myoblast city
The fusion of myoblasts leading to the formation of myotubes is an integral part of skeletal myogenesis in many organisms. In Drosophila, specialized founder myoblasts initiate fusion through expression of the receptor-like attractant Dumbfounded (Duf: Kin of irre/Kirre), which brings them into close contact with other myoblasts. Rolling pebbles (Rols), a gene expressed in founders, is an essential component for fusion during myotube formation. During fusion, Rols localizes in a Duf-dependent manner at membrane sites that contact other myoblasts. These sites are also enriched with D-Titin, which functions to maintain myotube structure and morphology. When Rols is absent or its localization is perturbed, the enrichment of D-Titin fails to occur. Rols encodes an ankyrin repeat-, TPR repeat-, and RING finger-containing protein. Rols, which is expressed specifically in founder cells, interacts with the cytoplasmic domain of Dumbfounded, a founder cell transmembrane receptor, and with Myoblast city, a cytoskeletal protein, both of which are also required for myoblast fusion. Thus Rols integrates the initial event of myoblast attraction with the downstream event of myotube structural organization by linking Duf to D-Titin (Chen, 2001).
The aggregation of Rols in distinctive cytoplasmic locations in founder cells, and the presence of multiple protein-protein interaction motifs in the Rols protein prompted an examination of whether Rols plays a role during myoblast fusion by mediating interactions between molecules in the myoblast fusion pathway(s). To test whether Rols interacts with other fusion molecules, coimmunoprecipitation assays were performed in Drosophila S2 cells using MYC-tagged Rols and other fusion proteins, including Blow, Duf, Mbc, and Sns, tagged with the V5-epitope at their carboxyl termini. Rols interacts with the founder cell receptor Duf but not the fusion-competent cell receptor Sns, despite the high homology shared by Duf and Sns. This specific interaction between Rols and Duf is consistent with the founder cell-specific expression of Rols. A cleaved form of is generated when full-length Duf is expressed in S2 cells. This form migrates slightly slower than the Duf cytoplasmic domain alone, suggesting that it is likely to contain both the transmembrane and the cytoplasmic domains. Interestingly, this cleaved form also associates with Rols. However, when the Duf cytoplasmic domain alone was tested, no interaction was detected. These results suggest that the transmembrane domain of Duf is required for its interaction with Rols. In addition, protein-protein interaction was detected between an amino-terminal fragment of Mbc and Rols, while no interaction was detected between Blow and Rols. The interactions between full-length Mbc and Rols could not be tested, since the full-length Mbc was not expressed at a detectable level (Chen, 2001).
During myoblast fusion, cell-cell recognition along with cell
migration and adhesion are essential biological processes. The factors involved
in these processes include members of the immunoglobulin superfamily like Sticks
and stones (Sns), Dumbfounded (Duf) and Hibris (Hbs), SH3 domain-containing
adaptor molecules like Myoblast city (Mbc) and multidomain proteins like Rolling
pebbles (Rols). For rolling pebbles, two differentially expressed
transcripts have been defined (rols7 and rols6). However, to date,
only a muscle fusion phenotype has been described and assigned to the lack of
the mesoderm-specific expressed rols7 transcript. This study shows that a
loss of the second rolling pebbles transcript, rols6, which is
expressed from the early bud to later embryonic stages during Malpighian tubule
(MpT) development, leads to an abnormal MpT morphology that is not due to
defects in cell determination or proliferation but to aberrant morphogenesis. In
addition, when Myoblast city or Rac are knocked out, a similar phenotype is
observed. Myoblast city and Rac are essentially involved in the development of
the somatic muscles and are proposed to be interaction partners of Rols7.
Because of the predicted structural similarities of the Rols7 and Rols6
proteins, it is argued that genetic interaction of rols6, mbc and
rac might lead to proper MpT morphology. It is also proposed that these
interactions result in stable cell connections due to rearrangement of the
cytoskeleton (Putz, 2005).
