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 are internalized to form the salivary gland tubes. By analyzing immuno-stained histological sections and scanning electron micrographs of multiple stages of salivary gland development, it has been showm that internalization occurs in a defined series of steps, involves coordinated cell shape changes, and begins with the dorsal-posterior cells of the primordia. The ordered pattern of internalization is critical for the final shape of the salivary gland. In embryos mutant for huckebein (hkb), which encodes a transcription factor, or faint sausage (fas), which encodes a cell adhesion molecule, internalization begins in the center of the primordia, and completely aberrant tubes are formed. The sequential expression of hkb in selected cells of the primordia 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 that fas is required for hkb-dependent signaling events that coordinate internalization (Myat, 2000).

Salivary gland cells in embryos carrying a null allele of faint sausage (fas) do not invaginate. Examination of salivary gland morphogenesis in fas mutant embryos reveals that the cells invaginate but show gross morphological defects that are very similar to those of hkb mutant embryos. By whole-mount analysis of fas mutant embryos stained with anti-dCREB-A, the placodes appear morphologically identical to placodes of WT and hkb mutant embryos. At the stage when the dorsal-posterior pit forms in WT embryos, a slight indentation is observed near the center of the placode of fas mutant embryos, suggestive of cell shape changes. Although the initial indentation occurs at slightly variable locations in different embryos of similar age, the pit observed in late-stage fas mutants is trough-shaped and uniformly located close to the center of the placode. At late stages of embryogenesis, the overall morphology of the salivary glands of fas mutants is similar to that of hkb mutants; the glands are dome-shaped, are fused at the ventral midline and remain close to the embryo surface (Myat, 2000).

Although the salivary gland placodes of fas mutants appear identical to those of WT embryos at a gross morphological level, histological sections reveal significantly altered cell morphology. Instead of the monolayer of uniformly elongated epithelial cells that is observed in sections of WT embryos, placode cells in fas mutants are found in multiple layers, and are variably elongated. At later stages, the pit that forms in fas mutants is not as deep or wide as the pits of WT or hkb mutant embryos. Cells at the center of the pit appear elongated with basally positioned nuclei; however, the surrounding cells in the pit are round and found in multiple 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. The internalized gland is comprised of a mixture of elongated and wedge-shaped cells. Cells in the anterior and posterior parts of the gland are multi-layered. After internalization, the fas mutant salivary glands fuse into one dome-shaped organ, which is located close to the ventral surface, 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 an epithelial monolayer and, instead, appear to have condensed into a single, multilayered organ with remnants of a potentially contiguous lumen. As in the hkb mutant embryos, potential secretory products, indicated by dark Methylene Blue staining, are found in the lumen of fas mutant embryos (Myat, 2000).

Since fas and hkb mutant embryos have similar salivary gland phenotypes at a gross morphological level, and hkb encodes a transcription factor, the expression of fas was examined in both WT and hkb mutant embryos. Prior to invagination, FAS mRNA and protein are expressed in all secretory cells in WT embryos. At the start of invagination, FAS mRNA levels decrease to the levels observed in surrounding epithelial cells, and it is no longer detected in cells that have been internalized. At this stage, higher levels of Fas protein are detectable in all secretory cells, including the invaginating dorsal-posterior cells, relative to surrounding non-salivary gland cells. Fas protein is detected in all secretory cells that have internalized, and this level is maintained throughout the remainder of embryogenesis. Fas protein levels appear highest at the apical membrane, although different fixation procedures alter the relative levels of protein detected. The early expression of FAS mRNA and protein are unchanged in embryos mutant for hkb. Later, when elevated levels of Fas protein are observed in the invaginating dorsal-posterior cells of WT embryos, such elevated levels are instead observed in cells at the center of the glands in hkb mutants, and these cells are the first to internalize. In the internalized secretory cells, the level of Fas appears equivalent in hkb mutants and WT embryos. Thus, hkb affects fas expression transiently and only indirectly, by specifying the order in which secretory cells are internalized (Myat, 2000).

During Drosophila embryogenesis the Malpighian tubules evaginate from the hindgut anlage and in a series of morphogenetic events form two pairs of long narrow tubes, each pair emptying into the hindgut through a single ureter. Some of the genes that are involved in specifying the cell type of the tubules have been described. Mutations of previously described genes were surveyed and ten were identified that are required for morphogenesis of the Malpighian tubules. Of those ten, four block tubule development at early stages; four block later stages of development, and two, rib and raw, alter the shape of the tubules without arresting specific morphogenetic events. Three of the genes, sna, twi, and trh, are known to encode transcription factors and are therefore likely to be part of the network of genes that dictate the Malpighian tubule pattern of gene expression (Jack, 1999).

Mutations of faint sausage (fas) also arrest development of the tubules at the early bud stage. fas encodes a member of the immunoglobulin superfamily that is most likely to be anchored to the cell membrane and function as an adhesion protein. The protein is present in the cell membranes of neurons and is required for proper neuronal migration and axon formation, suggesting the possibility that interactions between Fas protein and proteins on neighboring cells guide cell and axon migration. If the Fas protein is in fact an adhesion protein, adhesive interactions between the tubule cells might be required for the cells to adopt the proper position relative to one another and continue growth. However, no expression of the protein was reported in the Malpighian tubules, while high levels were reported in the nervous system. Therefore another possibility is that the tip cell, which has neuronal characteristics, expresses Fas and requires it in order to induce proliferation of the tubule cells (Jack, 1999).

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

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

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.

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.

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 a cell surface receptor for advanced glycosylation end products of proteins. J. Biol. Chem. 267: 14998-15004.

Ramos, R. G. P., Igloi, G. L., Lichte, B., Baumann, U., Maier, D., Schneider, T., Brandstätter, J. H., Fröhlich, 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.

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

date revised: 10 August 98

Home page: The Interactive Fly © 1997 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.