Gary R. Hime

D-Cbl, the Drosophila homologue of the c-Cbl proto-oncogene, interacts with the Drosophila EGF receptor in vivo, despite lacking C-terminal adaptor binding sites

The c-Cbl proto-oncogene encodes a multidomain phosphoprotein that has been demonstrated to interact with a wide range of signalling proteins. The biochemical function of c-Cbl in these complexes is, however, unclear. Recent studies with the C. elegans Cbl homologue, sli-1, have suggested that Cbl proteins may act as negative regulators of EGF receptor (EGFR) signalling. As the EGFR and other protein tyrosine kinase receptor signalling pathways are highly conserved between insects and vertebrates, we sought a Drosophila homologue of c-Cbl for a detailed genetic analysis. We report here that Drosophila melanogaster has a single gene, D-cbl, that is homologous to c-cbl. We find that D-cbl encodes a 52 kDa protein that has a high degree of similarity to c-Cbl and SLI-1 across novel phosphotyrosine-binding (PTB) and RING finger domains. Surprisingly, however, D-Cbl is C-terminally truncated relative to c-Cbl and SLI-1 and consequently is unable to bind SH3-domain containing adaptor proteins, including the Drosophila Grb2 homologue, Drk. Although the D-Cbl protein lacks Drk binding sites it can nevertheless associate with a tyrosine phosphorylated protein, or is itself tyrosine phosphorylated in an DER dependent manner and associates with activated Drosophila EGF receptors (DER) in vivo. Consistent with a role for D-Cbl in DER dependent patterning in the embryo and adult, D-Cbl is expressed at a high level in early embryos and throughout the imaginal discs in third instar larvae. This study forms the basis for future genetic analysis of D-Cbl, aimed at gaining insights into the role of Cbl proteins in signal transduction.

WNT/Frizzled signaling in eye development and disease.

The canonical Wnt/Fzd signaling pathway is highly conserved among various species. Increasing evidence is accumulating for non-canonical Wnt signaling pathways, analogous to those discovered in Drosophila, being operative in vertebrates. Similarly, the networks of genes involved in eye development show significant conservation during evolution. The amenability of Drosophila for genetic manipulation and analysis of ocular phenotypes has delivered a great deal of information about the roles of the Wnt/Fzd signaling pathways at various stages of ocular development and growth, particularly in regulating the formation and size of the eye field, cell proliferation, polarity and differentiation. In addition to the numerous recent studies that have identified the expression of various components of these signaling pathways in the developing vertebrate eye, functional studies have revealed significant parallels in the way that Wnt/Fz signals regulate the formation of the vertebrate eye field and also the proliferation and differentiation of cells, particularly in the lens and retina. Significant advances have also recently been made in identifying mutations in these signaling pathways that underlie or contribute to various ocular diseases such as exudative vitreoretinopathy, retinal degenerations, cataract, ocular tumors and various congenital ocular malformations. Combined with the mechanistic studies in vertebrate and invertebrate models, these studies point to important functional roles for Wnt/Fzd pathways in the human eye. Further investigation of how these pathways function during eye development and growth may yield important insights into novel therapeutic approaches to treat or prevent diseases that cause blindness.

Expression of hedgehog signalling components in adult mouse testis

Hedgehog (Hh) signalling is known to regulate many aspects of normal development as well as being upregulated in various cancers. Signalling is mediated by the Gli family of zinc finger transcription factors. Based on observations that deletion of one of the three Hh genes, Dhh, leads to male infertility, we hypothesized that regulated expression of Hh signalling components would be a feature of adult spermatogenesis. We used in situ hybridization to characterise Gli gene expression in juvenile and adult mouse testes. In the first wave of spermatogenesis, mRNAs encoding all three Glis are detected in spermatogonia and Sertoli cells. In adult mouse testes, these transcripts are observed in spermatogonia and spermatocytes, with reduced signal intensity in round spermatids. The mRNAs encoding key effectors of Hh signalling, Ptc2, Smo, and Fu, are also most apparent in spermatogonia, spermatocytes, and to a lower extent in round spermatids. In contrast, mRNA encoding SuFu, a negative regulator of Hh signalling, was most predominant in round spermatids and the protein is evident in round and elongating spermatids, suggesting that SuFu protein may switch off Hh signalling in haploid germ cells. Overall, the coordinated expression pattern of these genes in adult mouse testis indicates a role for Hh signalling in spermatogenesis.Developmental Dynamics 235:3063–3070, 2006.