The Malpighian tubules (MpTs) of Drosophila arise as four buds from
the hindgut anlage close to its boundary with the posterior midgut primordium.
The cells of the four buds are characterised by the expression of the
transcription factor Cut (Ct) at stage 10 of embryogenesis.
During germ band extension at stage 11, the cells of the four tubule primordia
undergo cell proliferation, and the tubules begin to bud out. By stage 13,
proliferation is complete and short tubules have formed. From stage 13
onwards, cells from the caudal mesoderm join the MpT primordia and later the
stellate cells (SCs). From the end of germ band retraction, the tubules begin to
elongate due to cell rearrangement. In stage 15 and 16 embryos, the
characteristic stereotypic course of the four renal tubules through the
embryonic body is clearly visible. The paired posterior tubules span the
posterior abdominal and terminal segments of the embryo. The anterior tubules
extend forwards into abdominal segments 2/3 where the tubule
loops back on itself so that the tips of both anterior tubules lie more
posteriorly within the abdomen (Putz, 2005).
Since Rols6 is expressed in the Malpighian tubules (MpTs) throughout their
development, the role of Rols6 in the generation of this tissue was investigated.
For this purpose, a rols6-specific mutant was generated, in which the
majority of the putative promoter region of rols6 was deleted, and thereby
rols6 transcription was knocked out, while rols7 expression persisted as in the wild type. In this rols6-specific mutant, the early phase of
organogenesis is the same as in wild type, i.e. the MpTs consist of two cell
types, the principal cells (PCs) and the SCs. As the SCs originate from the mesoderm, one might
expect that they would be affected in rols mutants. However, in the
specific rols6 mutation generated, the SCs are able to migrate and
integrate between the PCs as observed in the wild type. However, the PCs and SCs
do not arrange correctly, and therefore, the typical MpT arrangement as found in
wild-type embryo is not observed for stage 15 embryos onwards. The anterior
tubules often show abnormal curves and lasso-like structures and fail to extend
through the abdominal cavity. These navigation defects might well result from
incorrect cell rearrangements, indicated by thickened regions of the tubules,
whereas other parts seem to have a typical wild-type organisation (Putz, 2005).
Evidence is presented that correct cell rearrangement is dependent
on Rols6 and proteins such as Mbc and Rac. These factors have been proposed to
act with Rols7 in a common signalling cascade during myoblast fusion. An
additional defect is the disorientation of MpTs in the body cavity, which again
is characteristic for rols6, mbc and rac mutants (Putz, 2005).
Homozygous rols6 mutants are viable, indicating that the physiological functions of principal cells and stellate cells are largely unaffected. Loss of rols6 expression only moderately affects embryonic viability. Furthermore, homozygous EP(3)3330*5a flies do not die prematurely in contrast to those lacking another gene essential for MpT formation, hibris. The strongest hibris allelic combination die early as adults. Also, in contrast to rols6 mutants, in hibris mutants, the number of SCs is strongly reduced. This might cause defects in excretory function of the tubules, and thus leads to the observed lethality (Putz, 2005).
rols6-specific mutants show no distortion in rols7
transcription and in muscle development indicating that rols6 is specific
for MpT development, while rols7 is essential for myogenesis. This is
consistent with the observatio that Rols6 is not able to rescue the myogenic defect in rols mutants (Putz, 2005).
