The activity of the DAF-2 insulin-like receptor is required for Caenorhabditis elegans reproductive growth and normal adult life span. Informatic analysis identified 37 C. elegans genes predicted to encode insulin-like peptides. Many of these genes are divergent insulin superfamily members, and many are clustered, indicating recent diversification of the family. The ins genes are primarily expressed in neurons, including sensory neurons, a subset of which are required for reproductive development. Structural predictions and likely C-peptide cleavage sites typical of mammalian insulins suggest that ins-1 is most closely related to insulin. Overexpression of ins-1, or expression of human insulin under the control of ins-1 regulatory sequences, causes partially penetrant arrest at the dauer stage and enhances dauer arrest in weak daf-2 mutants, suggesting that INS-1 and human insulin antagonize DAF-2 insulin-like signaling. A deletion of the ins-1 coding region does not enhance or suppress dauer arrest, indicating a functional redundancy among the 37 ins genes. Of five other ins genes tested, the only other one bearing a predicted C peptide also antagonizes daf-2 signaling, whereas four ins genes without a C peptide do not, indicating functional diversity within the ins family. Insulin and its related proteins define a superfamily of secreted proteins that share a structural motif stabilized by a set of stereotypical disulfide bonds (Blundell and Humbel 1980;Murray-Rust et al. 1992). Insulin superfamily genes are ubiquitous in vertebrates, and have been identified in invertebrates, including insects, molluscs, and the nematode Caenorhabditis elegans (Duret et al. 1998;Gregoire et al. 1998;Smit et al. 1998;Kawano et al. 2000). Seven members of the insulin superfamily have been identified in humans, including insulin (Brown et al. 1955), insulin-like growth factors (IGFs) I and II (Rinderknecht and Humbel 1978a,b), relaxins HI and HII (Bedarkar et al. 1977;Schwabe and McDonald 1977), early placenta insulin-like peptide (EPIL) (Chassin et al. 1995;Koman et al. 1996), and relaxin-like factor (Bullesbach and Schwabe 1995). These hormones mediate diverse functions. Insulin is a metabolic hormone that acts on target tissues to increase glucose uptake and energy storage, IGFs are mitogenic stimulators that control cell survival and proliferation, and relaxin causes dilation of the symphysis pubis before parturition and vasodilation. No function is yet known for either EPIL or relaxin-like factor. Bombyxin in silk moths (Satake et al. 1997), and the neurons that secrete locust and molluscan insulin-related proteins (Smit et al. 1988;Lagueux et al. 1990) regulate metabolism, implicating insulin-like proteins in metabolic control broadly in animal phylogeny. The insulin-like proteins that regulate metabolism, insulin in vertebrates, bombyxin from silk moths, molluscan MIP, and locust LIRP, appear to be processed proteolytically to remove an internal C peptide (Smit et al. 1988;Lagueux et al. 1990;Kondo et al. 1996). This proc...

The semaphorin family contains a large number of secreted and transmembrane proteins, some of which are known to act as repulsive axon guidance cues during development or to be involved in immune function. We report here on the identification of semaphorin K1 (sema K1), the first semaphorin known to be associated with cell surfaces via a glycosylphosphatidylinositol linkage. Sema K1 is highly homologous to a viral semaphorin and can interact with specific immune cells, suggesting that like its viral counterpart, sema K1 could play an important role in regulating immune function. Sema K1 does not bind to neuropilin-1 or neuropilin-2, the two receptors implicated in mediating the repulsive action of several secreted semaphorins, and thus it likely acts through a novel receptor. In contrast to most previously described semaphorins, sema K1 is only weakly expressed during development but is present at high levels in postnatal and adult tissues, particularly brain and spinal cord.The semaphorins constitute a large family of evolutionally conserved glycoproteins that are defined by a characteristic semaphorin domain of approximately 500 amino acids (1-3). The first vertebrate semaphorin, collapsin-1 in chick, was identified by its ability to induce growth cone collapse (4). Consistent with this function, its mammalian homologue, sema III, has been shown to repel specific subsets of sensory axons (5). As a result of these and other studies, Coll-1/sema III/D has been implicated in the patterning of sensory axon projections into the ventral spinal cord and cranial nerve projections into the periphery (6 -11).Several other semaphorins have also been implicated as repulsive and/or attractive cues in axon guidance, axon fasciculation, and synapse formation (1,(12)(13)(14)(15)(16)(17). In addition, members of the semaphorin family have been implicated in functions outside the nervous system, including bone skeleton and heart formation (9), immune function (18, 19), tumor suppression (20 -22), and conferring drug resistance to cells (23).Recent studies have identified the first semaphorin receptor as a member of the neuropilin family. Neuropilin-1 is a high affinity receptor for sema III, E, and IV, whereas neuropilin-2 binds differentially to the subfamily of secreted semaphorins (24 -27).The vertebrate semaphorin family can be classified into several phylogenetically distinct subfamilies (Ref. 15, Fig. 1B). Each subfamily has a unique structural arrangement of protein domains. The secreted members of the semaphorin family contain a characteristic semaphorin domain at the NH 2 terminus, followed by an immunoglobulin (Ig) domain and a stretch of basic amino acids in the carboxyl-terminal region. Between the NH 2 -terminal semaphorin domain and the transmembrane spanning region, the transmembrane semaphorins contain several alternative structural motifs, including either an Ig domain, a stretch of thrombospondin repeats, or a sequence with no obvious domain homology. Interestingly, semaphorin-like sequences have been identifi...

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An outstanding challenge in developmental biology is to reveal the mechanisms underlying the morphogenesis of complex organs. A striking example is the developing inner ear of the vertebrate, which acquires a precise three-dimensional arrangement of its constituent epithelial cells to form three semicircular canals, a central vestibule and a coiled cochlea (in mammals). In generating a semicircular canal, epithelial cells seem to ‘disappear’ from the center of each canal. This phenomenon has been variously explained as (i) transdifferentiation of epithelium into mesenchyme, (ii) absorption of cells into the expanding canal or (iii) programmed cell death. In this study, an in situ DNA-end labeling technique (the TUNEL protocol) was used to map regions of cell death during inner ear morphogenesis in the chicken embryo from embryonic days 3.5-10. Regions of cell death previously identified in vertebrate ears have been confirmed, including the ventromedial otic vesicle, the base of the endolymphatic duct and the fusion plates of the semicircular canals. New regions of cell death are also described in and around the sensory organs. Reducing normal death using retrovirus-mediated overexpression of human bcl-2 causes abnormalities in ear morphogenesis: hollowing of the center of each canal is either delayed or fails entirely. These data provide new evidence to explain the role of cell death in morphogenesis of the semicircular canals.

Attempts to use replicationcompetent retroviruses to target genes to the chick CNS have met with limited success for injections performed prior to stage 14 using A-or E-subgroup viruses. This study was aimed at improving CNS infection by varying the stage of injection, viral envelope subgroup, viral titer, and the presence or absence of a transgene and/or the polycation polybrene in the inoculum. RCASBP vectors were injected into the neural tube of stages 3-13 embryos and protein expression was determined 9-48 hr later for forebrain, hindbrain, retina, and inner ear. Optimal injection parameters were defined which balanced good survival rates with high levels of transgene expression at early stages. The results demonstrate nearly complete expression of virus-mediated transgenes in neural tissues at stages 15-21 following injection of B-envelope RCASBP with polybrene at stages 7.5-12. This technique can now be applied to study the roles of genes in cell-autonomous events such as cell connectivity, physiology, and differentiation, as well as neural patterning and regional identity. o 1996 Wiley-Liss, Inc.