previously quantified by morphometry; Fernandes, 1998). The smaller muscles that result are thus likely due to the smaller number of available myoblasts, and possibly to the absence of neuromuscular excitation following denervation (Fernandes, 2005).
Two alternate mechanisms were ruled out: there was no evidence for a change in the rate of myoblast cell death following denervation, indicating that the motoneuron does not provide an essential survival factor for the cells. Also, no significant change was observed in the migratory behavior of myoblasts following unilateral denervation, when examined at either the single cell or population level. This indicates that the reduced population size was not the result of myoblast emigration from denervated sites (Fernandes, 2005).
The second major effect of denervation was the gross disruption of normal muscle formation in the de novo arising DVMs. Normally, myoblasts coalesced into discrete primordia that prefigure the three sets of DVM muscle fibers. When denervated, the DVM myoblasts remain unpatterned, and the muscles fail to form. This may be due to a direct effect of the motoneuron on the myoblasts. However, denervation also disrupts the behavior of a potential intermediary player, the imaginal pioneer cells, that are thought to prefigure the DVM fibers as myoblast fusion targets (Fernandes, 2005).
BrDU birthdating experiments reveal a significant rise in the rate of DLM myoblast proliferation that normally occurs at 18-24 APF. In denervated regions, myoblast proliferation remains unchanged, holding steady at the earlier, basal level seen prior to the onset of myoblast fusion (at 12-16 h APF). Thus, DLM myoblast proliferation involves two components: a basal and nerve-independent phase (at 12-16 h APF) and a later incremental nerve-dependent phase (at 18-24 h APF). It is proposed that the nerve-dependent increase in myoblast proliferation regulates the number of myoblasts available for fusion, and thus is a way for motoneurons to control muscle size (Fernandes, 2005).
The nerve-dependent rise in DLM myoblast proliferation correlates with the expansion of motoneuronal terminal arbors on the muscle fiber surface. While it is possible that the expansion of motoneuron terminals and the change in myoblast proliferation are independent responses to exogenous hormone signals, the data argue that motoneurons strongly influence myoblast cell division, since proliferation is reduced following denervation. It is proposed that the growing motoneuron terminal either releases a factor that influences myoblast cell division, or alternatively potentiates myoblast responsiveness to available growth factors and mitogens. In either case, the nerve-dependent control of myoblast proliferation would in turn influence the growth of the muscle fiber (Fernandes, 2005).
The rise in myoblast proliferation observed during the nerve-dependent phase of DLM development resembles a feature seen during photoreceptor development in the Drosophila eye. During differentiation of the neuroepithelium, there is a rise in cell proliferation, referred to as the second mitotic wave (SMW), which produces additional precursors that are recruited to eventually form a complete ommatidium. It is likely that the rise in DLM myoblast proliferation similarly serves to maintain the size of the myoblast pool, so that cells can be continuously drawn from the pool until the desired muscle size is achieved (Fernandes, 2005).
That the two phases of DLM myogenesis differ in their nerve-dependence resembles events associated with vertebrate myogenesis. Mammalian skeletal muscles form in two waves: primary myotubes form first, and serve as scaffolds for secondary myotube formation. Primary myogenesis is independent of innervation, while secondary myogenesis is nerve dependent. This is due to the presence of a nerve-dependent population of myoblasts essential for secondary myotube formation (Fernandes, 2005).
The DVMs develop from the de novo fusion of myoblasts, and critically depend on the motoneuron for muscle fiber formation (Fernandes, 1998 and Fernandes, 1999). Denervation also results in a failure of myoblasts to segregate into distinct DVM primordia. Myoblast patterning and fiber development for the DVMs have been proposed to depend on specialized imaginal pioneer cells. The 'imaginal pioneer' (IP) cells lie in close association with motoneuron arbors, as demonstrated by EM analysis, and are thus potentially dependent on neurons for their normal function or survival. The IP cells are thought to serve as myoblast fusion targets, and thus to prefigure the mature muscle fibers. There have been, however, no reported molecular markers for these cells (Fernandes, 2005).
In the Drosophila embryo the mesodermal cells that generate the somatic muscles are critically dependent on specialized founder cells. Each embryonic founder cell is the precursor of a specific muscle fiber, and is the target of fusion-competent myoblasts. The founder cells each express Dumbfounded/Kirre, a key component of the cell fusion machinery. In loss of function duf mutations myoblast fusion is disrupted (Fernandes, 2005).
Intriguingly, it was found that there are Duf-positive cells within the DVM myoblast pool. Their location, number, and size indicate that they are likely to be the IP cells previously described. The Duf-positive cells are present in the DVM I and II primordia in direct correspondence to the final number of DVM fibers, as is the case for the IP cells. Duf-positive cells have also been reported for other pupal muscles, and a correlation exists between Duf-positive cells and the numbers of both IFM and abdominal muscle fibers. Significantly, it was found that denervation affects the Duf cells of the DVM primordia. Although Duf-positive cells are initially present in the denervated hemisegments in the normal pattern and number (at 12 h APF), following denervation they are no longer reliably observed by 18-20 h APF. By 24 h APF, when control hemisegments possess well patterned Duf-positive DVMs, Duf-positive cells on the denervated side are rarely observed (Fernandes, 2005).
These observations support a model where Duf-positive IP cells in the pupa serve as fusion targets of myoblasts, as is the case for the Duf-positive founder cells in the embryo. Since the Duf molecule is an essential component of the cell fusion machinery, its disappearance following denervation suggests that fusion events are severely disrupted and may explain the associated muscle patterning defects. It cannot as yet be determine whether the loss of Duf labeling is due to a loss of Duf expression in the IP cells, or due to apoptosis. Distinguishing between these scenarios will require in situ time lapse imaging of vitally labeled IP cells in denervated hemisegments (Fernandes, 2005).
DLM fibers arise from larval muscles that persist into the pupal stage. Like all embryonically established somatic muscle fibers, the persistent larval fibers also do not depend on the motoneuron for their formation or maintenance. When denervated, the larval fibers persist and DLM fibers still form, albeit at a slower rate (Fernandes, 1998; Fernandes. 1999). Duf expression is also detected in the persistent larval fibers, consistent with the fact that they function as myoblast fusion targets. However, unlike the DVMs, denervation does not result in a loss of Duf expression in the developing DLMs. The reason for this independence remains uncharacterized, but likely reflects the distinct origin of these cells from larval precursor muscles (Fernandes, 2005).
