InteractiveFly: GeneBrief

HLH54F: Biological Overview | References

HLH54F, the Drosophila ortholog of the vertebrate basic helix-loop-helix domain-encoding genes capsulin and musculin, is expressed in the founder cells and developing muscle fibers of the longitudinal midgut muscles. These cells descend from the posterior-most portion of the mesoderm, termed the caudal visceral mesoderm (CVM), and migrate onto the trunk visceral mesoderm prior to undergoing myoblast fusion and muscle fiber formation. HLH54F expression in the CVM is regulated by a combination of terminal patterning genes and snail. HLH54F mutations were generated and this gene was shown to be crucial for the specification, migration and survival of the CVM cells and the longitudinal midgut muscle founders. HLH54F mutant embryos, larvae, and adults lack all longitudinal midgut muscles, which causes defects in gut morphology and integrity. The function of HLH54F as a direct activator of gene expression is exemplified by analysis of a CVM-specific enhancer from the Dorsocross locus, which requires combined inputs from HLH54F and Biniou in a feed-forward fashion. It is concluded that HLH54F is the earliest specific regulator of CVM development and that it plays a pivotal role in all major aspects of development and differentiation of this largely twist-independent population of mesodermal cells (Ismat, 2010).

The Drosophila mesoderm forms from the ventral-most cells of the early embryo that invaginate during gastrulation. The expression and function of two transcription factors, the basic helix-loop-helix (bHLH) protein Twist (Twi) and the zinc-finger protein Snail (Sna), in ventral cells located between ~15% and 85% egg length are essential for their invagination and for subsequent mesodermal tissue development. Although the absence of either twi or sna activity results in similar phenotypes, the molecular roles of the two genes in this pathway differ. twi functions in activating a variety of mesoderm-specific target genes, including several that are known to regulate the invagination, patterning and differentiation of the mesoderm. By contrast, sna is thought to act largely, if not exclusively, as a repressor of a number of neuroectodermal targets, permitting mesoderm formation by restricting the expression of these genes to areas outside the presumptive mesoderm (Ismat, 2010).

Notably, however, there is at least one group of mesodermal cells that requires sna, but not twi, for its initial phase of development. This cell group is located ventrally within the domain of early twi and sna expression, but is restricted to the posterior tip of the mesoderm between ~7.5% and 15% egg length. Because it is fated to develop into the longitudinal muscles of the midgut, it has been termed the caudal visceral mesoderm (CVM) primordium (Nguyen, 1998; Kusch, 1999). In light of the apparent lack of a requirement for twi for the initial development of the CVM, it is interesting that the cells of the CVM primordium are marked by the expression of another bHLH-encoding gene, HLH54F (Georgias, 1997). This situation raises the possibility that, in the caudal-most portion of the mesoderm, HLH54F instead of twi cooperates with sna to control early CVM development. However, until now, specific mutants for HLH54F have not been available to test this possibility (Ismat, 2010).

The Drosophila midgut musculature consists of syncytial fibers that arise through myoblast fusion between gut muscle founder cells and fusion-competent myoblasts and forms a meshwork of circular and longitudinal muscles around the endodermal layer. The CVM appears to be the sole source of founder cells of the longitudinal midgut muscles (Kusch and Reuter, 1999; San Martin, 2001). After their ingression, these cells migrate anteriorly and spread over the future midgut, where they fuse with resident fusion-competent cells to form the multinucleated longitudinal muscle fibers of the midgut. The fusion-competent cells for this event come from a different source, the so-called trunk visceral mesoderm (TVM), which provides a second (and major) contribution of precursors to the musculature of the midgut (San Martin, 2001). The primordia of the TVM are arranged bilaterally as 11 metameric cell clusters within the dorsal mesoderm, which subsequently merge with each other into the contiguous band of the TVM. The TVM primordia are marked by the expression of the NK homeodomain gene bagpipe (bap) and the FoxF gene biniou (bin), both of which are essential for TVM formation. Within the TVM, the founder cells of the circular midgut muscles are induced by Jelly belly (Jeb) signals acting through the receptor tyrosine kinase Alk. The founder myoblasts from the TVM fuse one-to-one with adjacent fusion-competent myoblasts into binucleated syncytia that form the circular midgut muscles. Subsequently, after the migrating CVM-derived founder cells have arrived at their destinations, each fuses with multiple fusion-competent cells from the TVM left over from the TVM founder cell fusions (San Martin, 2001). It is these multinucleated syncytia that will then differentiate into the longitudinal visceral muscles, which run perpendicularly to the circular muscles along the entire length of the midgut (Klapper, 2001). The longitudinal gut muscle fibers from the outer layer of the developing visceral musculature are tightly interwoven with the circular gut muscle fibers from the inner layer (Ismat, 2010).

