BIOLOGICAL OVERVIEW

Myo-D proteins direct muscle differentiation in vertebrates. The discovery of a MyoD homolog in Drosophila has been anti-climactic. For a time it seemed that nautilus served in a linear pathway of muscle cell determination, but several observations refute that attractively simple view. First, nautilus is expressed later than myocyte specific enhancer factor 2 (mef2), the fly homolog of the vertebrate MADS box gene. nautilus expression has been shown to be independent of mef2. In addition, ectopic expression of muscle segment homeobox-1 (a gene different from tinman), which is normally expressed in both ectoderm and mesoderm, results in altered expression of nau leading to a loss of some muscles and defects in the patterning of others.

nautilus is not expressed uniformly in all muscle precursors, but only in somatic muscle precursors. tinman, expressed in cardiac and visceral muscles requires dpp from overlying ectoderm for its expression (Staehling-Hampton, 1994). The homeobox gene H2 is also exclusively expressed in visceral mesoderm. It too requires contact with ectoderm for full expression (Baker, 1995).

Therefore, somatic and visceral muscles require independent cell autonomous systems for differentiation. Although mef-2 is expressed earlier than nautilus, nautilus expression is independent of mef-2. In addition, non-cell autonomous signals are required for nautilus expression, including cell contact with overlying ectoderm. In gastrulation-arrested embryos, Nautilus is produced only in cells adjacent to ectoderm, indicating that a signal from ectoderm is required for Nautilus production (Baker, 1995).

DFR1, an FGF receptor homolog (different from breathless ) is expressed in a segmentally reiterated pattern within mesoderm and prefigures the ventral, lateral and dorsal somatic clusters (Shishido, 1993). DFR1 is expressed in large clusters of mesodermal cells in a pattern similar to the wild type distribution of nautilus expressing cells. A disruption of the DRF1 locus results in severly reduced nautilus expression (Shishido, 1993).

A second population of nautilus expressing cells is present in a medial position in ventral mesoderm, and these cells are dependent on wingless, expressed either in ectoderm or in mesoderm. Transgenic expression of wg solely in the ectoderm of wg mutants is sufficient to rescue nautilus expression in medial cells. Thus, wingless function is required during and after gastrulation for the formation of nau-expressing medial muscle precursor cell clusters (Ranganayakulu, 1996).

Additional evidence for the importance of non-cell autonomous signaling in muscle cell differentation is found in the effects of stripe mutants. stripe is a muscle development gene expressed not in muscles but rather in segment border cells of the overlying ectoderm. Stripe is a homolog of the mammalian early growth response transcription factor. In stripe mutants, muscle cell organization is distrupted due to defective guidance of migrating muscle fibers to their sites of insertion (Volk, 1994 and Lee, 1995).

Therefore, each of the mesodermal muscle types (visceral somatic and heart) have independent lineages and independent mechanisms for their determination. Both cell autonomous and non-cell autonomous signals are required to carry out muscle fate determination. The problem with expecting nautilus to qualify as a unified determinant of muscle differentiation lies in expectations of linear pathways.

Stereotypic founder cell patterning and embryonic muscle formation in Drosophila require nautilus (MyoD) gene function

nautilus is the only MyoD-related gene in Drosophila. Nautilus expression begins around stage 9 at full germ-band extension in a subset of mesodermal cells organized in a stereotypic pattern in each hemisegment. The muscle founder cell marker Duf-LacZ, produced by the enhancer trap line rP298LacZ, is coexpressed in numerous Nautilus-positive cells when founders first appear. Founders entrain muscle identity through the restricted expression of transcription factors such as S59, eve, and Kr, all of which are observed in subsets of the nautilus expressing founders. The nautilus gene was inactivated using homology-directed gene targeting and Gal4/UAS regulated RNAi to determine whether loss of nautilus gene activity affects founder cell function. Both methods produced a range of defects that include embryonic muscle disruption, reduced viability and female sterility, which could be rescued by hsp70-nautilus cDNA transgenes. These results demonstrate Nautilus expression marks early founders that give rise to diverse muscle groups in the embryo, and that nautilus gene activity is required to seed the correct founder myoblast pattern that prefigures the muscle fiber arrangement during embryonic development (Wei, 2007).

nau^armGFP homozygous embryos were stained with antibody to myosin heavy chain raised to exon-17, an exon conserved in all skeletal muscle myosins. Approximately one-third of the null embryos showed a severely disrupted muscle phenotype reminiscent of embryos injected with nautilus dsRNAs. In severe disruptions, the muscles often appeared as rounded balls or thin disorientated fibers, whereas in the less severe cases, various subsets of fibers were missing (Wei, 2007).

