RESUMO
BACKGROUND: SPARC is a collagen-binding glycoprotein whose functions during early development are unknown. We previously reported that SPARC is expressed in Drosophila by hemocytes and the fat body (FB) and enriched in basal laminae (BL) surrounding tissues, including adipocytes. We sought to explore if SPARC is required for proper BL assembly in the FB. RESULTS: SPARC deficiency leads to larval lethality, associated with remodeling of the FB. In the absence of SPARC, FB polygonal adipocytes assume a spherical morphology. Loss-of-function clonal analyses revealed a cell-autonomous accumulation of BL components around mutant cells that include collagen IV (Col lV), Laminin, and Perlecan. Ultrastructural analyses indicate SPARC-deficient adipocytes are surrounded by an aberrant accumulation of a fibrous extracellular matrix. CONCLUSIONS: Our data indicate a critical requirement for SPARC for the proper BL assembly in Drosophila FB. Since Col IV within the BL is a prime determinant of cell shape, the rounded appearance of SPARC-deficient adipocytes is due to aberrant assembly of Col IV.
Assuntos
Membrana Basal/fisiologia , Drosophila melanogaster/embriologia , Corpo Adiposo/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Adipócitos/metabolismo , Animais , Animais Geneticamente Modificados , Membrana Basal/metabolismo , Mapeamento Cromossômico , Colágeno Tipo IV/metabolismo , Corpo Adiposo/metabolismo , Genoma de Inseto , Glicoproteínas/metabolismo , Proteoglicanas de Heparan Sulfato/metabolismo , Laminina/metabolismo , Larva/fisiologia , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Mutação , Sistema Nervoso/embriologia , Osteonectina/metabolismo , FenótipoRESUMO
Bazooka/Par-3 (Baz) is an evolutionarily conserved scaffold protein that functions as a master regulator for the establishment and maintenance of cell polarity in many different cell types. In the vast majority of published research papers Baz has been reported to localize at the cell cortex and at intercellular junctions. However, there have also been several reports showing localization and function of Baz at additional subcellular sites, in particular the nuclear envelope and the neuromuscular junction. In this study we have re-assessed the localization of Baz to these subcellular sites in a systematic manner. We used antibodies raised in different host animals against different epitopes of Baz for confocal imaging of Drosophila tissues. We tested the specificity of these antisera by mosaic analysis with null mutant baz alleles and tissue-specific RNAi against baz. In addition, we used a GFP-tagged gene trap line for Baz and a bacterial artificial chromosome (BAC) expressing GFP-tagged Baz under control of its endogenous promoter in a baz mutant background to compare the subcellular localization of the GFP-Baz fusion proteins to the staining with anti-Baz antisera. Together, these experiments did not provide evidence for specific localization of Baz to the nucleus or the neuromuscular junction.
Assuntos
Núcleo Celular , Proteínas de Drosophila , Drosophila melanogaster , Junção Neuromuscular , Animais , Núcleo Celular/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Junção Neuromuscular/metabolismo , Transporte Proteico , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismoRESUMO
Apico-basal polarity is the defining characteristic of epithelial cells. In Drosophila, apical membrane identity is established and regulated through interactions between the highly conserved Par complex (Bazooka/Par3, atypical protein kinase C and Par6), and the Crumbs complex (Crumbs, Stardust and PATJ). It has been proposed that Bazooka operates at the top of a genetic hierarchy in the establishment and maintenance of apico-basal polarity. However, there is still ambiguity over the correct sequence of events and cross-talk with other pathways during this process. In this study, we reassess this issue by comparing the phenotypes of the commonly used baz(4) and baz(815-8) alleles with those of the so far uncharacterized baz(XR11) and baz(EH747) null alleles in different Drosophila epithelia. While all these baz alleles display identical phenotypes during embryonic epithelial development, we observe strong discrepancies in the severity and penetrance of polarity defects in the follicular epithelium: polarity is mostly normal in baz(EH747) and baz(XR11) while baz(4) and baz(815) (-8) show loss of polarity, severe multilayering and loss of epithelial integrity throughout the clones. Further analysis reveals that the chromosomes carrying the baz(4) and baz(815-8) alleles may contain additional mutations that enhance the true baz loss-of-function phenotype in the follicular epithelium. This study clearly shows that Baz is dispensable for the regulation of polarity in the follicular epithelium, and that the requirement for key regulators of cell polarity is highly dependent on developmental context and cell type.
RESUMO
SPARC is an evolutionarily conserved collagen-binding extracellular matrix (ECM) glycoprotein whose morphogenetic contribution(s) to embryonic development remain elusive despite decades of research. We have therefore used Drosophila genetics to gain insight into the role of SPARC during embryogenesis. In Drosophila embryos, high levels of SPARC and other basal lamina components (such as network-forming collagen IV, laminin and perlecan) are synthesized and secreted by haemocytes, and assembled into basal laminae. A SPARC mutant was generated by P-element mutagenesis that is embryonic lethal because of multiple developmental defects. Whereas no differences in collagen IV immunostaining were observed in haemocytes between wild-type and SPARC-mutant embryos, collagen IV was not visible in basal laminae of SPARC-mutant embryos. In addition, the laminin network of SPARC-mutant embryos appeared fragmented and discontinuous by late embryogenesis. Transgenic expression of SPARC protein by haemocytes in SPARC-mutant embryos restored collagen IV and laminin continuity in basal laminae. However, transgenic expression of SPARC by neural cells failed to rescue collagen IV in basal laminae, indicating that the presence of collagen IV deposition requires SPARC expression by haemocytes. Our previous finding that haemocyte-derived SPARC protein levels are reduced in collagen-IV-mutant embryos and the observation that collagen-IV-mutant embryos showed a striking phenotypic similarity to SPARC-mutant embryos suggests a mutual dependence between these major basal laminae components during embryogenesis. Patterning defects and impaired condensation of the ventral nerve cord also resulted from the loss SPARC expression prior to haemocyte migration. Hence, SPARC is required for basal lamina maturation and condensation of the ventral nerve cord during Drosophila embryogenesis.