BIOLOGICAL OVERVIEW

A hallmark of mature skeletal muscles is the presence of multinucleate muscle fibers. In Drosophila, the formation of muscle syncytia requires the cooperative participation of two types of myoblasts, founder cells and fusion-competent myoblasts. A newly identified gene, lame duck (lmd), has an essential regulatory role in the specification and function of fusion-competent myoblasts. Embryos that lack lmd function show a loss of expression of two key differentiation and fusion genes, Mef2 and sticks-and-stones, in fusion-competent myoblasts. These embryos are completely devoid of multinucleate muscle fibers. By contrast, founder cells are specified and retain their capability to differentiate into mononucleate muscle cells. lmd encodes a novel member of the Gli superfamily of transcription factors and is expressed in fusion-competent myoblasts and their precursors in a Wingless- and Notch-dependent manner. The activity of the Lmd protein appears to be additionally controlled by its differential cytoplasmic versus nuclear localization. Results from an independent molecular screen for binding factors to a myoblast-specific Mef2 enhancer further demonstrate that Lmd is a direct transcriptional regulator of Mef2 in fusion-competent myoblasts. lame duck is another name for the gene gleeful, which was isolated in DNA microarray experiments for twist target genes (Duan, 2001; Furlong, 2001).

Two mutant alleles, lmd¹ and lmd², came from an EMS-induced screen for lethal mutations on the third chromosome which showed an aberrant Eve expression pattern in either the mesoderm or nervous system (James Skeath and Chris Q. Doe, unpublished - cited in Duan, 2001). These two alleles also disrupt Mef2 expression. To assess the degree of muscle differentiation in lmd mutants, homozygous lmd¹ and lmd² embryos, as well as embryos trans-heterozygous for lmd¹ and Df(3R)M95A, a deficiency that deletes the entire lmd gene locus, were examined for Mef2 and Myosin heavy chain (MHC) expression. This analysis revealed that embryos homozygous for either lmd¹ or lmd² exhibit identical phenotype as lmd¹/Df(3R)M95A embryos, indicating that both lmd alleles are genetically null mutations. In lmd mutant embryos, the early pan-mesodermal Mef2 expression pattern is normal. However, specific defects were observed within the somatic muscle lineage starting from late stage 12. In particular, Mef2 expression is significantly reduced in somatic myoblasts although its expression in cardioblasts is not affected. Expression of markers of visceral mesoderm specification and differentiation, such as Mef2, Bagpipe (Bap) and Fasciclin III (FasIII), is also not affected. Consistent with the reduced Mef2 expression in the somatic mesoderm, lmd mutant embryos do not exhibit multinucleate MHC-expressing muscle fibers. Only mononucleate, elongated MHC-positive muscle cells are detectable in the mutant embryos. As observed for Mef2, MHC expression is not affected in the cardiac and gut musculatures. Gut constrictions also do not appear to be defective. These observations indicate that lmd function is critical for somatic myogenesis, while its function appears dispensible for cardiac and visceral muscle development (Duan, 2001).

To address the possibility that reduced Mef2 expression in lmd mutant embryos reflects a general loss of somatic myoblasts, a twist-dependent Mef2-lacZ enhancer line that generates ß-gal protein in the somatic muscle lineage, which perdures until late stage 13, was crossed into the lmd¹ mutant background. Wild-type and mutant embryos harboring this Mef2 enhancer insertion were double-labeled with antibodies against ß-gal and Mef2. lmd mutant embryos exhibit a comparable amount of ß-gal-marked somatic mesoderm as do wild-type embryos. However, the majority of ß-gal-positive somatic mesodermal cells fail to express Mef2, suggesting that lmd function is needed for activating Mef2 expression in a particular subset of myoblasts (Duan, 2001).

The presence of MHC-positive muscle cells in lmd mutant embryos suggests that founder cells are not affected and are capable of differentiating into mononucleate mini muscles. To examine this possibility, an enhancer trap insertion in the duf locus, rP298-lacZ, which marks a large number of founder cells, was crossed into the lmd¹ background. Wild-type and mutant embryos with this enhancer construct were double-labeled for Mef2 and lacZ expression. In a wild-type background, both Mef2-positive/lacZ-positive founders and Mef2-positive/lacZ-negative fusion-competent myoblasts were found. By contrast, Mef2-positive/lacZ-negative fusion-competent myoblasts are absent in lmd mutant embryos, while Mef2-positive/lacZ-positive founder cells are similar in number and position to those observed in wild-type embryos. These data suggest that loss of lmd function results in a specific loss of Mef2 expression in fusion-competent myoblasts (Duan, 2001).

