极性蛋白Crumbs胞内域功能保守性研究

跨膜蛋白Crumbs(Crb)是细胞顶部的决定因子,对上皮细胞顶-底极性的建立和维持起着关键的作用。其胞内域虽然仅有37个氨基酸,但对Crb的功能必不可少。在果蝇(Drosophila melanogaster)中,如果胞内域发生突变,将造成胚胎发育异常、上皮细胞顶底极性丧失等严重后果。Crb胞内域从果蝇到小鼠(Mus musculus)和人类(Homo sapiens)具有很高的同源性,但线虫(Caenorhabditis elegans)两个Crb蛋白的胞内域与果蝇和哺乳动物却较为不同。为验证线虫Crb蛋白胞内域是否功能保守,本文利用基因组工程法(Genomic engineering),将果蝇基因组中Crb基因编码胞内域的部分替换为一致性和相似性较远的线虫Crb2基因的相应区段。与其他Crb胞内域突变果蝇不同,替换突变体胚胎发育正常,Crb及其他极性蛋白的表达和定位正常,胚胎上皮细胞顶底极性能够正确的建立和维持。这些结果证实虽然线虫和果蝇Crb蛋白胞内域之间存在大量序列变异,但重要的氨基酸位点和功能模块则完全保守。     

Expression of a novel secretory form (Crb1s) of mouse Crumbs homologue Crb1 in skin development

Drosophila Crumbs and the mammalian homologues encoded by the Crb genes are transmembrane proteins required for determination of retinal cell polarity. We cloned a novel variant of mouse Crb1 and termed it Crb1s. Since the 3'-end of exon 6 remained unspliced, Crb1s coded for a short secretory protein lacking the transmembrane and cytoplasmic domains required for the function of Crb1. The Crb1 expression was confined to brain and eye, whereas Crb1s was detectable in various tissues including skin, lung, and kidney in adult mice. Active expression of Crb1s, but not Crb1, was observed during the skin development, in which localization of the Crb1s protein was altered from the basal layer to the upper layers. Cultured mouse keratinocytes synthesized the Crb1s protein and secreted a 80 kDa processed form to the supernatant. After Ca(2+)-induced differentiation, Crb1s became associated with focal adhesions and cell-cell contacts. Crb1s may play a role distinct from that of Crb1 in epidermal tissue morphogenesis.     

Expression of Chx10 and Chx10-1 in the developing chicken retina

We have isolated full-length cDNAs of chick Chx10 and Chx10-1, two members of the paired type homeobox/CVC gene family. A comparison of sequences suggests that Chx10 is closely related to Alx/Vsx-2 and Vsx-2 of zebrafish and goldfish, respectively; while Chx10-1 is closely related to Vsx-1 of zebrafish and goldfish. Chx10 and Chx10-1 are expressed in the early retinal neuroepithelium, but not in the pigment epithelium and lens. The expression of Chx10 is present in most retinal neuroblasts, while Chx10-1 exhibits a novel pattern along the nasotemporal border. In the differentiating retina, both Chx10 and Chx10-1 are restricted to bipolar cells and are maintained at a low level in bipolar cells of the mature retina.     

FERM protein EPB41L5 is a novel member of the mammalian CRB-MPP5 polarity complex

Cell polarity is induced and maintained by separation of the apical and basolateral domains through specialized cell-cell junctions. The Crumbs protein and its binding partners are involved in formation and stabilization of adherens junctions. In this study, we describe a novel component of the mammalian Crumbs complex, the FERM domain protein EPB41L5, which associates with the intracellular domains of all three Crumbs homologs through its FERM domain. Surprisingly, the same FERM domain is involved in binding to the HOOK domain of MPP5/PALS1, a previously identified interactor of Crumbs. Co-expression and co-localization studies suggested that in several epithelial derived tissues Epb4.1l5 interacts with at least one Crumbs homolog, and with Mpp5. Although at early embryonic stages Epb4.1l5 is found at the basolateral membrane compartment, in adult tissues it co-localizes at the apical domain with Crumbs proteins and Mpp5. Overexpression of Epb4.1l5 in polarized MDCK cells affects tightness of cell junctions and results in disorganization of the tight junction markers ZO-1 and PATJ. Our results emphasize the importance of a conserved Crumbs-MPP5-EPB41L5 polarity complex in mammals.     

Vsx1, a rapidly evolving paired-like homeobox gene expressed in cone bipolar cells.

The paired-like homeodomain (HD) protein Chx10 is distinguished by the presence of the CVC domain, a conserved 56 amino acid sequence C-terminal to the HD. In mammals, Chx10 is essential both for the proliferation of retinal progenitor cells and for the formation or survival of retinal bipolar interneurons. We describe the cloning and characterization of a mouse Chx10 homologue, Vsx1; phylogenetic analysis suggests that Vsx1 and its putative vertebrate orthologues have evolved rapidly. Vsx1 expression in the adult is predominantly retinal. Whereas Chx10 is expressed both in retinal progenitors in the developing eye and apparently in all bipolar cells of the mature retina, Vsx1 expression is first detected in the eye at postnatal day 5, where it is restricted to cone bipolar cells.     