A dependence on motoneurons for the regulation of muscle size and patterning has been observed for several insect systems. When abdominal Drosophila muscles are denervated, the adult fibers are significantly reduced in mass. The most prominent effect involves the male-specific muscle (MSM) of the fifth abdominal segment of the adult. This muscle is larger than other body wall muscle fibers, a difference attributed to the enhanced recruitment of myoblasts from a common myoblast pool. When the abdominal myoblast pool is reduced experimentally through hydroxyurea treatment, a smaller muscle is present at the MSM location in segment A5. A BrDU labeling analysis remains to be performed to confirm the role of myoblast proliferation on MSM development (Fernandes, 2005).
Denervation studies in Manduca have similarly shown that proliferation of myonuclei is reduced in leg, abdominal, and DLM muscles. At the onset of metamorphosis, muscle precursors appear in the region of the future adult muscles and become associated with tendons (leg muscles) or persistent larval muscles (DLM). This accumulation is then followed by the appearance of proliferating 'myonuclei' within the developing primordia. By contrast, in the case of Drosophila DLMs, BrDU incorporation is restricted to myoblasts present outside the primordia, and there is no evidence of nuclear division within the muscle fibers (Fernandes, 2005).
In conclusion, it is proposed that the motoneuron critically influences the size of the myoblast pool through a direct effect on myoblast cell division, and that this helps regulate the final size of adult muscle fibers. The motoneuron has a second role in regulating the development of de novo forming fibers, where it is essential for the partitioning of myoblasts into muscle primordia. Moreover, continued Duf labeling within the primordia depends on the motoneuron's presence. Thus, the motoneuron influences both the number of cells available for fusion, as well as potentially regulates the fusion events themselves. This is an elegant mechanism for controlling muscle fiber differentiation during myogenesis, and may have evolved as a way to ensure that muscle primordia develop into muscles that meet the diverse demands placed on them by the nervous system (Fernandes, 2005).
The somatic muscles, the heart, the fat body, the somatic part of the gonad and most of the visceral muscles are derived from a series of segmentally repeated primordia in the Drosophila mesoderm. This work describes the early development of the fat body and its relationship to the gonadal mesoderm, as well as the genetic control of the development of these tissues. The first sign of fat body development is the expression of serpent in segmentally repeated clusters within the trunk mesoderm in parasegments 4-9. Segmentation and dorsoventral patterning genes define three regions in each parasegment in which fat body precursors can develop. The primary and secondary dorsolateral fat body primordia are formed ventral to the visceral muscle primoridium in each parasegment. The ventral secondary cluster forms more ventrally in the posterior portion of each parasegment. Fat body progenitors in these regions are specified by different genetic pathways. Two dorsolateral regions require engrailed and hedgehog (within the even-skipped domain) for their development while the ventral secondary cluster is controlled by wingless. Ubiquitous mesodermal en expression leads to an expansion of the primary clusters into the sloppy-paired domain, resulting in a continuous band of serpent-expressing cells in parasegments 4-9. The observed effect of en on fat body development is seen not only on mesodermal overexpression but also when en is overexpressed in the ectoderm. Loss of wingless leads to an expansion of the dorsolateral fat body primordium. decapentaplegic and one or more unknown genes determine the dorsoventral extent of these regions. High levels of Dpp repress serpent, resulting in the formation of visceral musculature, an alternative cell fate (Reichmann, 1998).
In each of parasegments 10-12 one of these primary dorsolateral regions generates somatic gonadal precursors instead of fat body. The balance between fat body and somatic gonadal fate in these serially homologous cell clusters is controlled by at least five genes. A model is suggested in which tinman, engrailed and wingless are necessary to permit somatic gonadal develoment, while serpent counteracts the effects of these genes and promotes fat body development. In wg mutant embryos, all dorsolateral mesodermal cells, including those in parasegments 10-12, acquire fat body fate. This phenotype can be interpreted as the combined effects of two separate functions of wg: (1) wg is necessary to repress fat body development in the dorsolateral mesoderm underlying the wg domain in all parasegments; (2) wg is required in the primary cluster to permit somatic gonadal precursor instead of fat body development in parasegments 10-12. Loss of engrailed results in the absence of demonstrable somatic gonadal precursors, similar to the situation in tinman mutants. Ubiquitous mesodermal en expression leads to the formation of additional somatic gonadal precursor cells in parasegments 10-12. The homeotic gene abdominalA limits the region of serpent activity by interfering in a mutually repressive feed back loop between gonadal and fat body development. It is unlikely that abdA represses srp directly, since srp can be expressed in cells in which abdA is active. abdA might prevent srp from inhibition of a somatic gonadal precursor competence factor (Riechmann, 1998).
In response to starvation, eukaryotic cells recover nutrients through autophagy, a lysosomal-mediated process of cytoplasmic degradation. Autophagy is known to be inhibited by TOR signaling, but the mechanisms of autophagy regulation and its role in TOR-mediated cell growth are unclear. Signaling through TOR and its upstream regulators PI3K and Rheb is necessary and sufficient to suppress starvation-induced autophagy in the Drosophila fat body. In contrast, TOR's downstream effector S6K promotes rather than suppresses autophagy, suggesting S6K downregulation may limit autophagy during extended starvation. Despite the catabolic potential of autophagy, disruption of conserved components of the autophagic machinery, including ATG1 and ATG5, does not restore growth to TOR mutant cells. Instead, inhibition of autophagy enhances TOR mutant phenotypes, including reduced cell size, growth rate, and survival. Thus, in cells lacking TOR, autophagy plays a protective role that is dominant over its potential role as a growth suppressor (Scott, 2004).
Autophagy likely evolved in single-cell eukaryotes to provide an energy and nutrient source allowing temporary survival of starvation. In yeast, Tor1 and Tor2 act as direct links between nutrient conditions and cell metabolism. These proteins sense nutritional status by an unknown mechanism, and effect a variety of starvation responses including changes in transcriptional and translational programs, nutrient import, protein and mRNA stability, cell cycle arrest, and induction of autophagy. Autophagy thus occurs in the context of a comprehensive reorganization of cellular activities aimed at surviving low nutrient levels (Scott, 2004).