HOW Is Required for Stem Cell Maintenance in the Drosophila Testis and for the Onset of Transit-Amplifying Divisions

The mechanisms by which germline stem cells (GSCs) in the Drosophila testis undergo asymmetric division to regenerate a stem cell as well as a daughter (gonialblast) that will only undergo a further four mitotic divisions prior to entering premeiotic S phase and differentiating into a cyst of spermatocytes are not fully resolved. Here we demonstrate that the HOW RNA-binding protein is required for maintenance of CycB and therefore mitotic progression in GSCs and gonialblasts as well as determining the timing of the spermatogonial divisions. HOW is normally expressed in a complementary pattern to Bam in the germline and bam mRNA is bound by HOW in vivo. Ectopic expression of the HOW(L) isoform is associated with a delay in accumulation of Bam to the level required for differentiation, resulting in extra mitotic divisions. Spatiotemporal regulation of HOW expression is therefore required to specify the four spermatogonial transit-amplifying divisions.

Drosophila Hfp negatively regulates dmyc and stg to inhibit cell proliferation.

Mammalian FIR has dual roles in pre-mRNA splicing and in negative transcriptional control of Myc. Here we show that Half pint (Hfp), the Drosophila orthologue of FIR, inhibits cell proliferation in Drosophila. We find that Hfp overexpression potently inhibits G1/S progression, while hfp mutants display ectopic cell cycles. Hfp negatively regulates dmyc expression and function, as reducing the dose of hfp increases levels of dmyc mRNA and rescues defective oogenesis in dmyc hypomorphic flies. The G2-delay in dmyc-overexpressing cells is suppressed by halving the dosage of hfp, indicating that Hfp is also rate-limiting for G2-M progression. Consistent with this, the cycle 14 G2-arrest of stg mutant embryos is rescued by the hfp mutant. Analysis of hfp mutant clones revealed elevated levels of Stg protein, but no change in the level of stg mRNA, suggesting that hfp negatively regulates Stg via a post-transcriptional mechanism. Finally, ectopic activation of the wingless pathway, which is known to negatively regulate dmyc expression in the wing, results in an accumulation of Hfp protein. Our findings indicate that Hfp provides a critical molecular link between the developmental patterning signals induced by the wingless pathway and dMyc-regulated cell growth and proliferation.

Cytoplasmic male sterility in Drosophila melanogaster associated with a mitochondrial CYTB variant

Somewhat buried in a paper investigating mitochondrial genetic variation and ageing in Drosophila melanogaster (Clancy, 2008) was a serendipitous finding; cytoplasmic male sterility involving naturally occurring mitonuclear incompatibility, associated with a single polymorphism in mitochondrial cytochrome b (CYTB) protein.

A Drosophila analogue of v-Cbl is a dominant-negative oncoprotein in vivo.

Cells rely on the ability to receive and interpret external signals to regulate growth, differentiation, and death. Positive transduction of these signals to the cytoplasm and nucleus has been extensively characterized, and genetic studies in Drosophila have made major contributions to the understanding of these pathways. Less well understood, but equally important, are the mechanisms underlying signal down-regulation. Here we report biochemical and genetic characterization of the Drosophila homologue of c-Cbl, a negative regulator of signal transduction with ubiquitin-protein ligase activity. A new isoform of D-Cbl, D-CblL, has been identified that contains SH3-binding and UBA domains previously reported to be absent. Genetic analysis demonstrates that Dv-cbl, analogous to the mammalian v-cbl oncogene, is a dominant negative mutation able to enhance signalling from the Drosophila Egfr and co-operate with activating mutations in the sevenless pathway to produce melanotic tumours. In addition, our data show genetic and biochemical links between D-Cbl and proteins involved in endocytosis and ubiquitination, suggesting that v-Cbl may exert its oncogenic effect by enhancing receptor signalling as a consequence of suppressing receptor endocytosis.

