BACKGROUND
in drosophila, the visceral midgut musculature consists of two layers of myofibers that derive from different embryonic primordia.
the inner layer of circular muscles originates from a subset of cells of the so-called trunk mesoderm and is characterized, e.g., by the expression of the bhlh factor hand  <cit> .
the outer lattice of longitudinal muscles arises from caudal mesoderm, located at the posterior tip of the blastoderm anlagen and is characterized by the expression of, e.g., bhlh54f  <cit> .
two cell types contribute to the formation of the circular muscles: founder cells  and fusion competent myoblasts .
during development, the founder cells fuse with the neighboring fusion competent myoblasts to form binucleated myofibers that elongate to surround the endodermal midgut later on  <cit> .
recently it was shown that fusion in the visceral mesoderm depends on receptor tyrosine kinase signaling  <cit> , whereas further differentiation depends on molecules including, e.g., blown fuse and kette  <cit> .
the visceral trunk mesoderm, as part of the early dorsal mesoderm, develops under inductive signals mediated by decapentaplegic   <cit> .
dpp is essential but not sufficient for the selection and differentiation of progenitors that give rise to cardioblasts, pericardial cells, several dorsal somatic muscles and the midgut circular muscles.
additional mesoderm-intrinsic factors are indispensable to enable cells to respond to the external signal.
a key player in the differentiating dorsal mesoderm cells is the nk homeobox transcription factor tinman , which is activated as a response to dpp signaling.
loss of tinman activity results in the absence of all derivatives of the dorsal mesoderm, including heart and circular visceral muscles  <cit> .
further development of the visceral trunk mesoderm requires the activity of the downstream factors bagpipe  and biniou , which are initially coexpressed in specific patches of cells in a segmental pattern along the anteroposterior axis of the dorsal mesoderm  <cit> .
tinman and bagpipe appear transiently in the visceral mesoderm and their activity diminishes during further visceral differentiation, indicating that both genes are responsible primarily for visceral mesoderm specification rather than differentiation.
biniou was shown to be crucial for further differentiation rather than cell specification.
biniou mutant embryos display visceral mesodermal cells but fail to form differentiated midgut musculature  <cit> .
the activity of several genes depends on biniou, including fasciclin iii, brokenheart, vimar, dpp and β3tubulin  <cit> .
regulation of dpp and β3tubulin in the visceral trunk mesoderm requires direct binding of biniou to specific enhancer elements, whereas the other downstream genes might be regulated indirectly.
in this report we examined the regulation of the bhlh transcription factor hand in the circular visceral mesoderm.
hand is expressed at stage  <dig> in the specified circular visceral muscle progenitors  <cit> , thus after the initial activity of the key regulators bap and bin.
using functional dissection assays in vivo, combined with a sequence comparison approach among hand loci of closely related drosophila species as well as protein/dna binding studies, we identified a highly conserved  <dig> bp element , located in the 3rd intron of the hand gene, which is crucial for activation of hand in circular visceral muscles.
our biochemical studies showed that the foxf-transcription factor biniou binds directly to the hv-element.
together with the observation that hand expression is abolished in the visceral mesoderm of bin mutant embryos whereas being normal in other expression domains, e.g., in the heart, our results indicate that hand is a direct target of biniou in the visceral trunk mesoderm.
embryos homozygous mutant for the hand gene show no morphological abnormalities in visceral mesoderm development, indicating that hand function is dispensable for the initial steps of visceral differentiation.
RESULTS
visceral expression of hand depends on a  <dig> bp intron sequence
within the visceral trunk mesoderm, hand is first detectable after the activation of the early key regulators bagpipe , tinman  and biniou   <cit> , raising the possibility that hand might be regulated by one of these factors.
to investigate this hypothesis we have searched for regulatory regions that are crucial for driving hand expression in the primordium of the circular visceral muscles.
the 3rd intron of the hand gene harbors all regulatory enhancers sufficient to drive hand expression in cardiac cells during embryogenesis and all postembryonic stages, as shown previously  <cit> .
in addition, the 3rd intron is capable to activate reporter gene expression in the visceral mesoderm .
reporter gene expression starts in stage  <dig> embryos in the progenitors of the circular visceral muscles, but is excluded from the visceral fusion competent cells  <cit> .