In multicellular organisms, TOR is thought to have retained its role as a nutrient sensor but has also adopted new functions in regulating and responding to growth factor signaling pathways and developmental programs. Thus in a variety of signaling, developmental, and disease contexts, TOR activity can be regulated independently of nutritional conditions. In these cases, autophagy may be induced in response to downregulation of TOR despite the presence of abundant nutrients and may potentially play an important role in suppressing cell growth rather than promoting survival. Identification of the tumor suppressors PTEN, and TSC1 and TSC2 as positive regulators of autophagy provides correlative evidence supporting such a role for autophagy in growth control. Alternatively, since TOR activity is required for proper expression and localization of a number of nutrient transporters, inactivation of TOR may lead to reduced intracellular nutrient levels, and autophagy may therefore be required under these conditions to provide the nutrients and energy necessary for normal cell metabolism and survival (Scott, 2004).
The results presented here provide genetic evidence that under conditions of low TOR signaling, autophagy functions primarily to promote normal cell function and survival, rather than to suppress cell growth. This conclusion is based on the finding that genetic disruption of autophagy does not restore growth to cells lacking TOR, but instead exacerbates multiple TOR mutant phenotypes. It is important to note that mutations in TOR do not disrupt larval feeding, and thus disruption of autophagy is detrimental in TOR mutants despite the presence of ample extracellular nutrients. The finding that autophagy is critical in cells lacking TOR further supports earlier studies suggesting that inactivation of TOR causes defects in nutrient import, resulting in an intracellular state of pseudo-starvation (Scott, 2004).
Can the further reduction in growth of TOR mutant cells upon disruption of autophagy be reconciled with the potential catabolic effects of autophagy? TOR regulates the bidirectional flow of nutrients between protein synthesis and degradation through effects on nutrient import, autophagy, and ribosome biogenesis. When TOR is inactivated, rates of nutrient import and protein synthesis decrease, resulting in a commensurate reduction in mass accumulation and cell growth. In addition, autophagy is induced to maintain intracellular nutrient and energy levels sufficient for normal cell metabolism. When autophagy is experimentally inhibited in cells lacking TOR, this reserve source of nutrients is blocked, leading to a further decrease in energy levels, protein synthesis, and growth. It is noted that autophagy may have additional functions in cells with depressed TOR signaling, including recycling of organelles damaged by the absence of TOR activity, or selective degradation of cell growth regulators, analogous to the regulatory roles of ubiquitin-mediated degradation (Scott, 2004).
Autophagy is required for normal developmental responses to inactivation of insulin/PI3K signaling in the nematode C. elegans. In response to starvation or disruption of insulin/PI3K signaling, C. elegans larvae enter a dormant state called the dauer. Autophagy has been observed in C. elegans larvae undergoing dauer formation: disruption of a number of ATG homologs interfers with normal dauer morphogenesis. Importantly, simultaneous disruption of insulin/PI3K signaling and autophagy genes results in lethality, similar to the results presented in this study. Thus despite significant differences in developmental strategies for surviving nutrient deprivation, autophagy plays an essential role in the starvation responses of yeast, flies, and worms (Scott, 2004).
The prevailing view that S6K acts to suppress autophagy was founded on correlations between induction of autophagy and dephosphorylation of rpS6 in response to amino acid deprivation or rapamycin treatment. However, the genetic data presented in this study argue strongly against a role for S6K in suppressing autophagy: unlike other positive components of the TOR pathway, null mutations in S6K do not induce autophagy in fed animals. It is suggested that the observed correlation between S6K activity and suppression of autophagy is due to common but independent regulation of S6K and autophagy by TOR. Thus, autophagy suppression and S6K-dependent functions such as ribosome biogenesis represent distinct outputs of TOR signaling (Scott, 2004).
How might TOR signal to the autophagic machinery, if not through S6K? In yeast, this is accomplished in part through regulation of Atg1 kinase activity and ATG8 gene expression (Kamada, 2000 and Kirisako, 1999). The demonstration of a role for Drosophila ATG1 and ATG8 homologs [see TG8a (CG32672) and ATG8b (CG12334)] in starvation-induced autophagy, and the genetic interaction observed between ATG1 and TOR, are consistent with a related mode of regulation in higher eukaryotes. However, it is noted that other components of the yeast Atg1 complex such as Atg17 and Atg13, whose phosphorylation state is rapamycin sensitive, do not have clear homologs in metazoans, indicating that differences in regulation of autophagy by TOR are likely (Scott, 2004).
In addition to excluding a role for S6K in suppression of autophagy, these results reveal a positive role for S6K in induction of autophagy. S6K may promote autophagy directly, through activation of the autophagy machinery, or indirectly through its effects on protein synthesis. The latter possibility is consistent with previous reports that protein synthesis is required for expansion and maturation of autophagosomes. Interestingly, despite being required for autophagy, S6K is downregulated under conditions that induce it, including chronic starvation and TOR inactivation. Consistent with this, it was found that lysotracker staining is significantly weaker in chronically starved animals or in TOR mutants than in wild-type animals starved 3-4 hr. Furthermore, expression of constitutively activated S6K has no effect in wild-type, but restores lysotracker staining in TOR mutants to levels similar to those of acutely starved wild-type animals. It is suggested that downregulation of S6K may limit rates of autophagy under conditions of extended starvation or TOR inactivation and that this may protect cells from the potentially damaging effects of unrestrained autophagy (Scott, 2004).
Co-culture and conditioned media experiments have shown that the Drosophila fat body is a source of diffusible mitogens. The fat body has also been shown to act as a nutrient sensor through a TOR-dependent mechanism and to regulate organismal growth through effects on insulin/PI3K signaling. The results in this study extend these findings by showing that this endocrine response is accompanied by the regulated release of nutrients through autophagic degradation of fat body cytoplasm. Preventing this reallocation of resources, either through constitutive activation of PI3K or through inactivation of ATG genes, results in profound nutrient sensitivity. Thus, in response to nutrient limitation, the fat body is capable of simultaneously restricting growth of peripheral tissues through downregulation of insulin/PI3K signaling and providing these tissues with a buffering source of nutrients necessary for survival through autophagy (Scott, 2004).