To analyze the development of founder cells and fusion-competent myoblasts in greater detail, the expression of Krüppel (Kr) and sticks-and-stone (sns) was followed in wild-type and mutant lmd¹ embryos. Kr marks a subset of founder cells, whereas sns is a marker for fusion-competent myoblasts and encodes an Ig-type protein that is essential for the fusion process. Until late stage 12, the number and position of Kr-positive founders are approximately normal in mutant embryos when compared with wild-type embryos. However, after stage 12, wild-type embryos show an increase of Kr-positive nuclei as a result of myoblast fusion, while lmd mutant embryos fail to show a similar increase. Examination with other founder markers, such as Nau and Lady bird, yields similar results. Thus, founder cells in lmd mutant embryos do not appear to undergo cell fusion as observed in wild-type embryos. Significantly, sns expression in fusion-competent myoblasts is completely abolished in lmd mutant embryos. Only residual expression is observed in cells in positions corresponding to garland cells (which function as nephrocytes). Together, these results strongly suggest that lmd is required for proper specification and development of fusion-competent myoblasts. During this process, lmd is essential for activating the expression of Mef2 and sns, two genes that regulate myoblast fusion (Duan, 2001).

The cellular distribution of the Lmd protein is consistent with its mutant phenotype. The expression of lmd in somatic mesodermal cells between late stage 11 and early stage 14 is compatible with the phenotype of lmd mutant embryos in which loss of Mef2 expression in fusion-competent myoblasts is first detected at late stage 12 and sns is never activated. Prominent levels of Lmd protein are detected in fusion-competent myoblasts, whereas extremely low, or undetectable, levels of Lmd expression are observed in rP298-lacZ positive founder cells. Although the possibility that the very low levels in founder cells could be functionally important cannot be ruled out conclusively, the presence of specified muscle founders in lmd mutant embryos that express Mef2 and can differentiate into elongated MHC-expressing muscle cells supports the conclusion that lmd function is dispensible in founder cells (Duan, 2001).

The data indicate that lmd activity is needed transiently during embryogenesis to trigger specific genetic programs that are critical for the generation of a fully functional somatic musculature. These lmd-dependent programs, which include activation of Mef2 and sns, are essential for proper specification of fusion-competent myoblasts and their subsequent differentiation, which includes cell fusion. The phenotype of Mef2-deficient embryos, in which cell fusion is not observed, underscores the importance of Mef2 function in fusion-competent myoblasts. However, it is currently not known whether Mef2 is needed to activate fusion-specific genes or to promote this process in an indirect manner by activating genes that generate a suitable milieu for cell fusion. The sns gene, which is expressed exclusively in fusion-competent myoblasts independently of Mef2, has been shown to be required for myoblast fusion. Whether its functional importance is limited to being an adhesion-type of molecule or includes a potential role in signaling between myoblasts remains to be determined. In the aggregate, the findings support the notion that fusion-competent myoblasts are subject to a unique determination and differentiation program and are not simply products of a default state of cells that fail to become muscle founders. Lmd appears to be a key regulator in establishing this program. Therefore, it will be important to identify additional targets of lmd that are essential for the generation of functional fusion-competent myoblasts (Duan, 2001).

In contrast to its critical role in fusion-competent myoblasts, the function of lmd in visceral mesodermal cells is not clear. Loss of lmd expression does not affect the expression of genes that are involved in visceral mesoderm specification and differentiation, such as bap, Mef2, FasIII and Mhc. In addition, sns is expressed in lmd mutant embryos albeit at reduced levels. Thus, it appears that lmd function in the visceral mesoderm could be partially compensated by other gene(s) and that loss of lmd activity results only in subtle defects that remain to be defined (Duan, 2001).