The EMS allele, nau¹⁸⁸, was placed over nau^armGFP as well as the Exelixis deficiency. The nau¹⁸⁸ allele is over a balancer marked with Sb^LacZ, so nau¹⁸⁸ can be followed easily by the loss of β-galactosidase expression. The nau¹⁸⁸ homozygous embryos showed minor to severe muscle disruptions, identical to the nau^armGFP homozygous embryos. As with the nau^armGFP mutant, the nau¹⁸⁸ allele was placed over the Exelixis deficiency to rule out effects of possible nonspecific mutations accumulated in the stock. There were no discernable differences in the either the pattern or the degree of muscle disruption between nau¹⁸⁸/nau^armGFP, nau^armGFP/Df, and nau¹⁸⁸/Df; ~22% of the nau¹⁸⁸/Df embryos showed severe muscle disruption. Less-severe muscle disruptions affecting fewer fibers were also observed in the nau¹⁸⁸ mutant, but the pattern of fiber loss was not specific. Approximately 50% of the nau¹⁸⁸ embryos did not survive to the pupal stage, similar to the percentage observed with the homozygous nau^armGFP mutant (Wei, 2007).

The viability of the nau^armGFP mutant was analyzed at different stages of development to determine when the loss of nautilus had the greatest impact. Most lethality, ~70%, occurred in the embryo and larval stages, as reflected by a significant reduction in the number of pupae. Larvae that survived were weak and moved in uncoordinated fashion, often staying in one position, oscillating back and forth. Slightly more than half of the pupae hatched with extended eclosion times of >12 h compared with a few minutes for normal pupae, whereas the remaining pupae did not open, or flies only partially exited the pupa case. Most of the flies that hatched remained at the bottom of the vial with shriveled wings, unable to move or stand, and eventually died. Temperature affected survival rates, because 64/108 pupae survived to adulthood at 25°C, but only 6/34 survived at 16°C, whereas embryo and larval survival percentages were minimally affected by temperature (Wei, 2007).

Surviving adult nau^armGFP and nau¹⁸⁸ females developed distended abdomens filled with eggs that remained held >20 days, even when they were mated in groups or in single crosses. The ovary from nautilus mutant females did not show the normal progression of egg development, and all of the egg chambers were of a similar but smaller size compared with normal mature egg chambers. This is observed in single ovarioles stained with phalloidin, where one can see the smaller, uniform-size egg chambers in the mutant (nau^armGFP), whereas there is a clear progression in egg chamber sizes in the normal ovariole. This likely reflects a reduced function in the musculature of the ovarian sheath but will have to be confirmed in future studies. The 'held egg' phenotype was also observed in nau¹⁸⁸/Df and nau^armGFP/nau¹⁸⁸ female populations. Surviving males are slow-moving and weak, but they are fertile. The loss of nautilus function affects all stages of Drosophila development, particularly somatic myogenesis in the embryo, maturation beyond the third instar larval stage, and female fertility (Wei, 2007).

An independent strategy was developed that did not disrupt the gene but targeted nautilus mRNA using an Gal4-inducible RNAi transgene. A modified pUAST vector was used, called pINT-1 (pUAST intron-1), containing the intron from the 5' UTR of the actin 5C gene as a spacer for inverted repeats, in this case consisting of the nautilus cDNA lacking the initiator codon. In S2 cells transfected with nau-pINT-1 and pACT-nautilus, nuclear Nautilus was evident but with the addition of pACT-Gal4, Nautilus protein was no longer detected, and silencing correlated with the appearance of nautilus-specific siRNAs (Wei, 2007).

In the absence of Gal4 induction, the homozygous nau-pINT-1 embryos displayed a normal embryonic muscle pattern. A homozygous nau-pINT-1 line on the third chromosome was crossed to a pACT Gal4/CyO stock to obtain progeny with one copy of the Gal4 inducer and one copy of the nau-pINT-1 RNAi transgene. This stock was then crossed back to the homozygous nau-pINT-1 stock to generate progeny with two copies of nau-pINT-1 and one copy of pACT-Gal4. Slightly >20% of the Gal4 induced embryos had severe muscle disruptions, identical to those observed with the nau^armGFP or nau¹⁸⁸ mutants. In addition, many of the adult females had enlarged abdomens and were unable to lay eggs, a phenocopy of the 'held egg' phenotype observed in the nau^armGFP mutant. In the surviving adult population from the first cross, there was also a 2-fold increase in the number of CyO compared with normal flies, indicating that approximately half of the induced nau-pINT-1 embryos died at earlier stages, similar to the early death observed in the nau^armGFP mutant. Importantly, the induced nau-pINT-1 resulted in a phenotype identical to the nau^armGFP mutant, as well as the previous phenotype observed with the injection of nautilus dsRNAs into the embryo (Wei, 2007).

