Whole-mount stainings of stage 10 embryos with labeled bin cDNA probes and Bin antibodies reveal that bin is expressed in progenitors of all three types of visceral musculature. These include the 11 bilateral patches of circular midgut muscle progenitors in the dorsal mesoderm in parasegments (PS)2-12 (also known as trunk visceral mesoderm progenitors), the hindgut and foregut visceral mesoderm, as well as the caudal visceral mesoderm which is located just anteriorly to the hindgut visceral mesoderm. Double stainings of early embryos with Bin and Bap antibodies show that the expression of Bin and Bap is activated almost simultaneously in the trunk visceral mesoderm progenitors. However, at early stage 10, cells were detected that express Bap but not Bin, indicating that bap expression initiates shortly before bin. bin is expressed in an additional, smaller group of dorsal mesodermal cells in PS 1, which upon coalescence of the metameric cell clusters also becomes part of the trunk visceral mesoderm. An important difference between bin and bap is that after the coalescence and segregation of the trunk visceral mesoderm, bap expression becomes segmental and then disappears, whereas bin is maintained uniformly and at constant levels. The circular midgut muscle precursors, which at stage 14 form a dorsal row and a ventral row and then surround the midgut, as well as the foregut and hindgut muscles, continue to express bin until late embryogenesis (Zaffran, 2001).

The early expression of bin in all visceral mesoderm derivatives allows an examination of the dynamic morphogenetic changes and a comparison of the gene activities in these developing tissues. Double stainings for Bin and Bap show that, in contrast to the trunk visceral mesoderm, the hindgut visceral mesoderm expresses bin shortly before bap, and the caudal visceral mesoderm does not express Bap at any time. The caudal visceral mesoderm splits bilaterally and then each group of cells moves towards and onto the posterior-most patch of trunk visceral mesoderm progenitors. The nuclei of the caudal visceral mesoderm cells retain Bin during their migration and dispersion along the trunk visceral mesoderm until they differentiate into longitudinal midgut muscles (Zaffran, 2001).

DmFoxF (Biniou) is a novel Drosophila fork head domain factor, which is expressed in the visceral mesoderm of the embryo. DmFoxF is the fly orthologue of the vertebrates FOXF1 and FOXF2 transcription factors. DmFoxF shares homology with FOXF1 and FOXF2 in its fork head domain, and it is able to specifically bind DNA sequences recognized by these vertebrate fork head factors. In stage 10-11 embryos, the DmFoxF protein is detected in the nuclei of cells of the presumptive visceral mesoderm. It localizes at the segmental cell clusters of the mesoderm, which will eventually develop to surround the midgut endoderm. DmFoxF is also expressed in the proctodeal mesoderm, which will develop into the visceral mesoderm of the hindgut (Perez Sanchez, 2002).

Effects of Mutation or Deletion

The bin gene maps to 65DE on the third chromosome and consists of four exons. Among lethal mutations that were obtained in a saturation screen at this location, one complementation group was identified with three EMS-induced alleles that correspond to bin. Sequencing of the bin loci from the respective mutant chromosomes confirmes that all three alleles, binI1, binS4, and binR22, carry mutations in the bin open reading frame (ORF). The mutations map to three different positions within the first exon and in each case introduce a transition from a CAG (Gln) to TAG (Stop) codon. Because the encoded polypeptides are truncated N-terminally to the forkhead domain and are expected to lack any DNA-binding activity, all three alleles are likely null alleles (Zaffran, 2001).

The function of bin in visceral mesoderm development was studied by staining bin mutant embryos with a variety of markers including the Ig-domain adhesion molecule Fasciclin III (FasIII), the Goalpha subunit Brokenheart (Bkh), and the Armadillo-repeat protein Vimar: these represent early and specific markers for the trunk visceral mesoderm and developing circular midgut musculature. In bin mutant embryos, none of these three markers are expressed in the trunk visceral mesoderm at any stage of development. The specificity of this phenotype is underscored by the observation that the progenitors and differentiated cells of the heart, in which bin is not expressed, show normal expression of all tested markers in bin mutant embryos. In addition, FasIII expression and tissue morphology can be restored in bin mutant embryos to almost normal levels by expressing wild-type bin in the trunk visceral mesoderm primordia under the control of a bap enhancer. The disruptions in the trunk visceral mesoderm in bin mutant embryos are slightly stronger than those found in bap null mutants, which show residual FasIII expression in thoracic segments and trace amounts in abdominal segments (coinciding with residual bin expression in these areas. The observed partial rescue of FasIII expression in abdominal segments of bap null mutants upon forced bin expression with a bap enhancer suggests that bin is a major effector of bap activity in trunk visceral mesoderm development (Zaffran, 2001).

