faint sausage


DEVELOPMENTAL BIOLOGY

Embryonic

During stage 10, a widespread but weak expression of FAS mRNA is observed in all germ layers. In addition to the widespread expression, localized regions with higher expression levels are observed. In particular, in the dorsal, lateral and ventral ectoderm, in the middle of each segment, there is a circular spot of FAS expression corresponding to the region from which many sensillum precursor cells (SOPs) segregate. During later stages (stage 12 onward) FAS expression becomes concentrated in the ganglion mother cells and neurons forming the CNS; at the same time, FAS expression disappears in all other tissues except for the heart, which, like the CNS, expresses fas at a high level until late embryonic stages. In the developing CNS, most ganglion mother cells and neurons express FAS at some level throughout embryonic development into the larval period. In each neuromere, at the level of the two commissures, there is a coherent population of cells expressing FAS more strongly than the remainder of the cells of the neuromere. Apart from this distinction of a "high level" and "low level" FAS domain, the expression appears mottled, with individual or small groups of cells expressing at a higher level than their neighbors (Lekven, 1998).

A polyclonal antiserum raised against a portion of the Fas protein was used on western blots of embryonic extracts and for whole-mount immunohistochemical stainings of wild-type and fas mutant embryos. The embryonic Fas expression pattern was studied with this antiserum in whole-mount stainings; Fas protein expression closely corresponds to the pattern described above for the mRNA, with the exception that protein expression is seen in the somatic cells of the gonads in late embryos, but in situ hybridization fails to detect FAS mRNA in those cells. Following a weak, widespread expression during stages 10 and 11, Fas is expressed at fairly high levels in the CNS and heart. During early stage 12, there is a distinct expression in multiple clusters of early myoblasts, as well in the mesodermal precursors of hemocytes, which are located in the head mesoderm. In all tissues, Fas is localized in a somewhat 'punctate' pattern at the cell surface. Since Fas contains a signal sequence and no transmembrane domain, it is hypothesized that this staining represents protein localized to the outer cell surface. Interestingly, in the CNS, there is intense staining of the cortex, which contains neuron cell bodies and glia, while there was no detectable staining in the neuropile, which contains axons. Double labeling experiments, combining anti-Faint sausage antibody with antibodies against Repo (alias RK2, a homeodomain-containing protein expressed exclusively in glial cells) indicate that glial cells do not express significant levels of Fas. Thus, Faint sausage shows a very dynamic expression pattern, and in the CNS faint sausage appears to be expressed only on neuronal cell bodies.The fact that Fas is detected on neuronal cell bodies suggests that the observed axonal pathfinding defects are due to improper cell body migration during CNS condensation (Lekven, 1998).

Effects of Mutation or Deletion

The most dramatic effect caused by loss of fas function can be seen in the axonal patterning of the CNS. In wild-type embryos, early differentiating pioneer neurons form a scaffold of longitudinal tracts (connectives) and transverse tracts (commissures) along which later axons fasciculate. fas null mutants are characterized by the virtual absence of connectives. The ontogeny of this phenotype has been followed using the FasII antibody, which recognizes most of the pioneer neurons of the connectives. In the wild type, the first pioneer neurons are aCC (projecting posteriorly and then into the periphery), pCC and vMP2 (projecting anteriorly and forming a medial longitudinal tract), and MP1 and dMP2 (projecting posteriorly and forming a lateral longitudinal tract). All of these cells develop with their cell bodies in close contact with longitudinal glial cells (LGCs) along which they project their axons. In fas mutants, these pioneer neurons develop at abnormal positions and project their axons abnormally. The MPs project their axons peripherally, instead of longitudinally. Also, both pCC and aCC, which can be recognized by their early expression of FasII and by their expression of Even skipped, project their axons straight, but laterally, instead of longitudinally. Later forming axons follow this abnormal trajectory, leaving the CNS devoid of any orderly longitudinal tracts. In addition, the overall amount of axons (i.e. the number and the length integrated) in a fas mutant embryo appears largely reduced. Thus, fas function is required for the correct temporal differentiation of neurons in the CNS and for correct pathfinding by pioneer and follower axons (Lekven, 1998).