To confirm that the nau^armGFP mutant phenotype was because of the loss of nautilus protein expression, a Casper hsp-70-nau cDNA transgene was used as a rescue construct, because the extent of the fully active nautilus promoter is not known. The nautilus cDNA was used previously to rescue female sterility attributed to nautilus loss of function in strains with overlapping nautilus deficiencies. With the rescue transgene and nau^armGFP balanced over a third chromosome marked with TM6B, the ratio of Tb:Tb+ pupae could be followed as a measure of lethality in the embryo and larval stages. A ratio of 2:1 for Tb:Tb+ pupae was expected in the absence of early lethality, but a ratio of 4.6:1 was observed, indicating significant death at earlier stages. The viability of embryos and larvae increased 2-fold with the introduction of heat-shock nautilus transgene, going from 35% to 77%. Approximately 90% of the rescued mutant females had normal abdomens (41 of 46), and roughly half of these (22 of 41) were fertile and produced normal armGFP-positive progeny when mated to wild-type males. Embryonic muscle disruption was greatly reduced in the rescued stock. Similar results were obtained for hsp-70-nau cDNA on the second chromosome. Rescue was achieved in the absence of heat shock, because overexpression of Nautilus was lethal, as reported for overexpression using the twist driver, 24B-GAL4, and UAS-nau cDNA. This confirmed that the loss of nautilus gene function in nau^armGFP was responsible for the range of phenotypes affecting myogenesis, viability, and fertility (Wei, 2007).

Injection of dsRNA representing the entire ORF or subregions of nautilus mRNA into the developing embryo has been shown to result in severe disruption of the embryonic muscle pattern. Another genetic study of EMS-induced nautilus loss-of-function alleles concluded nautilus had a minimal zygotic role in embryonic myogenesis and viability and was responsible for the specification of a subset of dispensable embryonic muscles, DA3 and DO4. This was an extension of an earlier study where transheterozygous flies for overlapping deficiencies that remove the nautilus gene survived to adulthood at rates similar to their heterozygous siblings (Wei, 2007 and references therein).

A comparison between the nau¹⁸⁸ EMS allele and the nau^armGFP mutant, either in combination or with the Exelixis nautilus deficiency, has now demonstrated that both null mutants have the same range of defects at all stages of development: there is severe disruption of the embryonic muscle pattern in roughly one-quarter to one-third of the embryos, and less severe disruptions do not correlate with the loss of a distinct set of muscle fibers; in the nau¹⁸⁸ and nau^armGFP mutants, 50%-70% of the embryos die by the pupal stage; surviving adults show reduced mobility, and female progeny are infertile with enlarged abdomens and unable to oviposit. This result is difficult to reconcile with previous observations that females transheterozygous for the overlapping nautilus deficiencies were able to oviposit at rates equivalent to wild-type females. This study never observed this with either the nau^armGFP or nau¹⁸⁸ mutants, and the basis for this discrepancy is not clear (Wei, 2007).

It was also possible to phenocopy both the nau^armGFP and nau¹⁸⁸ mutations using a Gal-4-inducible nautilus RNAi transgene. Although off-target effects in RNAi have become a concern, nautilus dsRNA has not been reported to have off-target issues in genomic wide screens (see Drosophila RNAi Screening Center), and the similarity between the genetic and RNAi data supports this interpretation (Wei, 2007).

The loss of nautilus gene function impacts all stages of development and demonstrates the Drosophila MyoD homolog is essential for normal myogenesis and viability. The select fiber loss for muscles DA3 and DO4 previously reported for the nau¹⁸⁸ allele was not observed in either the nau^armGFP or nau¹⁸⁸ mutants and is not representative of the range of the phenotypes shown in this study (Wei, 2007).

The appearance of a small number adult flies in nautilus nulls indicates there are redundant or compensatory mechanisms that can sustain a low level of survival, even though these flies have movement and coordination problems, and the females are sterile. Recent evidence for redundancy comes from C. elegans where SRF and HAND proteins work in concert with the worm nautilus homolog, hlh-1, as additional myogenic factors, because overexpression of either can convert naïve blastomeres to muscle, and HAND alone can trigger myogenic conversion in the absence of hlh-1 function (Fukushige, 2006). Alternatively, survival may reflect a stochastic outcome that selects for the minimal disruption in the founder cell pattern (Wei, 2007).