As in many other organisms, the blood of Drosophila consists of several types of hemocytes, which originate from the mesoderm. By lineage analyses of transplanted cells, two separate anlagen have been defined that give rise to different populations of hemocytes: embryonic hemocytes and lymph gland hemocytes. The anlage of the embryonic hemocytes is restricted to a region within the head mesoderm between 70% and 80% egg length. In contrast to all other mesodermal cells, the cells of this anlage are already determined as hemocytes at the blastoderm stage. Unexpectedly, these hemocytes do not degenerate during late larval stages, but have the capacity to persist through metamorphosis and are still detectable in the adult fly. A second anlage, which gives rise to additional hemocytes at the onset of metamorphosis, is located within the thoracic mesoderm at 50% to 53% egg length. After transplantation within this region, clones were detected in the larval lymph glands. Labeled hemocytes are released by the lymph glands not before the late third larval instar. The anlage of these lymph gland-derived hemocytes is not determined at the blastoderm stage, as indicated by the overlap of clones with other tissues. These analyses reveal that the hemocytes of pupae and adult flies consist of a mixture of embryonic hemocytes and lymph gland-derived hemocytes, originating from two distinct anlagen that are determined at different stages of development (Holz, 2003).
The origin of the embryonic hemocytes (EH) can be traced back to the head mesoderm of late stage 11 embryos by morphological criteria. Owing to the fact that srp is expressed in a narrow stripe within the cephalic mesoderm at the blastoderm stage and that a loss of srp function leads to a complete loss of embryonic hemocytes, this domain is considered to be the primordium of the EH. By homotopic single-cell transplantations it was possible to restrict the anlage to a sharply delimitated region located at 70% to 80% EL within the mesoderm, exactly corresponding to the cephalic expression domain of srp. The fact that none of the EH clones overlapped with other tissues indicates that the hemocytes are already determined at the blastoderm stage. This was confirmed by heterotopic transplantations from the EH anlage into the abdominal mesoderm; these transplanted cells give rise to hemocytes. Since mesodermal cells transplanted into the EH anlage do not transform into embryonic hemocytes, the determining factor is not able to induce a hemocyte fate within these cells and seems to function cell-autonomously. A good candidate for such a factor is Srp. However, since srp is also expressed in many other tissues that do not give rise to hemocytes, there must be additional genes that lead to a determination of the EH at the blastoderm stage. The early determination of the EH is quite unusual, since all other mesodermal tissues analyzed to date -- including the anlage of the lymph gland-derived hemocytes -- are not restricted to a tissue-specific fate prior to the second postblastodermal mitoses. This might be a developmental adaptation of the EH, which at stage 12 are already differentiated into functional macrophages and are responsible for the removal of apoptotic cells within developing tissues (Holz, 2003).
It is commonly believed that in Drosophila during larval development the EH population is entirely replaced by hemocytes that have been released by the larval lymph glands. However, it is possible to trace hemocytes originating from the head mesoderm through all stages of development until 14-day-old adult flies. The number of hemocytes progressively rises during larval life, from less than 200 to more than 5000 per individual. Cell lineage analyses unambiguously demonstrate that this increase is due to postembryonic proliferation of the EH. The contribution of the lymph glands to the hemocyte population was determined by means of cell lineage analyses. These studies reveal that the lymph glands do not release blood cells into the hemocoel during all larval stages but exclusively at the end of the third larval instar (Holz, 2003).
With the onset of metamorphosis, additional hemocytes are released from the lymph glands. Although the lymph glands do not persist through metamorphosis, the marked hemocytes released by the labeled lymph glands are still detectable in adult flies. Hence, all hemocytes found throughout larval life originate solely from the EH anlage, whereas the pupal and imaginal blood is made up of two different populations: EH and LGH (Holz, 2003).
The two populations of hemocytes share many functional, morphological and genetic similarities. In both cases, the determination of hemocytes depends on srp, while the specification towards the distinct blood cell types is induced by the expression of lozenge (lz) glia cells missing (gcm) and the gcm homolog gcm2. Both EH and LGH differentiate into podocytes, crystal cells and plasmatocytes. Hemocytes of both populations have the capability to adopt macrophage characteristics. However, despite all similarities, the history of the two populations is quite different, since they originate from two different mesodermal regions and are determined at different developmental stages. In view of the fact that the lymph glands do not release hemocytes before the onset of metamorphosis under nonimmune conditions, all hemocytes found in the larval hemocoel represent EH (Holz, 2003).
The many similarities between EG and LGH raise the question why there are two populations at all. A massive release of hemocytes by the lymph glands is seen just at the onset of pupation. The lymph glands additionally have the capacity to differentiate and release a special type of hemocytes, the lamellocytes, under immune conditions even before the onset of metamorphosis. Thus, because under nonimmune conditions the lymph glands do not release any cells before the onset of pupation, it might be their primary role to provide a reservoir of immune defensive hemocytes. The massive apoptosis and accumulation of cell debris might be a secondary trigger to stimulate proliferation and release of the lymph gland hemocytes (Holz, 2003).
Drosophila hematopoiesis occurs in a specialized organ called the lymph gland. In this systematic analysis of lymph gland structure and gene expression, the developmental steps in the maturation of blood cells (hemocytes) from their precursors are defined. In particular, distinct zones of hemocyte maturation, signaling and proliferation in the lymph gland during hematopoietic progression are described. Different stages of hemocyte development have been classified according to marker expression and placed within developmental niches: a medullary zone for quiescent prohemocytes, a cortical zone for maturing hemocytes and a zone called the posterior signaling center for specialized signaling hemocytes. This establishes a framework for the identification of Drosophila blood cells, at various stages of maturation, and provides a genetic basis for spatial and temporal events that govern hemocyte development. The cellular events identified in this analysis further establish Drosophila as a model system for hematopoiesis (Jung, 2005).
