In the Drosophila embryo, nautilus is expressed in a subset of muscle precursors and differentiated
fibers and is capable of inducing muscle-specific transcription, as well as myogenic transformation. In
this study, an examination was made of the consequences of nautilus loss-of-function on the development of the
somatic musculature. Genetic and molecular characterization of two overlapping deficiencies,
Df(3R)nau-9 and Df(3R)nau-11a4, reveal that both of these deficiencies remove the nautilus gene
without affecting a common lethal complementation group. Individuals transheterozygous for these
deficiencies survive to adulthood, indicating that nautilus is not an essential gene. These embryos are,
however, missing a subset of muscle fibers, that includes the dorsal oblique, dorsal acute and lateral longitudinals, providing evidence that (1) some muscle loss can be
tolerated throughout larval development and (2) nautilus does play a role in muscle development.
In addition to the absence of particular muscle fibers in transheterozygotes, novel muscle fibers are occasionally present in these embryos. The appearance of these fibers provides support for the hypothesis that, in the absence of nau, precursors to muscles such as 3 and 19 can undergo further myogenic differentiation. In some cases, these novel fibers have features reminiscent of specific muscles. For example, a novel muscle fiber is seen in a position and orientation similar to that of muscle 2. It is enticing to consider the possibility that this muscle arises from a precursor to muscle 3 and its differentiation program has been diverted by the lack of Nau expression.
Examination of muscle precursors in these embryos reveals that nautilus is not required for the
formation of muscle precursors, but rather plays a role in their differentiation into mature muscle fibers.
It is suggested that nautilus functions in a subset of muscle precursors to implement their specific
differentiation programs (Keller, 1998).
The expression of the MyoD gene homolog, nautilus (nau), in the Drosophila embryo defines a subset of
mesodermal cells known as the muscle 'pioneer' or 'founder' cells. These cells are thought to establish
the future muscle pattern in each hemisegment. Founders appear to recruit fusion-competent mesodermal
cells to establish a particular muscle fiber type. In support of this concept every somatic muscle in the
embryo is associated with one or more nautilus-positive cells. However, because of the lack of known
(isolated) nautilus mutations, no direct test of the founder cell hypothesis has been possible. Toxin ablation and genetic interference by double-stranded RNA (RNA interference or RNA-i) have been used to
determine both the role of the nautilus-expressing cells and the nautilus gene, respectively, in embryonic
muscle formation. In the absence of nautilus-expressing cells muscle formation is severely disrupted or
absent. A similar phenotype is observed with the elimination of the nautilus gene product by genetic
interference upon injection of nautilus double-stranded RNA (Misquitta, 1999).
To test whether the direct ablation of nautilus mRNA would result in a disrupted muscle
phenotype in the Drosophila embryo, the gal4/UAS system was used to express nautilus antisense RNA
throughout the mesoderm. Females from the gal4 enhancer trap line 24B containing the
twi-gal4 transgene were crossed with males from four independent lines homozygous for a gal4 UAS
antisense nautilus transgene containing only the coding region in reverse orientation. The degree of
disruption in the muscle pattern of the progeny flies depends partially on the particular antisense transgenic
line used in the cross. Previous studies with the
overexpression of nautilus gave a phenotype that includs the formation of some additional muscles and a
disruption of the heart tube, presumably because of the formation of skeletal muscle cells in the heart tube
itself. With the additional results from the antisense induction experiments it is concluded that nautilus
plays a major role in the formation of the muscle pattern and may be involved in the determination of the
muscle founder cell lineage in the embryo, because the muscle phenotypes resulting from the ricin
ablation of the nautilus-positive cells and the nautilus antisense expression are so similar.
These results define a crucial role for
nautilus in embryonic muscle formation. The application of RNA interference to a variety of known
Drosophila mutations as controls gives phenotypes essentially indistinguishable from the original mutation.
RNA-i provides a powerful approach for the targeted disruption of a given genetic function in Drosophila (Misquitta, 1999).