with beginning fusion, hand-gfp expression is detectable in syncytial visceral myofibers and persists until end of embryogenesis.
in addition, visceral expression of hand, as mimicked by the reporter gene activity, is present in larvae, pupae and adult flies .
to identify the enhancer elements crucial for the expression of hand in the visceral mesoderm, we compared hand 3rd intron sequences of orthologous loci in closely related drosophila species, an approach which was shown previously to be highly efficient to identify conserved regulatory elements  <cit> .
therefore we amplified the 3rd intron from d. erecta, d. mauritiana, d. simulans, d. teissieri, and d. yakuba, which was possible because the 3rd intron is flanked by highly conserved exons, allowing us the use of slightly degenerated primers .
in addition, the complete sequence of the hand locus from several drosophilids became available as part of recent genome sequencing projects.
sequence comparison of the hand 3rd intron from various species revealed two highly conserved sequence blocks .
to test whether one or both of these conserved elements have functional relevance we compared the capability of full-length 3rd intron and of mutated 3rd intron sequences to drive reporter gene expression in transgenic d. melanogaster flies.
the first construct harbors a deletion of  <dig> bp , thus removing the first highly conserved sequence block.
transgenic flies carrying the deletion construct exhibit a total loss of embryonic reporter gene expression in cardiomyoblasts, pericardial and lymph gland cells but not in visceral trunk mesoderm , indicating that the first conserved sequence block is required for hand expression in heart cells.
these findings are consistent with the observation of han and coworkers, who recently identified a cardiac and hematopoietic enhancer  in the same gene region  <cit> .
han and coworkers showed that activation of the hand gene in heart and hematopoietic cells is regulated, at least partially, through the activity of the transcription factors tinman, pannier and serpent that bind directly to the hch enhancer.
the second construct we tested carries a deletion of  <dig> bp corresponding to the second large conserved sequence block in the hand 3rd intron .
transgenic flies carrying this construct exhibit a total loss of reporter gene expression in the visceral trunk mesoderm, whereas cardiac expression remains unaffected .
furthermore, fusion of the visceral enhancer element to a reporter gene shows that it is capable to drive reporter gene activity in a pattern identical to that of endogenous hand expression in the visceral mesoderm.
thus, the hv-enhancer is not only necessary but also sufficient to drive reporter gene activity in the visceral trunk mesoderm and, later on in development, in the circular visceral muscles .
we conclude that the regulation of hand in the visceral mesoderm is accomplished by regulatory elements located in a conserved  <dig> bp sequence element .
biniou activates hand in the visceral mesoderm by direct binding to the hv regulatory region
the experiments described above have confirmed that the highly conserved non-coding sequences located in the 3rd intron of the hand gene are functional as cis-regulatory elements.
furthermore, the 3rd intron of hand loci isolated from related drosophilids are capable to drive reporter gene expression in transgenic drosophila melanogaster, as shown by han and coworkers for the hch enhancer from d. virilis  <cit>  and by us for the full-length intron isolated from d. mauritiana .
an additional observation indicates that the regulation of hand in cardiac and visceral mesoderm is conserved among drosophilids.
d. erecta and d. yakuba wildtype embryos, which we selected as examples, were probed with a labeled hand specific probe isolated from these species .
in both species, hand expression is detectable in the visceral and cardiac mesoderm .
our observations prompted us to search for upstream transcriptional activators responsible for visceral expression of hand by searching for conserved binding site motifs within the identified visceral enhancer.
alignment of sequences with homology to the hand visceral enhancer revealed the presence of four putative motifs with homology to the hfh- <dig> consensus binding site a/g c/t a a a c/t a  <cit> , which is recognized by the forkhead domain transcription factor biniou  <cit> .
the location of the four potential biniou binding sites is depicted in figure 3b.
to verify whether the expression of hand indeed depends on the activity of biniou, we examined hand expression and hand driven gfp reporter gene expression in mutant embryos.
in embryos lacking biniou, hand expression is totally abolished in the visceral mesoderm .
the embryos we tested carried a bap-lacz transgene, which serves as a marker for early visceral mesoderm  <cit> .