In the peripheral nervous system (PNS) of fas mutants, neurons fail to delaminate from the ectodermal epithelium. The sensory nervous system of wild-type embryos is composed of sensilla, small clusters of specialized cells distributed in an invariant pattern over the entire epidermis. The majority of sensilla, specialized for mechanoreception and chemoreception, are visible at the outer surface of the epidermis and are therefore called external sensilla. Each external sensillum consists of one or more subepidermal neurons and a group of accessory cells, all of which are formed by the mitotic division of a sensory organ precursor cell (SOP) located within the epidermis. Following SOP division, the presumptive sensory neuron moves from the epidermis into the interior of the embryo, whereas the accessory cells remain within the epidermis and form concentric sheaths around the sensory dendrite. Apical processes of the outer two accessory cells (trichogen cell and tormogen cell, respectively) form the stimulus-receiving apparatus of the sensillum. In fas mutant embryos the movement and shape of sensillum cells are defective. After a period of normal SOP division, many sensory neurons as visualized by the antibody mAb22C10 are located within the epidermis, instead of subepidermally. Epidermal cells surrounding the sensilla often do not assemble into regular monolayered sheets, as in wild-type, with an apical and basal surface, but pile up into 2-3 layers of irregularly shaped cells. Accessory cells of the sensilla fail to form lateral processes that wrap around the sensory dendrite, nor do they form apical processes that become the shaft and socket of the sensillum. Thus, fas is necessary for the delamination of the sensory neuron precursor and for the proper differentiation of the sensilla accessory cells (Lekven, 1998).

During Drosophila development, the salivary primordia areinternalized to form the salivary gland tubes. By analyzingimmuno-stained histological sections and scanning electronmicrographs of multiple stages of salivary glanddevelopment, it has been showm that internalization occurs in adefined series of steps, involves coordinated cell shapechanges, and begins with the dorsal-posterior cells of theprimordia. The ordered pattern of internalization is criticalfor the final shape of the salivary gland. In embryos mutantfor huckebein (hkb), which encodes a transcription factor,or faint sausage (fas), which encodes a cell adhesionmolecule, internalization begins in the center of theprimordia, and completely aberrant tubes are formed. Thesequential expression of hkb in selected cells of theprimordia presages the sequence of cell movements. It is proposed that hkb dictates the initial site of internalization,the order in which invagination progresses and,consequently, the final shape of the organ. It is proposed thatfas is required for hkb-dependent signaling events thatcoordinate internalization (Myat, 2000).

Salivary gland cells in embryos carrying a null allele of faint sausage (fas) do not invaginate. Examination of salivary gland morphogenesisin fas mutant embryos reveals that the cells invaginate butshow gross morphological defects that are very similar to thoseof hkb mutant embryos. By whole-mount analysis of fasmutant embryos stained with anti-dCREB-A, the placodes appearmorphologically identical to placodes of WT and hkb mutantembryos. At the stage when the dorsal-posterior pitforms in WT embryos, a slight indentation is observed near thecenter of the placode of fas mutant embryos, suggestive of cellshape changes. Although the initial indentationoccurs at slightly variable locations in different embryos ofsimilar age, the pit observed in late-stage fas mutants is trough-shapedand uniformly located close to the center of the placode. At late stages of embryogenesis, the overallmorphology of the salivary glands of fas mutants is similar tothat of hkb mutants; the glands are dome-shaped, are fused atthe ventral midline and remain close to the embryo surface (Myat, 2000).

Although the salivary gland placodes of fas mutants appearidentical to those of WT embryos at a gross morphologicallevel, histological sections reveal significantly altered cellmorphology. Instead of the monolayer of uniformly elongatedepithelial cells that is observed in sections of WT embryos,placode cells in fas mutants are found in multiple layers, andare variably elongated. At later stages, the pit thatforms in fas mutants is not as deep or wide as the pits of WTor hkb mutant embryos. Cells at the center of thepit appear elongated with basally positioned nuclei; however,the surrounding cells in the pit are round and found inmultiple layers. Cells in the anterior and posterior parts of the gland also form multi-layered placodes.The salivary glands of fas mutants are eventually internalized,despite gross abnormalities in cell shape. Theinternalized gland is comprised of a mixture of elongated andwedge-shaped cells. Cells in the anteriorand posterior parts of the gland are multi-layered.After internalization, the fas mutant salivary glands fuse intoone dome-shaped organ, which is located close to the ventralsurface, like the glands of hkb mutant embryos.Unlike the salivary gland cells of WT and hkb mutants,salivary gland cells of fas mutant embryos are not in anepithelial monolayer and, instead, appear to have condensedinto a single, multilayered organ with remnants of apotentially contiguous lumen. As in the hkb mutant embryos, potential secretory products, indicated by darkMethylene Blue staining, are found in the lumen of fas mutantembryos (Myat, 2000).