Previous studies have demonstrated that Mef2 expression within the somatic muscle lineage is controlled by a modular-type of regulation, suggesting that specific activators exist that differentially exert regulatory effects on the various somatic muscle enhancers. lmd is identified in the present study as a direct upstream regulator of Mef2 expression in fusion-competent myoblasts. A notable feature of the lmd mutant phenotype is the loss of Mef2 expression in fusion-competent myoblasts beginning at mid-embryogenesis, whereas the earlier ubiquitous Twist-dependent Mef2 expression in the forming mesoderm is not affected. Muscle founders also express Mef2 normally and are capable of differentiating into mononucleate muscle cells. These observations suggest that, after the disappearance of Twist and other unknown early regulators of Mef2, lmd activity is required to activate Mef2 expression in the fusion-competent myoblasts. Indeed, direct and independent support for lmd as a direct transcriptional regulator of Mef2 in these myoblasts was derived from a yeast one-hybrid screen. In this unbiased approach, Lmd was identified as a DNA binding factor that can activate the particular enhancer I-E_D5, which directs Mef2 expression in fusion-competent myoblasts. Based upon the present data, Mef2 expression in founder cells must require yet unknown regulators that would function primarily through the two distinct founder cell enhancers that were previously defined (Duan, 2001).

The development of fusion-competent myoblasts is likely to be regulated in a two-step process. The first step involves the activation of lmd transcription, which provides cells with the potential to become fusion competent, and the second step promotes nuclear translocation of the Lmd protein, which allows Lmd to make the cells functional for fusion (Duan, 2001).

lmd expression in fusion-competent myoblasts is regulated by Notch and wg, as well as through wg-independent pathways. The regulation by wg is reminiscent of the wg-dependent formation of the majority of S59- and Nautilus-expressing muscle founders and the wg-independence of a subset of them. These observations suggest a coordinate regulation of both founder and fusion-competent myoblasts through Wg-dependent events. By contrast, Notch signaling has a reciprocal effect on the expression of regulatory genes in prospective muscle progenitors (which will form founders) and fusion-competent cells and, as a consequence, on the formation of these two types of myoblasts. Previous studies have established that the formation of muscle progenitors requires the absence of Notch signaling and loss of Notch function leads to increased numbers of muscle founders. Conversely, Notch function is essential for lmd expression and hence the formation of fusion-competent myoblasts. This result also explains the reported Notch dependence of sns, a downstream gene of lmd. Interestingly, E(spl) function is also required for lmd activation, thus suggesting that E(spl) could function as an activator of lmd or that it allows lmd transcription by downregulating a repressor in precursors of fusion-competent cells. Altogether, it appears that lmd may be the first example of a regulatory gene that is turned on by Notch in cells that fail to be singled out from a pre-cluster. lmd also serves to specify cell identity, which in this particular case is that of fusion-competent cells (Duan, 2001).

The nuclear/cytoplasmic distribution of the Lmd protein is reminiscent of the related Drosophila Ci and vertebrate Gli proteins, which are effectors of Hh signaling. The function of Ci/Gli proteins as transcriptional activators or repressors has been shown to be regulated by protein proteolysis, subcellular localization and levels. The analysis suggests that some of the post-transcriptional events described for ci/Gli gene products could also contribute to the regulation of Lmd activity. High resolution analysis has shown that Mef2 expression is correlated with elevated levels of nuclear-localized Lmd protein whereas exclusive cytoplasmic-localized Lmd expression is correlated with an absence of Mef2 expression. These observations suggest that subcellular localization and, by analogy to Ci/Gli proteins, regulated processing of the Lmd protein, may be required for activating Mef2 and other target genes. The presence of two putative PKA phosphorylation sites in the C-terminal region of the Lmd protein invokes the possibility that phosphorylation could have a regulatory role, as with Ci/Gli proteins. However, it does not appear that hh is needed for regulating Lmd activity because relatively well-developed muscle fibers are present in mutant embryos in which Hh activity has been removed during the relevant stages. Nevertheless, if modulation of Lmd activity were to involve cell-cell communication through other pathways, then this could provide a mechanism to coordinate the final stage of development of the fusion-competent myoblasts with that of neighboring muscle founders (Duan, 2001).