anti-βgalactosidase staining demonstrates that visceral mesodermal cells are present in biniou mutant embryos.
therefore, the absence of hand expression is not caused by the absence of these cells.
to examine whether biniou acts as a direct regulator on the hv-enhancer, we performed in vitro dna-binding experiments .
our results from gel mobility shift assays show that all four oligonucleotides  corresponding to the four putative biniou binding sites in the hv-element can compete for binding of biniou, although with different specificity.
oligonucleotides, in which the forkhead core-binding motif was mutated, failed to compete.
we next analyzed the functionality of the four biniou binding sites in vivo.
for this purpose we established constructs carrying the hand 3rd intron, but with individually mutated biniou binding sites, fused to a reporter gene.
we found that none of the tested mutations causes a total loss of reporter gene activity in the visceral mesoderm .
nevertheless, a mutated bin <dig> site results in a reduced reporter gene expression in the trunk mesoderm.
reporter gene expression in the cardiac mesoderm, serving as an internal control, appears normal.
next we tested a construct in which the two binding sites that reveal highest binding specificity in the gel mobility shift assay, bin <dig> and bin <dig>  were simultaneously mutated.
this results in the total loss of enhancer activity in the embryonic visceral mesoderm, while expression in other tissues remains unaffected .
our observations indicate that the combined binding of the bin <dig> and bin <dig> sites is critical for transcriptional activation of hand in the embryonic visceral mesoderm.
the high conservation of the  <dig> bp visceral enhancer element among drosophilids prompted us to search for additional regulators.
therefore we searched for transcription-factor binding sites by in silico analysis using various web-based algorithms  and a candidate approach.
the only potentially interesting binding site, predicted by such algorithms, indicates binding of hairy.
thus, we analyzed the expression of hand in the corresponding hairy mutant and found that hand expression is not altered.
because of their known expression in visceral cells, we considered other transcription factors as being potential regulators of hand, although binding sites were not predicted in the visceral enhancer.
when analyzing embryos homozygous mutant for the gli-like transcription factor lame duck , we observed a strong expansion of hand expression in the visceral mesoderm .
lmd is described as one of the major factors crucial for myoblast fusion  <cit> .
in the visceral mesoderm lmd is expressed in the fusion competent cells.
in contrast to its function in the somatic mesoderm as a positive regulator of fusion, essentially by promoting downstream factors like sticks and stones , it is dispensable for fusion in the visceral mesoderm  <cit> .
our results indicate that lmd acts directly or indirectly as a repressor of hand at early stages within the fusion competent cells prior to fusion.
at developmental stages when hand is normally restricted to a single row of visceral founder cells we found hand to be generally expressed in all cells of the visceral mesoderm in the lmd mutant .
in a recent study, varshney and palmer provided evidence that tyrosine kinase anaplastic lymphoma kinase -mediated signaling in the developing drosophila gut positively drives hand transcription  <cit> , although through an unknown effector.
in alk mutant embryos hand expression is abolished in the visceral mesoderm while being normal in other tissues, for instance the heart.
overexpression of jelly belly , the corresponding alk ligand  <cit> , within the entire mesoderm, induces alk signaling in all cells of the visceral mesoderm, and therefore leads to an expanded expression of hand in these cells.
the authors point out that several candidate molecules exist which might regulate the transcription of hand in the visceral mesoderm in response to alk activation, among them is biniou.
our present work clearly demonstrates that biniou is indeed critical for hand regulation in the visceral mesoderm.
hand function during visceral mesoderm differentiation
since the absence of biniou activity affects differentiation of the midgut visceral mesoderm, and expression of hand is lost in the trunk mesoderm as a consequence of the loss of biniou function, we were interested to analyze whether hand is required for midgut visceral muscle differentiation.
we utilized the recently described hand specific mutant generated by homologous recombination  <cit>  as well as the molecularly characterized deficiency dfexel <dig>  <cit>  that deletes  <dig> genes including hand .
when homozygous mutant embryos were examined for visceral mesoderm development, no detectable abnormalities were observed.
circular muscles, visualized by β3tubulin expression, are visible and the gut constrictions are formed normally as well .