Drosophila Lin-7 is a component of the Crumbs complex in epithelia and photoreceptor cells and prevents light-induced retinal degeneration

The Drosophila Crumbs protein complex is required to maintain epithelial cell polarity in the embryo, to ensure proper morphogenesis of photoreceptor cells and to prevent light-dependent retinal degeneration. In Drosophila, the core components of the complex are the transmembrane protein Crumbs, the membrane-associated guanylate kinase (MAGUK) Stardust and the scaffolding protein DPATJ. The composition of the complex and some of its functions are conserved in mammalian epithelial and photoreceptor cells. Here, we report that Drosophila Lin-7, a scaffolding protein with one Lin-2/Lin-7 (L27) domain and one PSD-95/Dlg/ZO-1 (PDZ) domain, is associated with the Crumbs complex in the subapical region of embryonic and follicle epithelia and at the stalk membrane of adult photoreceptor cells. DLin-7 loss-of-function mutants are viable and fertile. While DLin-7 localization depends on Crumbs, neither Crumbs, Stardust nor DPATJ require DLin-7 for proper accumulation in the subapical region. Unlike other components of the Crumbs complex, DLin-7 is also enriched in the first optic ganglion, the lamina, where it co-localizes with Discs large, another member of the MAGUK family. In contrast to crumbs mutant photoreceptor cells, those mutant for DLin-7 do not display any morphogenetic abnormalities. Similar to crumbs mutant eyes, however, DLin-7 mutant photoreceptors undergo progressive, light-dependent degeneration. These results support the previous conclusions that the function of the Crumbs complex in cell survival is independent from its function in photoreceptor morphogenesis.     

Comparative analysis of structure, expression and PSD95-binding capacity of Lrfn, a novel family of neuronal transmembrane proteins

Leucine-rich repeat and fibronectin III domain-containing (Lrfn) has five members in mouse and human (Lrfn1, Lrfn2, Lrfn3, Lrfn4, Lrfn5), and homologues in other vertebrates. Lrfn proteins share leucine-rich repeat (LRR)–immunoglobulin-like (Ig)–fibronectin type III (Fn)–transmembrane domain structure, which is also found in LRR–Ig–Fn superfamily proteins. Mouse Lrfn genes were expressed at adult stage predominantly in the brain. In the course of development, expression of Lrfn1, Lrfn3, and Lrfn4 started from immature neural cells, whereas that of Lrfn2 and Lrfn5 was limited to mature ones. Lrfn1–5 commonly encode glycoproteins spanning the plasma membrane, with their N-terminus located on the extracellular side. C-termini of Lrfn1, Lrfn2 and Lrfn4 were bound by PDZ domains of postsynaptic protein PSD95, re-distributing PSD95 to cell periphery where the Lrfn proteins were detected. These results suggest that Lrfn proteins are neuronal components with a role in the developing or mature vertebrate nervous system.     

DaPKC-dependent phosphorylation of Crumbs is required for epithelial cell polarity in Drosophila

Both in Drosophila and vertebrate epithelial cells, the establishment of apicobasal polarity requires the apically localized, membrane-associated Par-3-Par-6-aPKC protein complex. In Drosophila, this complex colocalizes with the Crumbs-Stardust (Sdt)-Pals1-associated TJ protein (Patj) complex. Genetic and molecular analyses suggest a functional relationship between them. We show, by overexpression of a kinase-dead Drosophila atypical PKC (DaPKC), the requirement for the kinase activity of DaPKC to maintain the position of apical determinants and to restrict the localization of basolateral ones. We demonstrate a novel physical interaction between the apical complexes, via direct binding of DaPKC to both Crb and Patj, and identify Crumbs as a phosphorylation target of DaPKC. This phosphorylation of Crumbs is functionally significant. Thus, a nonphosphorylatable Crumbs protein behaves in vivo as a dominant negative. Moreover, the phenotypic effect of overexpressing wild-type Crumbs is suppressed by reducing DaPKC activity. These results provide a mechanistic framework for the functional interaction between the Par-3-Par-6-aPKC and Crumbs-Sdt-Patj complexes based in the posttranslational modification of Crb by DaPKC.     