Although the high degree of sequence identity within the Zn-finger domain and the spacing of the Cys and His residues puts Lmd closest to Ci/Gli proteins, several notable differences exist. (1) Lmd has no additional homology outside of the Zn-finger domain, as observed among Ci/Gli proteins; (2) there is a striking divergence between Lmd and Ci/Gli proteins in the first and part of the second finger, although the terminal three fingers are highly conserved; (3) in vivo data indicate that Lmd recognizes a novel sequence, suggesting an involvement of the first two fingers in DNA-binding specificity. This would contrast with Gli proteins, in which binding has been shown to be mediated through the two C-terminal fingers. (4) ci/Gli genes have important roles in a variety of Hh-dependent patterning events during Drosophila development, and patterning of the neural ectoderm and somites in vertebrates. By contrast, Lmd appears to function only within the mesoderm and to regulate specification and differentiation events. This mesoderm-restricted feature is shared with macho-1 (Nishida, 2001), a Zic-related gene that has been shown to encode an mRNA that functions as a localized determinant of muscle fate in ascidians (Duan, 2001).

Taken together, these features identify Lmd as the first representative of a new type of protein family within the Gli superfamily of transcription factors. The results indicate that lmd function in the specification of fusion-competent myoblasts requires Wingless and Notch signaling for its initial expression and yet unknown signals for its transition into the nucleus. Nuclear Lmd then activates a spectrum of downstream genes, including the Mef2 and sns genes, which have critical roles in the development and functioning of fusion-competent myoblasts. Given the critical role of lmd in myogenesis, it will be interesting to identify vertebrate homologs of lmd to determine whether analogous mechanisms of muscle cell specification and development have been conserved during evolution (Duan, 2001).

GENE STRUCTURE

cDNA clone length - 1.3 kb

Bases in 5' UTR - 248

Exons - 5

Bases in 3' UTR - 333

PROTEIN STRUCTURE

Amino Acids - 866

Structural Domains

lmd was initially mapped between ebony and claret. Complementation tests with deficiencies further localized lmd to the region defined by the distal and proximal breakpoints of E226 and Df(3R)hh-GW2, respectively. The candidate region, demarcated by the 3' ends of the klingon and hh genes, was examined for potential transcripts by in situ hybridization. An approximately 1.3 kb genomic fragment detected RNA expression exclusively in mesodermal cells between late stage 11 and early stage 14, and encoded sequences for a protein with homology to a novel Zn-finger type of transcription factor. This information was used to identify a group of EST clones (LD47926, LD22708, LD23050, LD34514, LD39035) from the Berkeley Drosophila Genome Project for further analysis. Further sequencing of these clones showed that they correspond to overlapping cDNAs, and all contain the coding sequences present in the 1.3 kb genomic fragment. This analysis also revealed that the reported partial sequence of the 'K' gene (Casal, 1996) is included in these cDNA clones. The predicted ORF encodes a C₂H₂-type of Zn-finger protein, which shares sequence homology within the Zn-finger domain with proteins belonging to the Gli superfamily. The observed homology between Lmd and members of the Gli superfamily does not extend beyond the Zn-finger domain (Duan, 2001).

To determine whether this novel Zn-finger protein is in fact encoded by the lmd locus, the entire ORF of the Zn-finger protein in wild-type and lmd¹ mutant embryos was sequenced. This analysis shows that the Zn-finger protein contains a nucleotide change (C to T) that converts the Gln residue at amino acid 127 to a nonsense codon on the lmd¹ mutant chromosome. It has been concluded that lmd encodes a Zn-finger protein of the Gli superfamily. Of note, the truncation of the mutant Lmd¹ polypeptide is upstream of the putative DNA-binding domain, which is consistent with the identification of lmd¹ as a genetically null mutation (Duan, 2001).

A more detailed comparison of the Zn-finger domain from Lmd and representatives of the Gli superfamily, such as vertebrate Gli proteins, Drosophila Ci, C. elegans Tra-1, mouse Zic4 protein and ascidian Macho-1 indicates that Lmd bears strongest homology to Ci/Gli proteins. Although a high degree of sequence identity exists throughout the Zn-finger domain among the Ci/Gli proteins, identity between Lmd and Ci/Gli proteins is restricted to the third, fourth and fifth fingers, and a high level of divergence exists in the first and part of the second fingers. Thus, Lmd has been classified as a new and distinct member of the Gli superfamily (Duan, 2001).

date revised: 20 December 2001

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