our findings are consistent with the recent observations of varshney & palmer who used the deficiency dfexel <dig> for a phenotypic analysis  <cit> .
they show that neither alk, nor dumbfounded  or sns, typical markers for visceral mesoderm, are misregulated in the absence of hand.
a lack of a visible morphological phenotype in the gut formation argues clearly against an early role of hand in specification or initial differentiation of visceral mesoderm development, but leaves the possibility for a potential role of hand as a transcriptional regulator in this tissue, presumably later throughout life cycle.
this assumption is consistent with the high level of hand activity in postembryonic stages  <cit> .
examples of such physiological functions are mutations in the vimar and the β3tubulin gene  <cit> .
mutations in these genes cause lethality during larval stages without visible morphological abnormalities.
CONCLUSIONS
to get further insights into the network of transcriptional control we have analyzed the activation of the bhlh transcription factor hand.
we found that the expression of hand in cells that give rise to the circular visceral muscles is directly controlled by the foxf transcription factor biniou.
we show that two out of four biniou binding sites, located within a  <dig> bp visceral enhancer element are crucial for hand regulation.
examination of hand mutant embryos revealed that hand is not required for initial steps of circular muscle formation during embryogenesis.
furthermore, the gli-like transcription factor lame duck is needed to restrict hand expression to the founder cells of the visceral mesoderm at early stages.
a model for the regulation of hand in the visceral mesoderm is provided in figure 3e.

 in drosophila, the visceral midgut musculature consists of two layers of myofibers that derive from different embryonic primordia. the inner layer of circular muscles originates from a subset of cells of the so-called trunk mesoderm and is characterized, e.g., by the expression of the bhlh factor hand. the outer lattice of longitudinal muscles arises from caudal mesoderm, located at the posterior tip of the blastoderm anlagen and is characterized by the expression of, e.g., bhlh54f. two cell types contribute to the formation of the circular muscles: founder cells and fusion competent myoblasts. during development, the founder cells fuse with the neighboring fusion competent myoblasts to form binucleated myofibers that elongate to surround the endodermal midgut later on. recently it was shown that fusion in the visceral mesoderm depends on receptor tyrosine kinase signaling, whereas further differentiation depends on molecules including, e.g., blown fuse and kette. the visceral trunk mesoderm, as part of the early dorsal mesoderm, develops under inductive signals mediated by decapentaplegic. dpp is essential but not sufficient for the selection and differentiation of progenitors that give rise to cardioblasts, pericardial cells, several dorsal somatic muscles and the midgut circular muscles. additional mesoderm-intrinsic factors are indispensable to enable cells to respond to the external signal. a key player in the differentiating dorsal mesoderm cells is the nk homeobox transcription factor tinman, which is activated as a response to dpp signaling. loss of tinman activity results in the absence of all derivatives of the dorsal mesoderm, including heart and circular visceral muscles. further development of the visceral trunk mesoderm requires the activity of the downstream factors bagpipe and biniou, which are initially coexpressed in specific patches of cells in a segmental pattern along the anteroposterior axis of the dorsal mesoderm. tinman and bagpipe appear transiently in the visceral mesoderm and their activity diminishes during further visceral differentiation, indicating that both genes are responsible primarily for visceral mesoderm specification rather than differentiation. biniou was shown to be crucial for further differentiation rather than cell specification. biniou mutant embryos display visceral mesodermal cells but fail to form differentiated midgut musculature. the activity of several genes depends on biniou, including fasciclin iii, brokenheart, vimar, dpp and β3tubulin. regulation of dpp and β3tubulin in the visceral trunk mesoderm requires direct binding of biniou to specific enhancer elements, whereas the other downstream genes might be regulated indirectly. in this report we examined the regulation of the bhlh transcription factor hand in the circular visceral mesoderm. hand is expressed at stage in the specified circular visceral muscle progenitors, thus after the initial activity of the key regulators bap and bin. using functional dissection assays in vivo, combined with a sequence comparison approach among hand loci of closely related drosophila species as well as protein/dna binding studies, we identified a highly conserved bp element, located in the 3rd intron of the hand gene, which is crucial for activation of hand in circular visceral muscles. our biochemical studies showed that the foxf-transcription factor biniou binds directly to the hv-element. together with the observation that hand expression is abolished in the visceral mesoderm of bin mutant embryos whereas being normal in other expression domains, e.g., in the heart, our results indicate that hand is a direct target of biniou in the visceral trunk mesoderm. embryos homozygous mutant for the hand gene show no morphological abnormalities in visceral mesoderm development, indicating that hand function is dispensable for the initial steps of visceral differentiation.  