The development of the caudal visceral mesoderm in bin mutant embryos was examined using the expression of the FoxC gene crocodile (croc) and the bHLH54F gene as markers. The normal expression of both genes in the early caudal visceral mesoderm in bin mutants shows that bin is not required for the formation of these cells. However, in bin mutants, anterior migration of these cells stalls during stage 11. Because bin is expressed in the longitudinal gut muscle precursors, the observed disruption of the migration and differentiation of these cells could reflect a cell-autonomous function of bin. However, the requirement of bin for normal trunk visceral mesoderm differentiation and the known function of the trunk visceral mesoderm in guiding caudal visceral mesoderm migration could also suggest that the observed phenotype is nonautonomous. To distinguish between these two possibilities, bHLH54F expression was examined in bin mutant embryos in which bin expression was brought back exclusively in the trunk visceral mesoderm via a bap enhancer. In these embryos, bHLH54F expression as well as the migration and morphology of the longitudinal muscle precursors appears normal, thus indicating that the disruption of these processes in the complete absence of bin activity is, at least predominantly, a nonautonomous effect. Presumably, the abnormal or absent trunk visceral mesoderm in bin mutants is unable to support proper guidance and differentiation of longitudinal gut muscle precursors. A similar effect is observed for the endoderm, which has also been shown to require normal trunk visceral mesoderm as a substrate for migration. In summary, the bin phenotypes in the trunk and caudal visceral mesoderm as well as the endoderm are almost identical to the ones that have been observed in tin and bap null mutant embryos. In contrast to the midgut musculature, no abnormalities are detected in the morphology or gene expression patterns of the foregut and hindgut visceral mesoderm in either bin or bap mutants (Zaffran, 2001).

The fate of the primordial cells of the trunk visceral mesoderm in bin or bap mutant embryos was further studied with a bap-lacZ marker. In wild-type as well as bin and bap mutant backgrounds, bap3-lacZ expression initiates during stage 10 in a normal metameric pattern, and ß-gal protein perdures in descendants of these cells until late embryogenesis. Therefore, bap-driven ß-gal marks the trunk visceral mesoderm and, at late stages, the circular gut muscle cells of wild-type embryos. In bin mutant embryos carrying bap3-lacZ, ß-gal stainings show that the presumptive trunk visceral mesoderm primordia segregate towards the interior and coalesce into a band as in wild-type embryos. However, there are irregularities in the arrangement of the visceral mesoderm cells, which become much more pronounced after stage 11. At stage 13, these cells fail to become columnar, are not tightly attached to the endoderm, and become clustered segmentally instead of maintaining a continuous band. During stages 14-17, only a few of the bap3-lacZ expressing cells are attached to the endoderm, whereas the majority of them are scattered in areas within or underneath the somatic mesoderm. In addition, the midgut fails to undergo any constrictions. bap null mutants show a similar although more severe phenotype. More specifically, the presumptive visceral mesoderm cells internalize, but coalescence of the clusters during stage 11 is incomplete, and at later stages, very few of the bap3-lacZ expressing cells are attached to the endoderm (Zaffran, 2001).

Because of the apparent intermingling of bap3-lacZ-positive cells with somatic mesoderm, a test was performed to see whether cells derived from the trunk visceral mesoderm primordia become incorporated into body wall muscle fibers in bin and bap mutant embryos. Syncytial muscle fibers stained for myosin heavy chain (MHC) are positive for ß-gal, whereas in wild-type backgrounds, bap3-lacZ does not produce any significant signals in the somatic musculature. Because the particular ß-gal from this construct is both nuclear and cytoplasmic, it is inferred that the ß-gal-positive nuclei seen in the syncytial muscle fibers in bin and bap mutant embryos are the ones that are derived from cells that would normally form midgut muscles. In bin mutant embryos, the transformation from a visceral into a somatic muscle fate appears to affect a large proportion although not all of the bap3-lacZ-positive cells, whereas in bap null mutants, essentially all of them appear to be transformed (Zaffran, 2001).