HLH54F is the earliest known marker of the CVM primordia and its expression is maintained throughout the development and differentiation of the longitudinal midgut muscles (Georgias, 1997). To test whether HLH54F plays an important role in the development of the CVM and longitudinal midgut musculature, loss-of-function mutations for this gene were generated by imprecise P-excision and EMS mutagenesis screens. This study demonstrates that in the absence of HLH54F activity, no longitudinal gut muscle founder cells are formed. The absence of all tested CVM markers and the observed apoptotic death of the cells that would normally be destined to form CVM in HLH54F mutants, show that HLH54F has an essential role in determining the CVM and in specifying the founder cells of the longitudinal gut musculature. This function includes feed-forward regulation and direct binding to target enhancers (e.g., from the Dorsocross genes). It was also shown that ectopic expression of HLH54F can interfere with normal somatic muscle, cardiac and TVM development. Further, the known pathway of CVM development has been extended by showing that the initiation of HLH54F expression is largely independent of twi, but depends critically on the combined activities of sna and terminal patterning genes, particularly the synergistic activities of fork head (fkh) and brachyenteron (byn). Hence, the CVM primordia are determined at the intersection of the domains of these mesodermal and terminal regulators (Ismat, 2010).

This study has shown that HLH54F is a key regulator in the CVM, a population of cells in which the bHLH gene twi appears to have only minor functions. Although twi is initially co-expressed with HLH54F in these cells, it makes only a small contribution to activating HLH54F expression, and the expression of both bHLH genes rapidly becomes mutually exclusive. Instead of twi, the activation of HLH54F in the CVM primarily involves the combined activities of mesodermal sna and the terminal genes fkh and byn. As sna is generally thought to act as a ventral repressor of non-mesodermal genes in early mesoderm development, it will be interesting to determine whether the positive requirement for sna in the activation of HLH54F expression is direct, which would be unique to date. Alternatively, HLH54F might be activated by high levels of nuclear Dorsal and repressed by lateral genes that are repressed by sna ventrally. Along the anteroposterior axis, the posterior border of HLH54F expression is apparently defined by the posterior expression border of sna, which is delineated by the repressive action from hkb. It is proposed that the anterior border of HLH54F is determined by near-maximal threshold levels of tll, the expression of which declines steeply in the area anterior to the HLH54F domain. However, tll acts largely indirectly, through the combined activities of its downstream genes byn and fkh, in activating HLH54F. The low residual levels of HLH54F mRNA in fkh byn double mutants suggest the involvement of direct inputs from additional posterior activities, possibly tll or maternal torso. It appears that high-level expression of zfh1 in the CVM largely depends on tll and sna, whereas HLH54F and zfh1 do not depend on one another (Ismat, 2010).

Notably, neither twi nor HLH54F is required individually for the internalization of the CVM cells during gastrulation, although a redundant function cannot be excluded. The posterior portion of the mesoderm, which includes the CVM and portions of the presumptive HVM, bends around during gastrulation to form a second, internal mesodermal layer. It is conceivable that this movement is a passive process brought about by the invagination of the PMG rudiment. However, for subsequent migrations of CVM cells from these positions, the activity of HLH54F, but not twi, is crucial. In addition, byn, zfh1 and fkh are required for normal migration after stage 10. Whereas their respective functions are likely to be cell-autonomous, the observed requirement of twi for normal pathfinding of CVM cells is likely to be due to the absence of the migration substrate normally formed from the trunk mesoderm (Ismat, 2010).

The genetic data show that, after the caudal mesodermal cells have ingressed in this manner, they do not develop any further in the absence of HLH54F activity and undergo apoptosis. In the normal situation, HLH54F is needed for the activation of several transcription factor-encoding genes at this stage, including bin, croc and the Doc genes. Although the functions of these genes in CVM development have not been defined, it is likely that they regulate specific aspects of CVM development downstream of, and perhaps in combination with, HLH54F. The data from loss-of-function and ectopic expression analyses of HLH54F show that this gene is essential, but not sufficient, for specification of longitudinal gut muscle founders. Parallel inputs, albeit less pervasive, appear to come from high-level zfh1, which like HLH54F is required for croc/croc-lacZ expression (Kusch, 1999). Altogether, it is proposed that HLH54F is necessary for activating the vast majority of early CVM-specific genes, with one known exception being high-level zfh1, and that zfh1, byn and fkh in various combinations act together with HLH54F to activate certain targets during the specification and early migration of CVM cells (Ismat, 2010).