myoblast city mutants, rolling pebbles mutants and rac1/rac2 double mutants show late defects in Malpighian tubule differentiation. mbc mutants exhibit a MpT phenotype and it is proposed that this might be due to a failure to complete cell rearrangement; a phenomenon which is more apparent in mbc mutants than for rolling pebbles ones. Mbc, the homologue of vertebrate DOCK180 in Drosophila, associates with the adapter protein Crk. This interaction regulates cell migration and cytoskeleton organisation in a Rac-dependent manner. This agrees with the finding that rac1/rac2 double mutants exhibit the characteristic MpT defects as rols6 and mbc mutants do. Rols7 and Duf have been shown to interact in myogenesis. The strong similarity between the Rols proteins and their proposed functions leads to the hypothesis that Rols6 interacts with a so far unknown partner in the PCs. It is proposed that Rols6 initiates a signalling cascade via Mbc and Rac that leads to the correct rearrangement of cells, presumedly by rearranging the cytoskeleton, as has been proposed for Rols7 in the myogenic precursor cells. In the development of the
somatic musculature, rearrangement of cytoskeleton is mediated by Blown fuse
(Blow) and Kette in the second fusion wave (Putz, 2005).
Individual factors and protein complexes involved in cell migration and cytoskeleton arrangement have been described from many model organisms as well as from cell culture experiments. DOCK180/CED-5, the homologues of Drosophila Myoblast city (Mbc) in vertebrates and in C. elegans, form a complex with
ELMO1/CED-12 that functions as a guanine nucleotide exchange factor (GEF). This
functional GEF promotes Rac activation, and thus facilitates cell migration and
rearrangement of the cytoskeleton. In vertebrates, additional protein
complexes are built via DOCK180/p130Cas/Crk interaction and regulate
cell migration and cytoskeletal organisation in a Rac-dependent manner. From kidney
cells of human and mouse, the signalling molecule NEPHRIN is known to be of
major importance in the podocyte for slit-diaphragm formation. Mutations in the
nephrin gene are the major cause of congenital nephrotic syndrome in
humans. In Drosophila, the homologue of vertebrate Nephrin, Hibris (hbs), is expressed during
MpT development specifically in SCs. Therefore, it is likely that during MpT
differentiation, Hibris mediates cell adhesion and arrangement between the PCs
and the SCs, a mechanism comparable to myogenesis. In vertebrates, CMS/CD2AP has
been identified as an interaction partner for Nephrin. The CMS/CD2AP homologue
in Drosophila can be detected in silico as CG11316. CD2AP knock-out mice die due to kidney failure. Moreover, the Nephrin/CD2AP complex is able to bind to actin and to p130Cas (corresponding to CG1212). In Drosophila,
homologues have been identified for all the above-mentioned factors involved in
these protein complexes. However, little is known about their role in the
developmental processes taking place during MpT development (Putz, 2005).
In Drosophila, a group of immunoglobulin-like proteins act in
cell-cell recognition and attraction during myogenesis. These processes
are also of importance in MpT development. Rolling pebbles is a multidomain and adapter-like protein. It is proposed that Rols6 interacts in Malpighian
tubule development with proteins also involved in myogenesis such as Mbc and
Rac. It is assumed that Rolling pebbles interacts with Mbc, and thus activates Rac.
This hypothesis is supported by the observations that mbc and rac
mutants exhibit defects in MpT development which might be linked to cell
organisation in this tissue (Putz, 2005).
The mechanisms underlying the stereotypic course
of the MpTs through the body cavity are still unclear. However, studies of
phenotypes of early determination mutants like numb show that the tip
cell and its sibling might both play a critical role in controlling the spatial
arrangement of the growing tubules. This is indicated by the MpT phenotypes of
numb mutants and UAS-numb embryos, where numb is
overexpressed. These embryos lack either the tip cell or the sibling cell but
form elongated MpTs with normally rearranged PCs. Although the PCs rearrange normally in these numb alleles, the MpTs are misrouted through the body cavity, as has been observed for rols and rac mutants. This raises the question whether determination of the tip cells is affected in rols
mutants (Putz, 2005).