In the late embryo, the lymph gland consists of a single pair of lobes containing ~20 cells each. These express the transcription factors Srp and Odd skipped (Odd), and each cluster of hemocyte precursors is followed by a string of Odd-expressing pericardial cells that are proposed to have nephrocyte function. These lymph gland lobes are arranged bilaterally such that they flank the dorsal vessel, the simple aorta/heart tube of the open circulatory system, at the midline. By the second larval instar, lymph gland morphology is distinctly different in that two or three new pairs of posterior lobes have formed and the primary lobes have increased in size approximately tenfold (to ~200 cells. By the late third instar, the lymph gland has grown significantly in size (approximately another tenfold) but the arrangement of the lobes and pericardial cells has remained the same. The cells of the third instar lymph gland continue to express Srp (Jung, 2005).
The third instar lymph gland also exhibits a strong, branching network of extracellular matrix (ECM) throughout the primary lobe. This network was visualized using several GFP-trap lines in which GFP is fused to endogenous proteins. For example, line G454 represents an insertion into the viking locus, which encodes a Collagen IV component of the extracellular matrix. The hemocytes in the primary lobes of G454 (expressing Viking-GFP) appear to be clustered into small populations within pockets or chambers bounded by GFP-labeled branches of various sizes. Other lines, such as the uncharacterized GFP-trap line ZCL2867, also highlight this branching pattern. What role this intricate ECM network plays in hematopoiesis, as well as why multiple cells cluster within these ECM chambers, remains to be determined (Jung, 2005).
Careful examination of dissected, late third-instar lymph glands by differential interference contrast (DIC) microscopy revealed the presence of two structurally distinct regions within the primary lymph gland lobes that have not been previously described. The periphery of the primary lobe generally exhibits a granular appearance, whereas the medial region looks smooth and compact. These characteristics were examined further with confocal microscopy using a GFP-trap line G147, in which GFP is fused to a microtubule-associated protein. The G147 line is expressed throughout the lymph gland but, in contrast to nuclear markers such as Srp and Odd, distinguishes morphological differences among cells because the GFP-fusion protein is expressed in the cytoplasm in association with the microtubule network. Cells in the periphery of the lymph gland make relatively few cell-cell contacts, thereby giving rise to gaps and voids among the cells within this region. This cellular individualization is consistent with the granularity of the peripheral region observed by DIC microscopy. By contrast, cells in the medial region were relatively compact with minimal intercellular space, which is also consistent with the smoother appearance of this region by DIC microscopy. Thus, in the late third instar, the lymph gland primary lobes consist of two physically distinct regions: a medial region consisting of compactly arranged cells, which was termed the medullary zone; and a peripheral region of loosely arranged cells, termed the cortical zone (Jung, 2005).
Mature hemocytes have been shown to express several markers, including collagens, Hemolectin, Lozenge, Peroxidasin and P1 antigen. The expression of the reporter Collagen-gal4 (Cg-gal4), which is expressed by both plasmatocytes and crystal cells, is restricted to the periphery of the primary lymph gland lobe. Comparison of Cg-gal4 expression in G147 lymph glands, in which the medullary zone and cortical zone can be distinguished, reveals that maturing hemocytes are restricted to the cortical zone. In fact, the expression of each of the maturation markers mentioned above is found to be restricted to the cortical zone. The reporter hml-gal4 and Pxn, which are expressed by the plasmatocyte and crystal cell lineages, are extensively expressed in this region. Likewise, the expression of the crystal cell lineage marker Lozenge is restricted in this manner. The spatial restriction of maturing crystal cells to the cortical zone was verified by several means, including the distribution of melanized lymph gland crystal cells in the Black cells background and analysis of the terminal marker ProPOA1. The cortical zone is also the site of P1 antigen expression, a marker of the plasmatocyte lineage. The uncharacterized GFP fusion line ZCL2826 also exhibits preferential expression in the cortical zone. Last, it was found that the homeobox transcription factor Cut is preferentially expressed in the cortical zone of the primary lobe. Although the role of Cut in Drosophila hematopoiesis is currently unknown, homologs of Cut are known to be regulators of the myeloid hematopoietic lineage in both mice and humans. Cells of the rare third cell type, lamellocytes, are also restricted to the cortical zone, based upon cell morphology and the expression of a msn-lacZ reporter (msn06946). In summary, based on the expression patterns of several genetic markers that identify the three major blood cell lineages, it is proposed that the cortical zone is a specific site for hemocyte maturation (Jung, 2005).
The medullary zone was initially defined by structural characteristics and subsequently by the lack of expression of mature hemocyte markers. However, several markers have been identified that are exclusively expressed in the medullary zone at high levels but not the cortical zone. Consistent with the compact arrangement of cells in the medullary zone, it was found that Drosophila E-cadherin (DE-cadherin or Shotgun) is highly expressed in this region. No significant expression of DE-cadherin was observed among maturing cells in the cortical zone. E-cadherin, in both vertebrates and Drosophila, is a Ca2+-dependent, homotypic adhesion molecule often expressed by epithelial cells and is a crucial component of adherens junctions. Attempts to study DE-cadherin mutant clones in the medullary zone where the protein is expressed were unsuccessful since no clones were recoverable. The reporter lines domeless-gal4 and unpaired3-gal4 are preferentially expressed in the medullary zone. The gene domeless (dome) encodes a receptor molecule known to mediate the activation of the JAK/STAT pathway upon binding of the ligand Unpaired. The unpaired3 (upd3) gene encodes a protein with homology to Unpaired and has been associated with innate immune function. These gal4 lines are in this study only as markers that correlate with the medullary zone and, at the present time, there is no evidence that their associated proteins have a role in lymph gland hematopoiesis. Other markers of interest with preferential expression in the medullary zone include the molecularly uncharacterized GFP-trap line ZCL2897 and actin5C-GFP. Cells expressing hemocyte maturation markers are not seen in the medullary zone. It is therefore reasonable to propose that this zone is largely populated by prohemocytes that will later mature in the cortical zone. Prohemocytes are characterized by their lack of maturation markers, as well as their expression of several markers described as expressed in the medullary zone (Jung, 2005).