The results from the
injection of nautilus dsRNA point to a more general approach for the analysis of gene function during
Drosophila development and suggest that the RNA interference method essentially would mimic a gene
knock-out in the injected generation of Drosophila embryos. To test this idea a variety of
cDNA clones were obtained representing a maternal gene expressed in the embryo (daughterless); additional genes
involved in myogenesis (S59, DMEF2); homeobox genes (engrailed and S59); a gene important for
gastrulation (twist), and a gene expressed in the adult eye (white). This panel of genes covers most stages
of Drosophila development. twist was initiatially tested because the mutant has a clear phenotype that is easy to score when compared with
wild-type larva. The injection of twist dsRNA (the complete coding region) into embryos
produces a twisted larval phenotype that is indistinguishable from the original twist mutation. Similarly, injection of the first 1,200 bp
of engrailed dsRNA produces the compressed dentical belt pattern characteristic of an engrailed null
mutant. Daughterless
mRNA is both maternally loaded and expressed zygotically, and the mutant phenotype produces very
characteristic disruptions in the central nervous system (CNS) and peripheral nervous system (PNS). It has been shown previously that mex3, a maternally loaded RNA in C. elegans, can be ablated by
dsRNA injection into the gonads. daughterless dsRNA (complete coding region) was injected and the characteristic neuronal phenotypes were sought by using the mAb MAB 22C10. The CNS as well as
the PNS were disrupted to varying degrees in the injected embryos. The severity of the phenotype consistently shows a CNS disruption with a variable
PNS pattern, possibly reflecting the fact that the CNS is formed before the PNS. This result suggests that
maternally loaded as well as zygotically expressed RNA can be affected by RNA-i in Drosophila. The
homeobox gene S59 marks a subset of muscle founder cells for 5 of 29 muscles in each hemisegment of
the embryo corresponding to muscles 5, 18, 25, 26, and 27. Embryos with an S59 lacZ transgene
marking muscles 18 and 25 were injected with S59 dsRNA (complete coding region).
In this case, the S59-specific lacZ antibody-staining pattern is abolished. The total muscle pattern
for embryos injected with S59 dsRNA,
although disrupted, still shows the presence of poorly organized muscle groups in each hemisegment. This
is unlike the almost complete absence of muscle observed with the injection of nautilus dsRNA. DMEF2, a member of the MADS domain transcription factor family, is essential for muscle
formation in Drosophila. The DMEF2 / embryo has no muscle and is missing the characteristic
gut constrictions found in the uninjected embryo. Injection of DMEF2 dsRNA (complete
coding region) results in embryos that lack any detectable muscle and an absence of gut morphology (Misquitta, 1999).
Because particular RNA interference phenotypes are transferable to the next generation of C. elegans, it was particularly interesting to see whether genes expressed in the adult eye could be affected by
the injection of dsRNA into the embryo. The white gene was chosen, even though it is expressed
throughout embryogenesis: it was asked if any aspect of the white-eyed mutant phenotype could be observed
after the injection of white dsRNA (the first 500 bp from the P element minigene) into wild-type embryos
with red eyes. Phenotypes indicating interference with white gene function were observed in response to
the RNA interference with white dsRNA,
although the frequency of the mutant phenotype is extremely low (<3%) when compared with the level of
typical mutant phenotypes scored in the embryos injected with dsRNA (>75%). Similar to the results
reported in C. elegans, very few molecules of white dsRNA appear to be required to obtain some
evidence of interference with white gene function in the adult eye, because on the
order of only107 molecules were injected. This last result supports the idea that RNA interference is acting catalytically
because the transition from embryo to adult fly would substantially dilute the injected dsRNA (Misquitta, 1999).
nautilus (nau), the single Drosophila member of the bHLH-containing myogenic regulatory family of genes, is expressed in a subset of muscle precursors and differentiated fibers. It is capable of inducing muscle-specific transcription as well as myogenic transformation, and plays a role in the differentiation of a subset of muscle precursors into mature muscle fibers. The nau zygotic loss-of-function phenotype has been determined using genetic deficiencies in which the gene is deleted. This genetic loss-of-function phenotype differs from the loss-of-function phenotype determined using RNA interference (Misquitta, 1999). The present
study re-examines this loss-of-function phenotype using EMS-induced mutations that specifically alter the nau gene, and extends the genetic analysis to include the loss of both maternal and zygotic nau function. In brief, embryos lacking nau both maternally and zygotically are missing a distinct subset of muscle fibers, consistent with its apparent expression in a subset of muscle fibers. The muscle loss is tolerated, however, such that the loss of nau both maternally and zygotically does not result in lethality at any stage of development (Balagopalan, 2001).
The subtle muscle phenotype exhibited by the
deficiency embryos represents the zygotic loss-of-function phenotype.