Drad21, a Drosophila rad21 homologue expressed in S-phase cells.

Cohesin is an evolutionarily conserved multiprotein complex required to establish and maintain sister chromatid cohesion. Here, we report the cloning and initial characterization of the Drosophila homologue of the fission yeast rad21 cohesin subunit, called Drad21. The Drad21 coding region was localized to centromeric heterochromatin and encodes a 715 amino acid (aa) protein with 42% aa identity to vertebrate Rad21p-homologues, 25% with Scc1p/Mcd1p (S. cerevisiae) and 28% with Rad21p (S. pombe). Sequences with similarity to the sites of proteolytic cleavage identified in Scc1p/Mcd1p are not evident in DRAD21. Northern blot and mRNA in-situ studies show that Drad21 is developmentally regulated, with high levels of expression in early embryogenesis, in S-phase cells of proliferating imaginal tissues, and in the early endocycling cells of the embryonic gut.

Snai1 regulates cell lineage allocation and stem cell maintenance in the mouse intestinal epithelium

Snail family members regulate epithelial‐to‐mesenchymal transition (EMT) during invasion of intestinal tumours, but their role in normal intestinal homeostasis is unknown. Studies in breast and skin epithelia indicate that Snail proteins promote an undifferentiated state. Here, we demonstrate that conditional knockout of Snai1 in the intestinal epithelium results in apoptotic loss of crypt base columnar stem cells and bias towards differentiation of secretory lineages. In vitro organoid cultures derived from Snai1 conditional knockout mice also undergo apoptosis when Snai1 is deleted. Conversely, ectopic expression of Snai1 in the intestinal epithelium in vivo results in the expansion of the crypt base columnar cell pool and a decrease in secretory enteroendocrine and Paneth cells. Following conditional deletion of Snai1, the intestinal epithelium fails to produce a proliferative response following radiation‐induced damage indicating a fundamental requirement for Snai1 in epithelial regeneration. These results demonstrate that Snai1 is required for regulation of lineage choice, maintenance of CBC stem cells and regeneration of the intestinal epithelium following damage.

Wnt signaling regulates Snai1 expression and cellular localization in the mouse intestinal epithelial stem cell niche.

Snail genes are transcriptional repressors well known to play important roles in epithelial to mesenchymal transitions during both embryogenesis and cancer metastasis. Although they are generally regarded as markers of mesenchymal cells, Snail genes have also recently been implicated in regulating stem cell populations in both Drosophila and vertebrates. In this study we investigate Snai1, a member of the mouse Snail family, in the intestinal stem cell niche and examine the relationship between canonical Wnt signaling, a key regulatory pathway of intestinal stem cells, and expression and cellular localization of Snai1. Strong nuclear expression of Snai1 was detected in the crypt base columnar stem cells in the adult small intestine while Snai1 was mostly found in the cytoplasm of differentiated enterocytes and enteroendocrine cells. Expression and cellular localization of Snai1 in the intestinal epithelium appears to be regulated by the canonical Wnt signaling pathway as Snai1 expression was dramatically reduced after conditional deletion of β-catenin. Conversely, significant nuclear Snai1 was detected in polyps derived from Apc(min) mice and in intestinal villi after conditional mutation of Apc in AhCre, Apc(f/f) mice, indicating that upregulation of the Wnt pathway in the intestinal epithelium induces both increased expression and nuclear localization of Snai1.