The posterior signaling center (PSC), a small cluster of cells at the posterior tip of each of the primary (anterior-most) lymph gland lobes, is defined by its expression of the Notch ligand Serrate and the transcription factor Collier. During this analysis, several additional markers were identified that exhibit specific or preferential expression in the PSC region. For example, it was found that the reporter Dorothy-gal4 is strongly expressed in this zone. The Dorothy gene encodes a UDP-glycosyltransferase, which belongs to a class of enzymes that function in the detoxification of metabolites. The upd3-gal4 reporter, which has preferential expression in the medullary zone, is also strongly expressed among cells of the PSC. Last, three uncharacterized GFP-gene trap lines, ZCL2375, ZCL2856 and ZCL0611 were found, that are preferentially expressed in the PSC. This analysis has made it clear that the PSC is a distinct zone of cells that can be defined by the expression of multiple gene products (Jung, 2005).
The PSC can be defined just as definitively by the characteristic absence of several markers. For example, the RTK receptor Pvr, which is expressed throughout the lymph gland, is notably absent from the PSC. Likewise, dome-gal4 is not expressed in the PSC, further suggesting that this population of cells is biased toward the production of ligands rather than receptor proteins. Maturation markers such as Cg-gal4, which are expressed throughout the cortical zone, are not expressed by PSC cells. Additionally, the expression levels of the hemocyte marker Hemese and the Friend-of-GATA protein U-shaped are dramatically reduced in the PSC when compared with other hemocytes of the lymph gland. Taken together, both the expression and lack of expression of a number of genetic markers defines the cells of the PSC as a unique hemocyte population (Jung, 2005).
In contrast to primary lobes of the third instar, maturing hemocytes are generally not seen in the secondary lobes. Correspondingly, secondary lobes often have a smooth and compact appearance, much like the medullary zone of the primary lobe. Consistent with this appearance, secondary lymph gland lobes also express high levels of DE-cadherin. The size of the secondary lobe, however, varies from animal to animal and this correlates with the presence or absence of maturation markers. Smaller secondary lobes contain a few or no cells expressing maturation markers, whereas larger secondary lobes usually exhibit groups of differentiating cells. Direct comparison of DE-cadherin expression in secondary lobes with that of Cg-gal4, hml-gal4 or Lz revealed that the expression of these maturation markers occurs only in areas in which DE-cadherin is downregulated. Therefore, although there is no apparent distinction between cortical and medullary zones in differentiating secondary lobes, there is a significant correlation between the expression of maturation markers and the downregulation of DE-cadherin, as is observed in primary lobes (Jung, 2005).
The relatively late 'snapshot' of lymph gland development in the third larval instar establishes the existence of spatial zones within the lymph gland that are characterized by differences in structure as well as gene expression. In order to understand how these zones form over time, lymph glands of second instar larvae, the earliest time at which it was possible to dissect and stain, were examined for the expression of hematopoietic markers. As expected, Srp and Odd are expressed throughout the lymph gland during the second instar since they are in the late embryo and third instar lymph gland. Likewise, the hemocyte-specific marker Hemese is expressed throughout the lymph gland at this stage, although it is not present in the embryonic lymph gland (Jung, 2005).
To determine whether the cortical zone is already formed or forming in second instar lymph glands, the expression of various maturation markers were examined in a pair-wise manner to establish their temporal order. Of the markers examined, hml-gal4 and Pxn are the earliest to be expressed. The majority of maturing cells were found to be double-positive for hml-gal4 and Pxn expression, although a few cells were found to express either hml-gal4 or Pxn alone. This indicates that the expression of these markers is initiated at approximately the same time, although probably independently, during lymph gland development. The marker Cg-gal4 is next to be expressed since it was found among a subpopulation of Pxn-expressing cells. Finally, P1 antigen expression is initiated late, usually in the early third instar. Interestingly, the early expression of each of these maturation markers is restricted to the periphery of the primary lymph gland lobe, indicating that the cortical zone begins to form in this position in the second instar. Whenever possible, each genetic marker was directly compared with other pertinent markers in double-labeling experiments, except in cases such as the comparison of two different gal4 reporter lines or when available antibodies were generated in the same animal. In such cases, the relationship between the two markers, for example dome-gal4 and hml-gal4, was inferred from independent comparison with a third marker such as Pxn (Jung, 2005).
By studying the temporal sequence of expression of hemocyte-specific markers, one can describe stages in the maturation of a hemocyte. It should be noted, however, that not all hemocytes of a particular lineage are identical. For example, in the late third instar lymph gland, the large majority of mature plasmatocytes (~80%) expresses both Pxn and hml-gal4, but the remainder express only Pxn (~15%) or hml-gal4 (~5%) alone. Thus, while plasmatocytes as a group can be characterized by the expression of representative markers, populations expressing subsets of these markers indeed exist. It remains unclear at this time whether this heterogeneity in the hemocyte population is reflective of specific functional differences (Jung, 2005).
In the third instar, Pxn is a prototypical hemocyte maturation marker, while immature cells of the medullary zone express dome-gal4. Comparing the expression of these two markers in the second instar reveals an interesting developmental progression. A group of cells along the peripheral edge of these early lymph glands already express Pxn. These developing hemocytes downregulate the expression of dome-gal4, as they do in the third instar. Next to these developing hemocytes is a group of cells that expresses dome-gal4 but not Pxn; these cells are most similar to medullary zone cells of the third instar and are therefore prohemocytes. Interestingly, there also exists a group of cells in the second instar that expresses neither Pxn nor dome-gal4. This population is most easily seen in the medial parts of the gland, close to the centrally placed dorsal. These cells resemble earlier precursors in the embryo, except they express the marker Hemese. These cells are called pre-prohemocytes. Interpretation of the expression data is that pre-prohemocytes upregulate dome-gal4 to become prohemocytes. As prohemocytes begin to mature into hemocytes, dome-gal4 expression is downregulated, while the expression of maturation markers is initiated. The prohemocyte and hemocyte populations continue to be represented in the third instar as components of the medullary and cortical zones, respectively (Jung, 2005).
The cells of the PSC are already distinguishable in the late embryo by their expression of collier. It was found that the canonical PSC marker Ser-lacZ is not expressed in the embryonic lymph gland and is only expressed in a small number of cells in the second instar. This relatively late onset of expression is consistent with collier acting genetically upstream of Ser. Another finding was that the earliest expression of upd3-gal4 parallels the expression of Ser-lacZ and is restricted to the PSC region. Finally, Pvr and dome-gal4 are excluded from the PSC in the second instar, similar to what is seen in the third instar (Jung, 2005).