Prolyl 4-hydroxylase alpha-related proteins in Drosophila melanogaster: tissue-specific embryonic expression of the 99F8-9 cluster

Three retinaldehyde dehydrogenases (RALDH1, RALDH2 and RALDH3), which catalyze the oxidation of retinaldehyde into retinoic acid, have been shown to be differentially expressed during early embryogenesis. Here, we report their differential expression patterns throughout later mouse organogenesis. Raldh1 is prominently expressed in developing lung (notably in bronchial and tracheal epithelia), and shows stage-specific expression in stomach and intestine epithelial and mesenchymal layers. Raldh3 expression is specific to the differentiating intestinal lamina propria. Raldh2 is expressed throughout the kidney nephrogenic zone, whereas Raldh1 and Raldh3 are mostly expressed in collecting duct epithelia. Raldh3 expression is more restricted than that of Raldh1 in the urogenital tract and sex gland epithelia, whereas Raldh2 expression is mesenchymal. Raldh1 is coexpressed with Raldh2 in the early heart epicardium, and is later specifically expressed in developing heart valves. All three genes exhibit distinct expression patterns in respiratory and olfactory epithelia and/or mesenchymes, and in developing teeth. Only Raldh1 expression is seen after birth in specific brain structures. These data indicate a requirement for regulated RA synthesis in various differentiating organs.     

Crumbs proteins stabilize the cone mosaics of photoreceptors and improve vision in zebrafish

Although the unique organization of vertebrate cone mosaics was first described long ago,both their underlying molecular basis and physiological significance are largely unknown.Here,we demonstrate that Crumbs proteins,the key regulators of epithelial apical polarity,establish the planar cellular polarity of photoreceptors in zebrafish.Via heterophilic Crb2a-Crb2b interactions,the apicobasal polarity protein Crb2b restricts the asymmetric planar distribution of Crb2a in photoreceptors.The planar polarized Crumbs proteins thus balance intercellular adhesions and tension between photoreceptors,thereby stabilizing the geometric organization of cone mosaics.Notably,loss of Crb2b in zebrafish induces a nearsightedness-like phenotype in zebrafish accompanied by an elongated eye axis and impairs zebrafish visual perception for predation.These data reveal a detailed mechanism for cone mosaic homeostasis via previously undiscovered apical-planar polarity coordination and propose a pathogenic mechanism for nearsightedness.     

The mouse Dlx-2 (Tes-1) gene is expressed in spatially restricted domains of the forebrain, face and limbs in midgestation mouse embryos

The pattern of RNA expression of the murine Dlx-2 (Tes-1) homeobox gene is described in embryos ranging in age from E8.5 through E11.5. Dlx-2 is a vertebrate homologue of the Drosophila Distal-less (Dll) gene. Dll expression in the Drosophila embryo is principally limited to the primordia of the brain, head and limbs. Dlx-2 is also expressed principally in the primordia of the forebrain, head and limbs. Within these regions it is expressed in spatially restricted domains. These include two discontinuous regions of the forebrain (basal telencephalon and ventral diencephalon), the branchial arches, facial ectoderm, cranial ganglia and limb ectoderm. Several mouse and human disorders have phenotypes which potentially are the result of mutations in the Dlx genes.     

bHLH Transcription factors and mammalian neuronal differentiation

The basic helix-loop-helix (bHLH) factor Mashl is expressed in the developing nervous system. Null mutation of Mash1 results in loss of olfactory and autonomic neurons and delays differentiation of retinal neurons, indicating that Mash1 promotes neuronal differentiation. Other bHLH genes, Math/NeuroD/Neurogenin, all expressed in the developing nervous system, have also been suggested to promote neuronal differentiation. In contrast, another bHLH factor, HES1, which is expressed by neural precursor cells but not by neurons, represses Mash1 expression and antagonizes Mash1 activity in a dominant negative manner. Forced expression of HES1 in precursor cells blocks neuronal differentiation in the brain and retina, indicating that HES1 is a negative regulator of neuronal differentiation. Conversely, null mutation of HES1 up-regulates Mash1 expression, accelerates neuronal differentiation, and causes severe defects of the brain and eyes. Thus, HES1 regulates brain and eye morphogenesis by inhibiting premature neuronal differentiation, and the down-regulation of HES1 expression at the right time is required for normal development of the nervous system. Interestingly, HES1 can repress its own expression by binding to its promoter, suggesting that negative autoregulation may contribute to down-regulation of HES1 expression during neural development. Recent studies indicate that HES1 expression is also controlled by RBP-J, a mammalian homologue of Suppressor of Hairless [Su(H)], and Notch, a key membrane protein that may regulate lateral specification through RBP-J during neural development. Thus, the Notch → RBP-J → HES1 ÷ Mash1 pathway may play a critical role in neuronal differentiation.     

Heart specific expression of mouse BMP-10 a novel member of the TGF-beta superfamily

Here we report the cloning and expression of murine BMP-10, a novel member of the TGF-β superfamily. In the mouse embryo, BMP-10 expression begins at 9.0 d.p.c. and is restricted to the developing heart. Initially, BMP-10 expression localizes to the trabeculated part of the common ventricular chamber and to the bulbus cordis region. After 12.5 d.p.c., additional BMP-10 expression is seen in the atrial wall. The data presented here suggest that BMP-10 plays an important role in trabeculation of the embryonic heart.