Although zygotic expression of nau is not essential for
survival to adulthood, the eggs of adult females lacking nau
exhibit severely reduced rates of fertilization. While a clear
explanation for this mutant phenotype remains to be determined,
it has facilitated the isolation of EMS-induced nau
specific mutations termed nau. Embryos with homozygous null mutations are missing a subset of muscle fibers. Individuals lacking these
muscles still survive to adulthood. Analysis of RNA from ovaries and unfertilized eggs did not reveal maternally provided nau transcripts that might
account for the difference between the zygotic loss-of-function phenotype and that observed with RNAi (Misquitta, 1999). Interpretation of these results as conclusive was prevented, however, by the potential for
alternative forms of the nau mRNA that may not be
detected by the chosen oligonucleotides. In addition, RT-PCR
analyses could not address whether nau protein was
provided to the egg. These explanations for the subtle
zygotic loss-of-function phenotype could be eliminated
only by the generation of null alleles and subsequent
analysis of germline clone embryos. These embryos were
derived from ovaries in which the germline cells, and
resulting eggs, were lacking nau genetically and were fertilized
by nau mutant sperm. The elimination
of any hypothetical maternally-provided nau does not alter
the loss-of-function phenotype or survival rate of embryos
lacking nau. These observations therefore do not support
the existence of a maternal contribution for nau, confirming
that nau is essential for the formation of only a subset
of muscle fibers but not adult viability (Balagopalan, 2001).
The subtle muscle phenotype observed in flies lacking
nau is in contrast to the more critical role that the vertebrate
Myogenic Regulatory Factors (MRFs) play in vertebrate myogenesis, and was not
anticipated at the time of its initial isolation. Such early
expectations might, however, be somewhat naive in the
context of current understanding of Drosophila myogenesis.
Specification of the elaborate pattern of larval body wall muscles actually
begins concurrent with the earliest stages of myogenesis in
the Drosophila embryo. Distinct equivalence groups composed
primarily of post-mitotic myoblasts segregate from
the mesoderm at specific locations. In a process of lateral inhibition mediated by Notch, a single muscle progenitor will then be selected from the
cells within each equivalence group. This single founder
cell, which may undergo one additional mitotic division,
then seeds the fusion process and controls the unique
features of the resulting muscle fiber. Thus, the larval body wall muscles that develop in a Drosophila embryo are not derived from a common pool of
homogeneous myoblasts, and appear to segregate from the
mesoderm with distinct features. The results presented here establish that
nau, the single Drosophila homolog of the MRFs, is not
required for determination of all embryonic myoblasts. Indeed, no factor has yet
been identified that is specifically required for the determination
of all myoblasts. In fact, based upon its pattern of
expression and ectopic behavior, Drosophila Twist may
serve such a function, making a
general role for nau unnecessary. Alternatively, one might
anticipate the existence of several factors that, through
individual and combinatorial mechanisms, are responsible
for differentiation of founder myoblasts. Consistent with
this prediction, several genes are expressed in subsets of
founder myoblasts and, in at least some cases, are essential
for the development of a subset of muscle fibers. It seems plausible that nau is simply another example of a gene that serves such a function (Balagopalan, 2001).
It is noted that the roles of other highly conserved proteins
in Drosophila myogenesis are in some contrast to their
roles in vertebrate myogenesis. For example, murine TWI is
a powerful negative regulator of skeletal muscle differentiation,
whereas a high level of Drosophila Twi is a critical
determinant of somatic myogenesis. In addition, although the C. elegans twi homolog hlh-8 is involved in the development of a subset of non-striated muscle, it is not required for formation of striated muscle. Definitive
comparisons of the functions of the vertebrate and
invertebrate MEF2 family members in skeletal and somatic
myogenesis remain incomplete or are precluded by early lethality.
However, even invertebrate family members play distinctly
different roles. For example, embryos lacking the
single Drosophila family member Dmef2 exhibit severe
defects in the differentiation of all three muscle lineages:
somatic, cardiac, and visceral, while the single C. elegans MEF2 homolog is not essential for myogenesis. Finally, distinct differences in function
have been observed among members of the MRF family
of bHLH proteins. In contrast to the critical role in all
muscle fibers revealed by mouse knockouts for the MRFs,
these data suggest a nonessential muscle fiber-specific role for
nau. Yet another role seems to be suggested by the analysis
of mutations in CeMyoD, the single C. elegans MRF
homolog. In brief, CeMyoD appears to be essential for the
integrity of the muscle fibers but is not necessary for their
formation (Balagopalan, 2001).