visceral expression of hand depends on a bp intron sequence within the visceral trunk mesoderm, hand is first detectable after the activation of the early key regulators bagpipe, tinman and biniou, raising the possibility that hand might be regulated by one of these factors. to investigate this hypothesis we have searched for regulatory regions that are crucial for driving hand expression in the primordium of the circular visceral muscles. the 3rd intron of the hand gene harbors all regulatory enhancers sufficient to drive hand expression in cardiac cells during embryogenesis and all postembryonic stages, as shown previously. in addition, the 3rd intron is capable to activate reporter gene expression in the visceral mesoderm. reporter gene expression starts in stage embryos in the progenitors of the circular visceral muscles, but is excluded from the visceral fusion competent cells. with beginning fusion, hand-gfp expression is detectable in syncytial visceral myofibers and persists until end of embryogenesis. in addition, visceral expression of hand, as mimicked by the reporter gene activity, is present in larvae, pupae and adult flies. to identify the enhancer elements crucial for the expression of hand in the visceral mesoderm, we compared hand 3rd intron sequences of orthologous loci in closely related drosophila species, an approach which was shown previously to be highly efficient to identify conserved regulatory elements. therefore we amplified the 3rd intron from d. erecta, d. mauritiana, d. simulans, d. teissieri, and d. yakuba, which was possible because the 3rd intron is flanked by highly conserved exons, allowing us the use of slightly degenerated primers. in addition, the complete sequence of the hand locus from several drosophilids became available as part of recent genome sequencing projects. sequence comparison of the hand 3rd intron from various species revealed two highly conserved sequence blocks. to test whether one or both of these conserved elements have functional relevance we compared the capability of full-length 3rd intron and of mutated 3rd intron sequences to drive reporter gene expression in transgenic d. melanogaster flies. the first construct harbors a deletion of bp, thus removing the first highly conserved sequence block. transgenic flies carrying the deletion construct exhibit a total loss of embryonic reporter gene expression in cardiomyoblasts, pericardial and lymph gland cells but not in visceral trunk mesoderm, indicating that the first conserved sequence block is required for hand expression in heart cells. these findings are consistent with the observation of han and coworkers, who recently identified a cardiac and hematopoietic enhancer in the same gene region. han and coworkers showed that activation of the hand gene in heart and hematopoietic cells is regulated, at least partially, through the activity of the transcription factors tinman, pannier and serpent that bind directly to the hch enhancer. the second construct we tested carries a deletion of bp corresponding to the second large conserved sequence block in the hand 3rd intron. transgenic flies carrying this construct exhibit a total loss of reporter gene expression in the visceral trunk mesoderm, whereas cardiac expression remains unaffected. furthermore, fusion of the visceral enhancer element to a reporter gene shows that it is capable to drive reporter gene activity in a pattern identical to that of endogenous hand expression in the visceral mesoderm. thus, the hv-enhancer is not only necessary but also sufficient to drive reporter gene activity in the visceral trunk mesoderm and, later on in development, in the circular visceral muscles. we conclude that the regulation of hand in the visceral mesoderm is accomplished by regulatory elements located in a conserved bp sequence element. biniou activates hand in the visceral mesoderm by direct binding to the hv regulatory region the experiments described above have confirmed that the highly conserved non-coding sequences located in the 3rd intron of the hand gene are functional as cis-regulatory elements. furthermore, the 3rd intron of hand loci isolated from related drosophilids are capable to drive reporter gene expression in transgenic drosophila melanogaster, as shown by han and coworkers for the hch enhancer from d. virilis and by us for the full-length intron isolated from d. mauritiana. an additional observation indicates that the regulation of hand in cardiac and visceral mesoderm is conserved among drosophilids. d. erecta and d. yakuba wildtype embryos, which we selected as examples, were probed with a labeled hand specific probe isolated from these species. in both species, hand expression is detectable in the visceral and cardiac mesoderm. our observations prompted us to search for upstream transcriptional activators