Essential transcription factors for Tip cell determination and PC
cell proliferation are the A-SC, Krüppel and Seven up. These factors could be required for rols6 expression in the MpTs. However, since rols6 is expressed in the rudimentary primordia of MpTs in Krüppel mutants and in seven up mutants, this is unlikely. It is
assumed, therefore, that Rolling pebbles is not a signalling molecule involved in
cell specification through direct regulation of early genes, but rather that it
plays a role as an adapter molecule in a protein complex connecting the cells in
the tissue as Rols7 does in myogenesis. Since rols6, mbc and rac
mutant embryos exhibit the described MpT phenotype, it is likely that they
belong to a group of genes that can be helpful in discovering the mechanisms in
MpT development that lead to the typical thin tubule morphology through cell
rearrangement (Putz, 2005).
See the embryonic expression pattern of mbc at the Berkeley Drosophila Genome Project Patterns of Gene Expression Site.
Northern analysis reveals that mbc is expressed early in
development, in embryos ~0-4 h after egg laying. mbc
transcript levels remain relatively high during embryogenesis, with the possible exception of a decline from 8-12 h
that may be, in part, an artifact of slightly degraded
mRNA. Expression is not evident during larval stages, but the transcript does reappear during pupation, suggesting a possible role in adult development. A
form of mbc with slightly altered mobility appears late in
metamorphosis. This transcript may reflect alternative
splicing and is under further investigation. PCR amplification of two different regions from the
mRNA of unfertilized embryos reveals a small but detectable signal, and suggests that the transcript is maternally provided. Finally, the transcript was expressed in
adult males and females, as evidenced by PCR analysis of
cDNA (Erickson, 1997).
The earliest expression of the mbc transcript is in the pole
cells. It is later found in lateral portions of the embryo during cellularization but is not evident
at the termini. Surprisingly, the ventral furrow, which will
invaginate during gastrulation to form the mesoderm,
shows no expression at this time. At germband
elongation, expression is still quite strong in the ectoderm. By late stage 12, the mRNA appears to be decreasing in the ectoderm, leaving a pattern of stripes. mbc is expressed in both the mesoderm and
endoderm during stage 12. Expression decreases in both the epidermal layer and the somatic mesoderm
during stage 14 but remains strong in the visceral musculature. Examination of
a stage 16 embryo reveals mRNA in both the cardial and
pericardial cells of the dorsal vessel. Of note, the
mbc transcript is not observed in mature muscle fibers (Erickson, 1997).
The expression pattern of MBC was analyzed by fluorescent immunohistochemistry and confocal microscopy
using an antiserum directed against the COOH-terminal
portion of the protein. While slight temporal
differences were evident between maximal levels of
mRNA and maximal levels of protein in the pole cells, the expression of the
protein essentially correlates with that of the mRNA.
Mbc appears to be localized in the cytoplasm,
consistent with its human counterpart DOCK180. Mbc is
also present in the visceral musculature
and the dorsal vessel. Cross reactivity of
the Mbc antiserum is observed in the filtzkorper but does not correlate with the presence of transcript.
Although mRNA was not evident in mature muscles, the
protein can be detected in mature muscle at a low level (Erickson, 1997).
Fluorescent immunohistochemistry and confocal microscopy were used to confirm that Mbc is present in myoblasts. For this analysis, the embryos were hybridized with
antibodies to both Mbc and Mef2. The mef2 gene encodes a transcription factor that appears to be expressed
throughout the mesoderm, including somatic muscle precursors and all muscle fibers. As anticipated from the expression pattern of mRNA,
Mbc is present in ectodermal and endodermal germ layers. Of note, expression in the ectoderm is concentrated in
the epidermal layer and appears to be absent from the underlying neuroectoderm. Mbc is also clearly present in
presumptive myoblasts, coincident with the Mef2-expressing nuclei (Erickson, 1997).
