The molecular mechanisms underlying cell fate determination from common progenitors in the vertebrate CNS remain elusive. We previously reported that the OTX2 homeoprotein regulates retinal photoreceptor cell fate determination. While Otx2 transactivation is a pivotal process for photoreceptor cell fate determination, its transactivation mechanism in the retina is unknown. Here, we identified an evolutionarily conserved Otx2 enhancer of ϳ500 bp, named embryonic enhancer locus for photoreceptor Otx2 transcription (EELPOT), which can recapitulate initial Otx2 expression in the embryonic mouse retina. We found that the RAX homeoprotein interacts with EELPOT to transactivate Otx2, mainly in the final cell cycle of retinal progenitors. Conditional inactivation of Rax results in downregulation of Otx2 expression in vivo. We also showed that NOTCH-HES signaling negatively regulates EELPOT to suppress Otx2 expression. These results suggest that the integrated activity of cell-intrinsic and -extrinsic factors on EELPOT underlies the molecular basis of photoreceptor cell fate determination in the embryonic retina.
Microphthalmia with limb anomalies (MLA) is a rare autosomal-recessive disorder, presenting with anophthalmia or microphthalmia and hand and/or foot malformation. We mapped the MLA locus to 14q24 and successfully identified three homozygous (one nonsense and two splice site) mutations in the SPARC (secreted protein acidic and rich in cysteine)-related modular calcium binding 1 (SMOC1) in three families. Smoc1 is expressed in the developing optic stalk, ventral optic cup, and limbs of mouse embryos. Smoc1 null mice recapitulated MLA phenotypes, including aplasia or hypoplasia of optic nerves, hypoplastic fibula and bowed tibia, and syndactyly in limbs. A thinned and irregular ganglion cell layer and atrophy of the anteroventral part of the retina were also observed. Soft tissue syndactyly, resulting from inhibited apoptosis, was related to disturbed expression of genes involved in BMP signaling in the interdigital mesenchyme. Our findings indicate that SMOC1/Smoc1 is essential for ocular and limb development in both humans and mice.
Dystroglycan (DG) is a key component of the dystrophin-glycoprotein complex (DGC)at the neuromuscular junction postsynapse. In the mouse retina, the DGC is localized at the presynapse of photoreceptor cells, however, the function of presynaptic DGC is poorly understood. Here, we developed and analyzed retinal photoreceptor-specific DG conditional knock-out (DG CKO) mice. We found that the DG CKO retina showed a reduced amplitude and a prolonged implicit time of the ERG b-wave. Electron microscopic analysis revealed that bipolar dendrite invagination into the photoreceptor terminus is perturbed in the DG CKO retina. In the DG CKO retina, pikachurin, a DG ligand in the retina, is markedly decreased at photoreceptor synapses. Interestingly, in the Pikachurin Ϫ/Ϫ retina, the DG signal at the ribbon synaptic terminus was severely reduced, suggesting that pikachurin is required for the presynaptic accumulation of DG at the photoreceptor synaptic terminus, and conversely DG is required for pikachurin accumulation. Furthermore, we found that overexpression of pikachurin induces formation and clustering of a DG-pikachurin complex on the cell surface. The Laminin G repeats of pikachurin, which are critical for its oligomerization and interaction with DG, were essential for the clustering of the DG-pikachurin complex as well. These results suggest that oligomerization of pikachurin and its interaction with DG causes DG assembly on the synapse surface of the photoreceptor synaptic terminals. Our results reveal that the presynaptic interaction of pikachurin with DG at photoreceptor terminals is essential for both the formation of proper photoreceptor ribbon synaptic structures and normal retinal electrophysiology.