Since fas and hkb mutant embryos have similar salivary glandphenotypes at a gross morphological level, and hkb encodes atranscription factor, the expression of fas was examined in bothWT and hkb mutant embryos. Prior to invagination, FAS mRNAand protein are expressed in all secretory cells in WT embryos. At the start of invagination, FASmRNA levels decrease to the levels observed in surroundingepithelial cells, and it is no longer detected in cellsthat have been internalized. At this stage,higher levels of Fas protein are detectable in all secretory cells,including the invaginating dorsal-posterior cells, relative tosurrounding non-salivary gland cells. Fas protein isdetected in all secretory cells that have internalized,and this level is maintained throughout the remainder ofembryogenesis. Fas protein levelsappear highest at the apical membrane, although differentfixation procedures alter the relative levels of protein detected.The early expression of FAS mRNA and protein are unchangedin embryos mutant for hkb. Later, whenelevated levels of Fas protein are observed in the invaginatingdorsal-posterior cells of WT embryos, such elevated levels areinstead observed in cells at the center of the glands in hkbmutants, and these cells are the first to internalize.In the internalized secretory cells, the level of Fas appearsequivalent in hkb mutants and WT embryos. Thus,hkb affects fas expression transiently and only indirectly, byspecifying the order in which secretory cells are internalized (Myat, 2000).

During Drosophila embryogenesis the Malpighiantubules evaginate from the hindgut anlage and ina series of morphogenetic events form two pairs of longnarrow tubes, each pair emptying into the hindgutthrough a single ureter. Some of the genes that are involvedin specifying the cell type of the tubules havebeen described. Mutations of previously describedgenes were surveyed and ten were identified that are required for morphogenesis of the Malpighian tubules.Of those ten, four block tubule development atearly stages; four block later stages of development, andtwo, rib and raw, alter the shape of the tubules without arresting specificmorphogenetic events. Three of the genes, sna, twi, andtrh, are known to encode transcription factors and aretherefore likely to be part of the network of genes thatdictate the Malpighian tubule pattern of gene expression (Jack, 1999).

Mutations of faint sausage (fas) also arrest development of the tubulesat the early bud stage. fas encodes a member of theimmunoglobulin superfamily that is most likely to be anchoredto the cell membrane and function as an adhesionprotein. The protein is present in thecell membranes of neurons and is required for properneuronal migration and axon formation, suggesting thepossibility that interactions between Fas protein and proteinson neighboring cells guide cell and axon migration.If the Fas protein is in fact an adhesion protein, adhesiveinteractions between the tubule cells might be requiredfor the cells to adopt the proper position relative to oneanother and continue growth. However, no expression ofthe protein was reported in the Malpighian tubules, whilehigh levels were reported in the nervous system. Therefore another possibility is that the tipcell, which has neuronal characteristics, expresses Fas and requires it in order to induce proliferation of the tubule cells (Jack, 1999).


REFERENCES

Aurivillius, M., Hansen, O. C., Lazrek, M. B., Bock, E. and Obrink, B. (1990). The cell adhesion molecule Cell-CAM 105 is an ecto-ATPase and a member of the immunoglobulin superfamily. FEBS letters 264: 267-269. PubMed Citation: 2141577

Jack, J. and Myette, G. (1999). Mutations that alter the morphology of the Malpighian tubulesin Drosophila. Dev. Genes Evol. 209: 546-554. PubMed Citation: 10502111

Lekven, A. C., Tepass, U., Keshmeshian, M. and Hartenstein, V. (1998). faint sausage encodes a novel extracellular protein of the immunoglobulin superfamily required for cell migration and the establishment of normal axonal pathways in the Drosophila nervous system. Development 125: 2747-2758. PubMed Citation: 9636088

Myat, M. M. and Andrew, D. J. (2000). Organ shape in the Drosophila salivary gland is controlled by regulated, sequential internalization of the primordia. Development 127: 679-691. PubMed Citation: 10648227.

Neeper, M., Schmidt, A. M., Brett, J., Yan, S. D., Wang, F., Pan, Y.-C. E.,Elliston, K., Stern, D. and Shaw, A. (1992). Cloning and expression of acell surface receptor for advanced glycosylation end products of proteins. J.Biol. Chem. 267: 14998-15004. PubMed Citation: 1378843.

Ramos, R. G. P., Igloi, G. L., Lichte, B., Baumann, U., Maier, D., Schneider, T., BrandstŠtter, J. H., Fršhlich, A. and Fischbach, K.-F. (1993). The irregular chiasm C-roughest locus of Drosophila, which affects axonal projections and programmed cell death, encodes a novel immunoglobulin-like protein. Genes Dev. 7: 2533-2547. PubMed Citation: 7503814

Ranscht, B. (1988). Sequence of contactin, a 130-kD glycoprotein concentrated in areasof interneuronal contact, defines a new member of the immunoglobulin supergene family in the nervous system. J. Cell Biol. 107(4): 1561-1573. 89008597


faint sausage: Biological Overview | Developmental Biology | Effects of Mutation | References

date revised: 10 February 2013

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