To determine whether maturing cortical zone cells are indeed derived from medullary zone prohemocytes, a lineage-tracing experiment was performed in which dome-gal4 was used to initiate the permanent marking of all daughter cell lineages. In this system, the dome-gal4 reporter expresses both UAS-GFP and UAS-FLP. The FLP recombinase excises an intervening FRT-flanked 'STOP cassette', allowing constitutive expression of lacZ under the control of the actin5C promoter. At any developmental time point, GFP is expressed in cells where dome-gal4 is active, while lacZ is expressed in all subsequent daughter cells regardless of whether they continue to express dome-gal4. In this experiment, cortical zone cells are permanently marked with ß-galactosidase despite not expressing dome-gal4 (as assessed by GFP), indicating that these cells are derived from a dome-gal4-positive precursor. This result is consistent with and further supports independent marker analysis that shows that dome-gal4-positive prohemocytes downregulate dome-gal4 expression as they initiate expression of maturation markers representative of cortical zone cells. As controls to the above experiment, the expression patterns of two other gal4 lines, twist-gal4 and Serrate-gal4 were determined. The reporter twist-gal4 is expressed throughout the embryonic mesoderm from which the lymph gland is derived. Accordingly, the entire lymph gland is permanently marked by ß-galactosidase despite a lack of twist-gal4 expression (GFP) in the third instar lymph gland. Analysis of Ser-gal4 reveals that PSC cells remain a distinct population of signaling cells that do not contribute to the cortical zone (Jung, 2005).
Genetic manipulation of Pvr function provides valuable insight into its involvement in the regulation of temporal events of lymph gland development. To analyze Pvr function, FLP/FRT-based Pvr-mutant clones were generated in the lymph gland early in the first instar and then examined during the third instar for the expression of maturation markers. It was found that loss of Pvr function abolishes P1 antigen and Pxn expression, but not Hemese expression. The crystal cell markers Lz and ProPOA1 are also expressed normally in Pvr-mutant clones, consistent with the observation that mature crystal cells lack or downregulate Pvr. The fact that Pvr-mutant cells express Hemese and can differentiate into crystal cells suggests that Pvr specifically controls plasmatocyte differentiation. Pvr-mutant cells do not become TUNEL positive but do express the hemocyte marker Hemese and can differentiate into crystal cells, all suggesting that the observed block in plasmatocyte differentiation within the mutant clone is not due to cell death. Additionally, Pvr-mutant clones were large and not significantly different in size from their wild-type twin spots. Thus, the primary role of Pvr is not in the control of cell proliferation. Targeting Pvr by RNA interference (RNAi) revealed the same phenotypic features, confirming that Pvr controls the transition of Hemese-positive cells to plasmatocyte fate (Jung, 2005).
Entry into S phase was monitored using BrdU incorporation and distinct proliferative phases were identified that occur during lymph gland hematopoiesis. In the second instar, proliferating cells are evenly distributed throughout the lymph gland. By the third instar, however, the distribution of proliferating cells is no longer uniform; S-phase cells are largely restricted to the cortical zone. This is particularly evident when BrdU-labeled lymph glands are co-stained with Pxn. Medullary zone cells, which can be identified by the expression of dome-gal4, rarely incorporate BrdU. Therefore, the rapidly cycling prohemocytes of the second instar lymph gland quiesce as they populate the medullary zone of the third instar. As prohemocytes transition into hemocyte fates in the cortical zone, they once again begin to expand in number. This is supported by the observation that the medullary zone in white pre-pupae does not appear diminished in size, suggesting that the primary mechanism for the expansion of the cortical zone prior to this stage is through cell division within the zone. Proliferating cells in the secondary lobes continue to be distributed uniformly in the third instar, suggesting that secondary-lobe prohemocytes do not reach a state of quiescence as do the cells of the medullary zone. These results indicate that cells of the lymph gland go through distinct proliferative phases as hematopoietic development proceeds (Jung, 2005).
This analysis of the lymph gland revealed three key features that arise during development. The first feature is the presence of three distinct zones in the primary lymph gland lobe of third instar larvae. Two of these zones, termed the cortical and medullary zones, exhibit structural characteristics that make them morphologically distinct. These zones, as well as the third zone, the PSC, are also distinguishable by the expression of specific markers. The second key feature is that cells expressing maturation markers such as Lz, ProPOA1, Pxn, hml-gal4 and Cg-gal4 are restricted to the cortical zone. The medullary zone is consistently devoid of maturation marker expression and is therefore defined as a region composed of immature hemocytes (prohemocytes). The finding of different developmental populations within the lymph gland (prohemoctyes and their derived hemocytes) is similar to the situation in vertebrates where it is known that hematopoietic stem cells and other blood precursors give rise to various mature cell types. Additionally, Drosophila hemocyte maturation is akin to the progressive maturation of myeloid and lymphoid lineages in vertebrate hematopoiesis. The third key feature of lymph gland hematopoiesis is the dynamic pattern of cellular proliferation observed in the third instar. At this stage, the vast majority of S-phase cells in the primary lobe are located in the cortical zone, suggesting a strong correlation between proliferation and hemocyte differentiation. Compared with earlier developmental stages, cell proliferation in the medullary zone actually decreases by the late third instar, suggesting that these cells have entered a quiescent state. Thus, proliferation in the lymph gland appears to be regulated such that growth, quiescence and expansion phases are evident throughout its development (Jung, 2005).
Drosophila blood cell precursors, prohemocytes and maturing hemocytes each exhibit extensive phases of proliferation. The competence of these cells to proliferate seems to be a distinct cellular characteristic that is superimposed upon the intrinsic maturation program. Based on the patterns of BrdU incorporation in developing primary and secondary lymph gland lobes, it is possible to envision at least two levels of proliferation control during hematopoiesis. It is proposed that the widespread cell proliferation observed in second instar lymph glands and in secondary lobes of third instar lymph glands occurs in response to a growth requirement that provides a sufficient number of prohemocytes for subsequent differentiation. The mechanisms promoting differentiation in the cortical zone also trigger cell proliferation, which accounts for the observed BrdU incorporation in this zone and serves to expand the effector hemocyte population. The quiescent cells of the medullary zone represent a pluripotent precursor population because they, similar to vertebrate hematopoietic precursors, rarely divide and give rise to multiple lineages and cell types (Jung, 2005).