Similar to the Drosophila Egfr and to the mammalian PDGFR family, stimulation of PDGF- and VEGF-receptor related (Pvr) activates the MAP-kinase pathway in Schneider cells as well as in border cells. However, it has been shown, by loss-of-function and gain-of-function experiments, that MAP-kinase signaling does not affect border cell migration. In addition, no effect of phospholipase C-gamma (PLC-gamma) or phosphatidylinositol 3' kinase (PI3K) has been demonstrated on this migration, using loss-of-function mutants (PLC-gamma) or border cell expression of dominant negative and dominant activated forms (PI3K). This was somewhat unexpected, since PLC-gamma and PI3K have been implicated in motility and guidance effects of RTKs (in particular PDGFR) in tissue culture cells. To address how Pvr signaling might be affecting cell migration in vivo, the effect of Pvr signaling on cell morphology and cytoskeleton was tested. In border cells as well as in other follicle cells, expression of lambda-Pvr has a dramatic effect on the actin cytoskeleton. Massive F-actin accumulation, actin-rich extensions, and changes in cell shape were produced in lambda-Pvr expressing follicle cells. The normal cells have modest cortical F-actin accumulation. This result was likely to be relevant to the guidance function of Pvr, because direct control of F-actin accumulation would allow receptor activation to control cell migration (Duchek, 2001).
The actin cytoskeleton has been shown to be affected by small GTPases of the Rho superfamily in many systems, with the exact effects depending on the cellular context. In the border cell migration system, Rac is an attractive candidate for mediating the effect of activated Pvr, since dominant negative Rac (RacN17) has been shown to inhibit border cell migration. Epistasis experiments could not be done by quantifying border cell migration because activated Pvr and dominant negative Rac have the same effect. Instead, whether Rac is required for the effect of Pvr on the actin cytoskeleton in follicle cells was tested. Coexpression of dominant negative Rac suppresses the effect of activated Pvr on the actin cytoskeleton. In addition, follicle cells expressing activated Rac (RacV12) have dramatic accumulation of F-actin, resembling that caused by activated Pvr. Finally, if Rac were directly downstream of Pvr, one would expect activated Rac to inhibit border cell migration, as observed for the activated receptor. Although a previous study reported that activated Rac does not affect border cell migration (Murphy, 1996), this was reexamined using the slboGal4 driver and it was found that activated Rac completely blocks border cell migration. These results are consistent with a role of Rac in the guidance pathway downstream of Pvr (Duchek, 2001).
In mammalian tissue culture cells, PDGF stimulation can cause Rac-dependent F-actin accumulation, suggesting that the effect observed in follicle cells may reflect a conserved pathway. PI3K has been implicated as a mediator of the effect of PDGFR on Rac in Swiss 3T3 cells. However, PI3K does not appear to play a key role in guidance of border cell migration as discussed above. To investigate how Pvr might lead to activation of Rac, two groups of Drosophila mutants were tested for their effect on border cell migration: mutants in genes shown to be downstream of receptor tyrosine kinases in other contexts, and mutants linked to Rac activation. Most mutations were homozygous lethal, so their effect in border cells was tested by generating mutant clones in a heterozygous animal (mosaic analysis). Of the 8 genes tested, only myoblast city (mbc) has a detectable effect on border cell migration. Mbc is homologous to mammalian DOCK180 and C. elegans CED-5. Mbc/DOCK180/CED-5 acts as an activator of Rac (Duchek, 2001 and references therein).
mbc has been independently identified in a screen for gain-of-function suppressors of the slbo mutant phenotype. slbo mutant border cells migrate poorly. Increased expression of mbc in slbo mutant border cells improves their migration, suggesting that mbc has a positive role in promoting border cell migration. Mbc protein is detected in follicle cells, including border cells, and is overexpressed upon induction of the EP element EPg36390 located upstream of mbc. Removing mbc function from border cells by generating mutant clones causes severe delays in their migration. At stage 10, when 100% of control (GFP) clones have reached the oocyte, only 10% of mbc mutant border cell clusters have done so, and these are the oldest egg chambers. Thus, mbc is not absolutely required for border cell migration, but, contrary to the other genes implicated in RTK and Rac signaling, loss of mbc function severely impairs this cell migration (Duchek, 2001).
myoblast city:
Biological Overview
| Evolutionary Homologs
| Regulation
| Effects of Mutation
| References
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