To test whether bin has the potential to cause cell fate transformations from somatic to visceral mesoderm, the effects of ectopic expression of bin in the entire early mesoderm under the control of a twist enhancer was examined. Ectopic bin causes ectopic expression of bap, which becomes expressed almost uniformly in the dorsal mesoderm. The expression of additional differentiation markers for trunk visceral mesoderm, FasIII, vimar mRNA, and vimar-lacZ, is also expanded upon ectopic bin expression to include ventral areas that are normally occupied by the somatic mesoderm. Stainings for Even-skipped (Eve) demonstrate that ectopic bin interferes with the specification of dorsal somatic muscle and pericardial cell progenitors. Furthermore, stainings for ß3-tubulin and muscle myosin show that ectopic bin disrupts somatic muscle differentiation, including myoblast fusion. Altogether, these observations strongly suggest that the presence of bin activity is able to force the development of somatic mesodermal cells towards the trunk visceral mesoderm pathway. However, the atypical morphology, the supernumerary cells that express visceral mesoderm markers, and the diminished ectopic marker expression at late stages indicate that, at least under these particular conditions of ectopic bin expression, the cell fate transformations in this direction are not complete (Zaffran, 2001).

The final overall shape of the salivary gland and its position within the developing embryo arise as a consequence of both its intrinsic properties and its interactions with surrounding tissues. This study focuses on the role of directed cell migration in shaping and positioning the Drosophila salivary gland. The salivary gland turns and migrates along the visceral mesoderm to become properly oriented with respect to the overall embryo. Salivary gland posterior migration requires the activities of genes that position the visceral mesoderm precursors, such as heartless, thickveins, and tinman, but does not require a differentiated visceral mesoderm. A role for integrin function in salivary gland migration is demonstrated. Although the mutations affecting salivary gland motility and directional migration cause defects in the final positioning of the salivary gland, most do not affect the length or diameter of the salivary gland tube. These findings suggest that salivary tube dimensions may be an intrinsic property of salivary gland cells (Bradley, 2003).

In htl, tkv, and tinman, the residual fragments of VM express Fas3, have a VM-like structure, and are able to direct salivary gland migration if present along its migratory path. Thus, the residual structures appeared to be differentiated VM with wild-type properties. To determine whether salivary gland migration requires a differentiated VM, embryos with mutations in the VM-specific gene biniou (bin) were examined. In bin mutant embryos, VM precursors segregate from dorsal mesoderm and move internally where they coalesce into the typical VM band; however, all tested VM-specific genes, including Fas3, fail to be expressed in bin mutants. Thus, an intact structure formed from VM precursors is present in bin mutants, but the VM precursor cells fail to express markers indicative of differentiation from a general mesodermal cell into a VM-specific cell. The salivary glands in bin mutants had no defects in turning or posterior migration, suggesting that guidance of salivary gland posterior migration by the VM requires neither the terminal differentiation of the precursors nor the function of any VM gene whose expression is bin-dependent (Bradley, 2003).

The VM forms a contiguous structure that may physically block salivary cells from further dorsal movement, thereby causing the cells to move posteriorly, in the path of least resistance. Alternatively or additionally, there may be a bin-independent factor (or factors) that guides salivary gland migration in a more instructive way, perhaps via a secreted signal or a transmembrane guidance molecule. If the mesodermal cue were informational, a signaling pathway functioning within salivary gland cells would have to be involved. A screen of several candidate pathways revealed that mutations disrupting the FGFR1-, FGFR2-, EGF-, DPP-, JNK-, or Wg-signaling pathway did not have phenotypes consistent with a role in the salivary cells for their migration. Thus, focus was placed on molecules known to have a more direct role in migration, specifically the integrin family of cell adhesion molecules, which are heterodimers of two transmembrane proteins, an alpha and a ß subunit. In Drosophila, each of the five identified alpha subunits (alphaPS1-5) is thought to dimerize with the ßPS subunit encoded by the myospheroid (mys) gene. The alpha subunit of alphaPS2ßPS (PS2) integrin is expressed in all mesodermal cells beginning at a very early stage, suggesting that PS2 integrin is likely to be present in the VM precursor cells prior to bin-dependent differentiation. Indeed, alphaPS2 RNA expression was observed in the mesoderm of binR22 homozygotes. In embryos mutant for inflated (if), the gene encoding the alphaPS2 subunit, migration of two tissues along the VM is affected, the endoderm and the tracheal visceral branch. Thus, the PS2 integrin is required to make the VM a suitable substrate for the migration of at least two distinct cell populations (Bradley, 2003).


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biniou: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 20 July 2012

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