The continuous expression of HLH54F in the CVM and longitudinal gut muscles suggests that this gene is not only required for specification, but is also directly involved in many other developmental processes, including the continued migration, myoblast fusion and differentiation of the CVM cells. Possible downstream targets of HLH54F in the promotion of proper cell migration include beat-IIa, which encodes an as yet uncharacterized membrane-anchored Ig domain protein, and the FGF receptor-encoding gene heartless (htl), which is known to be required for normal migration. In this context, it is interesting that the vertebrate orthologs of HLH54F are expressed prominently in specific migrating populations of mesodermal cells as well. For example, musculin is expressed in myoblasts at the myotomal lips that migrate into the developing limbs, and capsulin is expressed in the migrating pro-epicardial cells (e.g. von Scheven, 2006). Therefore, it is possible that parts of the regulatory circuit in the control of cell migration have been conserved, even though they occur in different mesodermal cell types. capsulin is also expressed prominently in the splanchnopleura and tissues derived from it, including the developing smooth muscles of the stomach and gut (Hidai, 1998; Robb, 1998; von Scheven, 2006). Therefore, HLH54F and capsulin might share some functions in the terminal differentiation of the respective gut musculatures in the different systems. Both Capsulin and Musculin have been characterized largely as repressors. However, their activity (and likewise that of HLH54F) as repressors versus activators might well be context specific with respect to the particular enhancer, tissue or developmental stage in question and might depend on the relative concentrations of particular heterodimerization partners (Ismat, 2010).

Based on the phenotype of byn mutants, a role of the CVM in promoting midgut constrictions has also been proposed (Kusch, 1999). The phenotype of HLH54F mutants confirms this effect, although it was found that partial constrictions can frequently occur and that the effect is variable. It is inferred that the physical interactions between developing longitudinal and circular muscle fibers are necessary to provide the full force required for the efficient constriction of the midgut endoderm at the well-defined signaling centers. In the fully developed midgut, scanning electron microscopy images have revealed that the longitudinal fibers are tightly interwoven with the web-shaped circular fibers, which may explain the mechanical strength of this meshwork. Indeed, it was found that the mechanical stability and integrity of the midgut, particularly in HLH54F mutant adults, are severely compromised (Ismat, 2010).

In summary, HLH54F appears to sit at the top of the regulatory hierarchy of CVM development and is likely to fulfill additional key roles during the course of development of the CVM and the longitudinal gut muscles. Future efforts need to be directed towards dissecting additional downstream events that regulate the different steps of cell migration, myoblast fusion, morphogenesis and terminal differentiation of the longitudinal midgut musculature (Ismat, 2010).

A basic-helix-loop-helix protein expressed in precursors of Drosophila longitudinal visceral muscles

Basic-helix-loop-helix (bHLH) transcription factors are involved in the control of many developmental processes in vertebrates and invertebrates. The HLH domain mediates formation of homo- or heterodimers. This study took advantage of these dimerisation properties to identify a novel Drosophila HLH protein using the yeast two-hybrid system. Expression of bHLH54F at the blastoderm stage is restricted to a small subpopulation of mesodermal cells near the posterior pole. During germ band retraction these cells spread along the future midgut region. Later bHLH54F-expressing cells make up the longitudinal portion of the visceral musculature. Characterisation of this expression pattern demonstrates that precursors of the outer, longitudinal muscles of the midgut are distinct in origin and morphology from precursors of the inner, circular muscles (Georgias, 1997).

Search PubMed for articles about Drosophila HLH54f

Georgias, C., Wasser, M., and Hinz, U. (1997). A basic-helix-loop-helix protein expressed in precursors of Drosophila longitudinal visceral muscles. Mech. Dev. 69: 115-124. PubMed ID: 9486535

Hidai, H., Bardales, R., Goodwin, R., Quertermous, T. and Quertermous E. E. (1998). Cloning of capsulin, a basic helix-loop-helix factor expressed in progenitor cells of the pericardium and the coronary arteries. Mech. Dev. 73: 33-43. PubMed ID: 9545526

Ismat, A., et al. (2010). HLH54F is required for the specification and migration of longitudinal gut muscle founders from the caudal mesoderm of Drosophila. Development 137(18): 3107-17. PubMed ID: 20736287

Klapper, R., et al. (2002). The formation of syncytia within the visceral musculature of the Drosophila midgut is dependent on duf, sns and mbc. Mech. Dev. 110: 85-96. PubMed ID: 11744371

Kusch, T. and Reuter, R. (1999). Functions for Drosophila brachyenteron and forkhead in mesoderm specification and cell signalling. Development 126: 3991-4003. PubMed ID: 10457009

Nguyen, H. T. and Xu, X. (1998). Drosophila mef2 expression during mesoderm development is controlled by a complex array of cis-acting regulatory modules. Dev. Biol. 204: 550-566. PubMed ID: 9882489

Robb, L., et al. (1998). epicardin: a novel basic helix-loop-helix transcription factor gene expressed in epicardium, branchial arch myoblasts, and mesenchyme of developing lung, gut, kidney, and gonads. Dev. Dyn. 213: 105-113. PubMed ID: 9733105

San Martin, B., Ruiz-Gomez, M., Landgraf, M. and Bate, M. (2001). A distinct set of founders and fusion-competent myoblasts make visceral muscles in the Drosophila embryo. Development 128: 3331-3338. PubMed ID: 11546749

von Scheven, G., Bothe, I., Ahmed, M. U., Alvares, L. E. and Dietrich, S. (2006). Protein and genomic organisation of vertebrate MyoR and Capsulin genes and their expression during avian development. Gene Expr. Patterns 6: 383-393. PubMed ID: 16412697

date revised: 10 August 2012

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