Based on this analysis a model is proposed by which hemocytes mature in the lymph gland. Hematopoietic precursors that populate the early lymph gland are first distinguishable as Srp+, Odd+ (S+O+) cells. These will eventually give rise to a primary lymph gland lobe where the steps of hemocyte maturation are most apparent. During the first or early second instar, these S+O+ cells begin to express the hemocyte-specific marker Hemese (He) and the tyrosine kinase receptor Pvr. Such cells can be called pre-prohemocytes and, in the second instar, cells expressing only these markers occupy a narrow region near the dorsal vessel. Subsequently, a subset of these Srp+, Odd+, He+, Pvr+ (S+O+H+Pv+) pre-prohemocytes initiate the expression of dome-gal4 (dg4), thereby maturing into prohemocytes. The prohemocyte population (S+O+H+Pv+dg4+) can be subdivided into two developmental stages. Stage 1 prohemocytes, which are abundantly seen in the second instar, are proliferative, whereas stage 2 prohemocytes, exemplified by the cells of the medullary zone, are quiescent. As development continues, prohemocytes begin to downregulate dome-gal4 and express maturation markers (M; becoming S+O+H+Pv+dg4lowM+). Eventually, dome-gal4 expression is lost entirely in these cells (becoming S+O+H+Pv+dg4-M+), found generally in the cortical zone. Thus, the maturing hemocytes of the cortical zone are derived from prohemocytes previously belonging to the medullary zone. This is supported by lineage-tracing experiments that show cells expressing medullary zone markers can indeed give rise to cells of the cortical zone. In turn, the medullary zone is derived from the earlier, pre-prohemocytes. Early cortical zone cells continue to express successive maturation markers (M) as they proceed towards terminal differentiation. Depending on the hemocyte type, examples of expressed maturation markers are Pxn, P1, Lz, L1, msn-lacZ, etc. These studies have shown that differentiation of the plasmatocyte lineage requires Pvr, while previous work has shown that the Notch pathway is crucial for the crystal cell fate. Both the JAK/STAT and Notch pathways have been implicated in lamellocyte production (Jung, 2005).
Previous investigations have demonstrated that similar transcription factors and signal transduction pathways are used in the specification of blood lineages in both vertebrates and Drosophila. Given this relationship, Drosophila represents a powerful system for identifying genes crucial to the hematopoietic process that are conserved in the vertebrate system. The work presented here provides an analysis of hematopoietic development in the Drosophila lymph gland that not only identifies stage-specific markers, but also reveals developmental mechanisms underlying hemocyte specification and maturation. The prohemocyte population in Drosophila becomes mitotically quiescent, much as their multipotent precursor counterparts in mammalian systems. These conserved mechanisms further establish Drosophila as an excellent genetic model for the study of hematopoiesis (Jung, 2005).
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).
Genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into human muscle physiology and disease. Studies in Drosophila have been particularly useful for discovering key genes involved in muscle specification, myoblast fusion, and sarcomere organization. The muscles of the Drosophila female reproductive system have received little attention despite extensive work on oogenesis. This study used newly available GFP protein trap lines to characterize of ovarian muscle morphology and sarcomere organization. The muscle cells surrounding the oviducts are multinuclear with highly organized sarcomeres typical of somatic muscles. In contrast, the two muscle layers of the ovary, which are derived from gonadal mesoderm, have a mesh-like morphology similar to gut visceral muscle. Protein traps in the Fasciclin 3 gene produced Fas3::GFP that localized in dots around the periphery of epithelial sheath cells, the muscle surrounding ovarioles. Surprisingly, the epithelial sheath cells each contain a single nucleus, indicating these cells do not undergo myoblast fusion during development. Consistent with this observation, the Flp/FRT system was used to efficiently generate genetic mosaics in the epithelial sheath, suggesting these cells provide a new opportunity for clonal analysis of adult striated muscle (Hudson, 2008).
Two protein trap lines showed a striped pattern in ovarian muscle. The GFP transposon in these lines was inserted into the genes for two uncharacterized proteins, CG30084 and CG6416, both of which contain a PDZ domain and a ZASP motif (ZM). CG30084 also contains four LIM domains in its C-terminus. Both CG30084 and CG6416 proteins show homology to the human Z-disc Alternatively Spliced Protein (ZASP) that localizes to the Z-disc in skeletal muscle. Like the ZASP gene, both CG30084 and CG6416 encode a number of alternative splice forms, almost all of which could be tagged based on the locations of the protein trap insertions. An additional isoform of CG30084 has been annotated that encodes only LIM domains and cannot be tagged. Due to the homology of these genes to ZASP, CG30084 was designated as Zasp52, and CG6416 as Zasp66 based on the cytological location of the genes (Hudson, 2008).
The availability of protein trap lines was especially useful for describing intercellular contacts (e.g., Fas3::GFP, Ilk::GFP inserted into Integrin linked kinase) and for showing the location of 'new' sarcomere components (e.g., Zasp52::GFP, Zasp66::GFP). The muscle cells of the oviducts, which are derived from genital disks, have highly organized sarcomeres and a myotube organization typical of somatic muscles in insects, and likely arise by myoblast fusion during development. In contrast, the visceral muscle layers surrounding ovarioles and ovaries have a mesh-like shape similar to the visceral muscles of the gut. Surprisingly, it was found that the epithelial sheath muscles contain one nucleus rather than two as in gut visceral muscles, making them the only known adult somatic or visceral muscle that does not arise by myoblast fusion. It remains to be determined whether the peritoneal sheath cells are mononuclear as well (Hudson, 2008).
The nature of the epithelial sheath muscle makes it ideally suited for analysis of muscle function. The morphology of the cells can be viewed in great detail with an array of available sarcomere and cellular markers. Importantly, the ability to produce cells with a defined genotype by mitotic clone induction provides a new opportunity for studying the phenotypes in adult muscles caused by homozygous mutations in muscle genes, even if the mutations are lethal during early development. Finally, it is straightforward to do short-term in vitro culturing of ovarioles with the epithelial sheath intact and contracting; thus, the behavior of cells bearing mutations can be studied in live tissue under controlled conditions (Hudson, 2008).
See also the Gonads site.
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