To determine whether the disorganization in the embryonic muscle observed in the nau^armGFP mutant was because of a change in the underlying stereotypic founder cell pattern, the nau^armGFP mutation was introduced into the enhancer trap line, rP298LacZ, that marks the expression of the founder cell-specific protein, Dumbfounded (Duf), with nuclear β-galactosidase beginning from stage 11. In normal rP298LacZ embryos, Duf-LacZ (red) was expressed in a highly organized, stereotypic pattern in a subset of mesodermal cells before MHC expression. In the presence of the nau^armGFP mutation, the normal early Duf-LacZ expression pattern was disrupted, even in stage 11 embryos. Founder disruption was more evident in slightly later stage embryos, still in the absence of MHC expression. Myoblast fusion occurs within a matter of a few hours and is coincident with the onset of muscle-specific protein synthesis, so the absence of myosin expression is a good correlate for minimal muscle differentiation. Twenty to thirty percent of the embryos showed severe disruption in the founder cell pattern in the absence of myosin synthesis, similar to the percentage of embryos that demonstrated prominent muscle defects. In general there was no discernable decrease in the number of Duf-LacZ founders when comparing nau^armGFP and normal rP298LacZ embryos, although the LacZ intensity was sometimes reduced. In wild-type stage 13 embryos, myosin expression and syncytia formation are first observed in the posterior segments in the ventral mesoderm adjacent to the CNS, particularly in abdominal segments 5–7. In the nau^armGFP mutant, embryo myosin synthesis first appeared around the misplaced Duf-LacZ-positive founder cells, demonstrating the absence of nautilus gene function correlates with a disruption in the founder cell pattern and the loss of muscle integrity in the embryo. Disruption of the Kr founder pattern, which marks at least 13 muscle groups in both dorsal and ventral regions of the embryo, was also evident from stages 11–13 in the nau^armGFP mutant embryos. This was not unexpected, because Kr and Duf-LacZ are coexpressed in numerous founders, and subsets of Nautilus-positive founders also express Kr (Wei, 2007).

The loss of founder cell patterning suggests the nautilus gene is required for proper cell–cell interaction, possibly involving cell adhesion components or transmembrane receptors that position founder cells at particular sites in the epidermis of each hemisegment. Extracellular matrix and cell adhesion components have been identified in a screen enriched for genes expressed in founder cells, including nidogen and tartan, but it has not yet been determined whether they are potential targets for nautilus regulation. Gut constrictions are often absent or abnormal in the nau^armGFP mutant. Nautilus protein has been shown to be expressed in the gut constrictions in wild-type embryos, and this fits with the subsequent findings that founders and fusion-competent myoblasts are involved in the formation of visceral muscle and gut constriction. The founder cell pattern also prefigures adult myotube locations, so the weakness and uncoordinated movements seen in nau^armGFP larvae and adults may reflect alterations in the normal muscle fiber organization and attachments because of a general disruption of the founder cells throughout development (Wei, 2007).

Nautilus protein expression commences in a stereotypic pattern just as myogenic competence is being established, around stage 9–10. The founder cell marker, Duf-LacZ, is not highly expressed until stage 11, when muscle progenitors and fusion-competent myoblasts are segregated, but weak expression can be seen in a few Nautilus-positive cells in the posterior segments in the early embryo. By stage 12, founders (Duf-LacZ) organized into a segmentally repeated pattern begin to appear in the posterior segments and by this stage, most founders are Nautilus-positive. In a slightly magnified view of stage 13 embryos, subsets of Nautilus-positive founders are also seen to coexpress diverse muscle identity genes specifying both dorsal and ventral muscle groups; Kr and Nautilus are coexpressed in numerous more dorsally positioned founders in each hemisegment; coexpression of Nautilus and Eve was seen only in a group of founders located near the dorsal edge of the mesoderm in a region corresponding to the eventual position of the Eve-positive dorsal muscle, DA1; S59-LacZ and Nautilus are coexpressed in founders destined to form the ventral muscle cluster II composed of muscles 26/27/19/VaP, which also express Kr, Msh, and Apterous. Thus Nautilus marks subsets of founders that specify diverse muscle groups throughout the hemisegment, not just the minor dorsal muscles, DA3 and DO4, and these are disrupted in the nautilus null, as evidenced by the altered Duf-LacZ and Kr patterns (Wei, 2007).

Even though a majority of Duf-expressing founders are Nautilus-positive at stage 11, by stage 13, Nautilus expression is more restricted to subsets of Kr, S59, and Eve-positive cells. However, the transient expression of Nautilus likely underestimates its role in the specification of founder identity, because every muscle in the embryo contains one or more nuclei that initially expressed Nautilus, suggesting the loss of nautilus function could impact most muscles. Regardless, Nautilus expression is not detected in all founders once the muscle identity genes are activated, and this may determine, in part, the degree of disruption in the nautilus null. It is interesting to note that the integrity of the more dorsal muscle groups, where Nautilus, Kr, and Eve are coexpressed, is most often affected first in the least-penetrant nautilus null embryos and presents a phenotype similar to the nau¹⁸⁸ mutant (Wei, 2007).

In summary, not only does nautilus gene function play an important role in Drosophila myogenesis and viability as a determinant in founder cell patterning, but Nautilus expression is also an early marker for muscle founders involved in the specification diverse muscle groups in the embryo (Wei, 2007).

GENE STRUCTURE

date revised: 26 Feb 97 

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