responsible for visceral expression of hand by searching for conserved binding site motifs within the identified visceral enhancer. alignment of sequences with homology to the hand visceral enhancer revealed the presence of four putative motifs with homology to the hfh- consensus binding site a/g c/t a a a c/t a, which is recognized by the forkhead domain transcription factor biniou. the location of the four potential biniou binding sites is depicted in figure 3b. to verify whether the expression of hand indeed depends on the activity of biniou, we examined hand expression and hand driven gfp reporter gene expression in mutant embryos. in embryos lacking biniou, hand expression is totally abolished in the visceral mesoderm. the embryos we tested carried a bap-lacz transgene, which serves as a marker for early visceral mesoderm. anti-βgalactosidase staining demonstrates that visceral mesodermal cells are present in biniou mutant embryos. therefore, the absence of hand expression is not caused by the absence of these cells. to examine whether biniou acts as a direct regulator on the hv-enhancer, we performed in vitro dna-binding experiments. our results from gel mobility shift assays show that all four oligonucleotides corresponding to the four putative biniou binding sites in the hv-element can compete for binding of biniou, although with different specificity. oligonucleotides, in which the forkhead core-binding motif was mutated, failed to compete. we next analyzed the functionality of the four biniou binding sites in vivo. for this purpose we established constructs carrying the hand 3rd intron, but with individually mutated biniou binding sites, fused to a reporter gene. we found that none of the tested mutations causes a total loss of reporter gene activity in the visceral mesoderm. nevertheless, a mutated bin site results in a reduced reporter gene expression in the trunk mesoderm. reporter gene expression in the cardiac mesoderm, serving as an internal control, appears normal. next we tested a construct in which the two binding sites that reveal highest binding specificity in the gel mobility shift assay, bin and bin were simultaneously mutated. this results in the total loss of enhancer activity in the embryonic visceral mesoderm, while expression in other tissues remains unaffected. our observations indicate that the combined binding of the bin and bin sites is critical for transcriptional activation of hand in the embryonic visceral mesoderm. the high conservation of the bp visceral enhancer element among drosophilids prompted us to search for additional regulators. therefore we searched for transcription-factor binding sites by in silico analysis using various web-based algorithms and a candidate approach. the only potentially interesting binding site, predicted by such algorithms, indicates binding of hairy. thus, we analyzed the expression of hand in the corresponding hairy mutant and found that hand expression is not altered. because of their known expression in visceral cells, we considered other transcription factors as being potential regulators of hand, although binding sites were not predicted in the visceral enhancer. when analyzing embryos homozygous mutant for the gli-like transcription factor lame duck, we observed a strong expansion of hand expression in the visceral mesoderm. lmd is described as one of the major factors crucial for myoblast fusion. in the visceral mesoderm lmd is expressed in the fusion competent cells. in contrast to its function in the somatic mesoderm as a positive regulator of fusion, essentially by promoting downstream factors like sticks and stones, it is dispensable for fusion in the visceral mesoderm. our results indicate that lmd acts directly or indirectly as a repressor of hand at early stages within the fusion competent cells prior to fusion. at developmental stages when hand is normally restricted to a single row of visceral founder cells we found hand to be generally expressed in all cells of the visceral mesoderm in the lmd mutant. in a recent study, varshney and palmer provided evidence that tyrosine kinase anaplastic lymphoma kinase -mediated signaling in the developing drosophila gut positively drives hand transcription, although through an unknown effector. in alk mutant embryos hand expression is abolished in the visceral mesoderm while being normal in other tissues, for instance the heart. overexpression of jelly belly, the corresponding alk ligand, within the entire mesoderm, induces alk signaling in all cells of the visceral mesoderm, and therefore leads to an expanded expression of hand in these cells. the authors point out that several candidate molecules exist which might regulate the transcription of hand in the visceral mesoderm in response to alk activation, among them is biniou. our present work clearly demonstrates that biniou is indeed critical for hand regulation in the visceral mesoderm. hand function during visceral mesoderm differentiation since the absence of biniou activity affects differentiation of the midgut visceral mesoderm, and expression of hand is lost in the trunk mesoderm as a consequence of the loss of biniou function, we were interested to analyze whether hand is required for midgut