To establish the genetic tools for conditional gene deletion in mouse retinal progenitors, we generated a Dkk3-Cre transgenic mouse line using bacterial artificial chromosome (BAC) transgenesis. Cre recombination efficiency in vivo was assayed by crossing this transgenic line, termed BAC-Dkk3-Cre, with the CAG-CAT-Z reporter line. This BAC-Dkk3-Cre line showed Cre recombinase activity in most retinal progenitors. Cre activity was detectable from embryonic day 10.5 (E10.5) and generally restricted to the retina during embryogenesis. To verify that BAC-Dkk3-Cre mice successfully circumvented lethality, we generated Otx2flox/flox/BAC-Dkk3-Cre+ mice as Otx2 conditional knockout mice. The Otx2flox/flox/BAC-Dkk3-Cre+ mice were viable, and their retina showed loss of mature cell-type markers of photoreceptor cells, bipolar cells, and horizontal cells, in contrast, amacrine-like cells noticeably increased. Thus, the BAC-Dkk3-Cre transgenic mouse line provides a powerful tool for generating conditional knockout mouse lines for studying loss of gene functions in the developing retina.
It has been demonstrated that Ras is involved in in-. Furthermore, the activation of fos gene promoter following IL-3 stimulation was almost completely abolished when Ras(S17N) was induced. Under these conditions, Ras(S17N) exhibited no inhibitory effect on IL-3-dependent proliferation assessed by the increase of cell numbers and a mitochondrial enzyme activity. The results indicate that Ras-dependent pathways, including the Raf/MAPK/Fos pathway, are dispensable for IL-3-induced growth stimulation. When BaF3 cells were treated with a tyrosine kinase inhibitor, herbimycin A, IL-3-dependent proliferation of the cells was impaired, suggesting that tyrosine kinase-mediated pathways are critical for growth promotion. On the other hand, apoptotic cell death caused by deprivation of IL-3 was prevented by the induction of the activated mutant Ras(G12V), although the rate of cell number increase was markedly reduced. Thus, it is likely that Rasindependent pathways play important roles to facilitate the proliferation although they may not be essential for IL-3-stimulated antiapoptotic signal transduction.In various types of cells, Ras functions as a molecular switch that regulates intracellular signaling pathways for proliferation, differentiation, and other physiological responses. Tyrosine kinase receptors, such as epidermal growth factor receptor and platelet-derived growth factor receptor, when stimulated by their specific ligands, form a complex with a variety of signal-transducing molecules including phosphatidylinositol 3-kinase, Ras-GTPase activating protein (Ras-GAP), 1 phospholipase C-␥1, and adaptor proteins (e.g. Grb-2, Nck, and Shc) through the interaction between specific phosphotyrosines on the receptor and Src homology 2 (SH2) domains of the binding molecules Schlessinger, 1993). Among the above components of the signal-transducing complex, Shc and Grb-2 are well characterized as elements that link the receptor and a Ras-guanine nucleotide exchange factors (Ras-GEFs), such as mSos-1. Adaptors and Ras-GEFs are known to participate also in Ras regulation through non-tyrosine kinase-type receptors including interleukin 2 (IL-2), IL-3, and T cell antigen receptors (Burns et al., 1993;Cutler et al., 1993;Ravichandran et al., 1993;Sato et al., 1993;Buday et al., 1994;Reif et al., 1994;Ravichandran and Burakoff, 1994;Welham et al., 1994). The active GTP-bound form of Ras specifically binds RasGAPs (Boguski and McCormick, 1993), c-Raf-1 (Avruch et al., 1994), B-Raf (Moodie et al., 1994;Vaillancourt et al., 1994), phosphatidylinositol 3-kinase (Rodriguez-Viciana et al., 1994), Ral-guanine nucleotide dissociation stimulator (Hofer et al., 1994;Kikuchi et al., 1994;Spaargaren and Bischoff, 1994), mitogen-activated protein kinase (MAPK)/extracellular signalregulated kinase (ERK) kinase (MEK) kinase (MEKK) (LangeCarter and Johnson, 1994), and Rin1 (Han and Colicelli, 1995), among which Raf proteins are best characterized as direct targets of Ras (Daum et al., 1994;Marshall, 1995). After the binding of Ras, the serine/t...
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