visceral muscle differentiation. we utilized the recently described hand specific mutant generated by homologous recombination as well as the molecularly characterized deficiency dfexel that deletes genes including hand. when homozygous mutant embryos were examined for visceral mesoderm development, no detectable abnormalities were observed. circular muscles, visualized by β3tubulin expression, are visible and the gut constrictions are formed normally as well. our findings are consistent with the recent observations of varshney & palmer who used the deficiency dfexel for a phenotypic analysis. they show that neither alk, nor dumbfounded or sns, typical markers for visceral mesoderm, are misregulated in the absence of hand. a lack of a visible morphological phenotype in the gut formation argues clearly against an early role of hand in specification or initial differentiation of visceral mesoderm development, but leaves the possibility for a potential role of hand as a transcriptional regulator in this tissue, presumably later throughout life cycle. this assumption is consistent with the high level of hand activity in postembryonic stages. examples of such physiological functions are mutations in the vimar and the β3tubulin gene. mutations in these genes cause lethality during larval stages without visible morphological abnormalities.  to get further insights into the network of transcriptional control we have analyzed the activation of the bhlh transcription factor hand. we found that the expression of hand in cells that give rise to the circular visceral muscles is directly controlled by the foxf transcription factor biniou. we show that two out of four biniou binding sites, located within a bp visceral enhancer element are crucial for hand regulation. examination of hand mutant embryos revealed that hand is not required for initial steps of circular muscle formation during embryogenesis. furthermore, the gli-like transcription factor lame duck is needed to restrict hand expression to the founder cells of the visceral mesoderm at early stages. a model for the regulation of hand in the visceral mesoderm is provided in figure 3e. 
the visceral trunk mesoderm in drosophila melanogaster develops under inductive signals from the ectoderm. in addition, we provide evidence that hand is dispensable for the initial differentiation of the embryonic visceral mesoderm. we found that hand expression in the circular visceral mesoderm is abolished in embryos mutant for the foxf domain containing transcription factor biniou. furthermore we demonstrate that biniou regulates hand expression by direct binding to a bp sequence element, located within the 3rd intron of the hand gene. in the present report we show that cross species sequence comparison of non-coding sequences between orthologous genes is a powerful tool to identify conserved regulatory elements. in our report we focus on the analysis of the transcriptional control of the highly conserved transcription factor hand in circular visceral muscle cells, providing evidence that the hand gene is a direct target of biniou. combining functional dissection experiments in vivo and protein/dna binding studies we identified hand as a direct target of biniou in the circular visceral muscles. this leads to the activation of the key regulators tinman, bagpipe and biniou that are crucial for specification of the circular visceral muscles. herein we describe the identification of a regulatory region in the hand gene essential and sufficient for the expression in the visceral mesoderm during embryogenesis.
 the visceral trunk mesoderm in drosophila melanogaster develops under inductive signals from the ectoderm. this leads to the activation of the key regulators tinman, bagpipe and biniou that are crucial for specification of the circular visceral muscles. how further differentiation is regulated is widely unknown, therefore it seems to be essential to identify downstream target genes of the early key regulators. in our report we focus on the analysis of the transcriptional control of the highly conserved transcription factor hand in circular visceral muscle cells, providing evidence that the hand gene is a direct target of biniou.  herein we describe the identification of a regulatory region in the hand gene essential and sufficient for the expression in the visceral mesoderm during embryogenesis. we found that hand expression in the circular visceral mesoderm is abolished in embryos mutant for the foxf domain containing transcription factor biniou. furthermore we demonstrate that biniou regulates hand expression by direct binding to a bp sequence element, located within the 3rd intron of the hand gene. this regulatory element is highly conserved in different drosophila species. in addition, we provide evidence that hand is dispensable for the initial differentiation of the embryonic visceral mesoderm.  in the present report we show that cross species sequence comparison of non-coding sequences between orthologous genes is a powerful tool to identify conserved regulatory elements. combining functional dissection experiments in vivo and protein/dna binding studies we identified hand as a direct target of biniou in the circular visceral muscles. 