Specification of the vertebrate eye by a network of eye field transcription factors
ABSTRACT Several eye-field transcription factors (EFTFs) are expressed in the anterior region of the vertebrate neural plate and are essential for eye formation. The Xenopus EFTFs ET, Rx1, Pax6, Six3, Lhx2, tll and Optx2 are expressed in a dynamic, overlapping pattern in the presumptive eye field. Expression of an EFTF cocktail with Otx2 is sufficient to induce ectopic eyes outside the nervous system at high frequency. Using both cocktail subsets and functional (inductive) analysis of individual EFTFs, we have revealed a genetic network regulating vertebrate eye field specification. Our results support a model of progressive tissue specification in which neural induction then Otx2-driven neural patterning primes the anterior neural plate for eye field formation. Next, the EFTFs form a self-regulating feedback network that specifies the vertebrate eye field. We find striking similarities and differences to the network of homologous Drosophila genes that specify the eye imaginal disc, a finding that is consistent with the idea of a partial evolutionary conservation of eye formation.
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- "Noggin has been shown to induce the EFTFs and anterior neural marker, otx2 (Zuber et al., 2003). If SB43+DM treatment is sufficient to mimic Noggin's ability to generate retina, then we would expect that SB43+DM treatment is sufficient to increase EFTF expression in animal caps. "
ABSTRACT: Retina formation requires the correct spatiotemporal patterning of key regulatory factors. While it is known that repression of several signaling pathways lead to specification of retinal fates, addition of only Noggin, a known BMP antagonist, can convert pluripotent Xenopus laevis animal cap cells to functional retinal cells. The aim of this study is to determine the intracellular molecular events that occur during this conversion. Surprisingly, blocking BMP signaling alone failed to mimic Noggin treatment. Overexpressing Noggin in pluripotent cells resulted in a concentration-dependent suppression of both Smad1 and Smad2 phosphorylation, which act downstream of BMP and Activin signaling, respectively. This caused a decrease in downstream targets: endothelial marker, xk81, and mesodermal marker, xbra. We treated pluripotent cells with dominant-negative receptors or the chemical inhibitors, dorsomorphin and SB431542, which each target either the BMP or Activin signaling pathway. We determined the effect of these treatments on retina formation using the Animal Cap Transplant (ACT) assay; in which treated pluripotent cells were transplanted into the eye field of host embryos. We found that inhibition of Activin signaling, in the presence of BMP signaling inhibition, promotes efficient retinal specification in Xenopus tissue, mimicking the affect of adding Noggin alone. In whole embryos, we found that the eye field marker, rax, expanded when adding both dominant-negative Smad1 and Smad2, as did treating the cells with both dorsomorphin and SB431542. Future studies could translate these findings to a mammalian culture assay, in order to more efficiently produce retinal cells in culture. © 2015. Published by The Company of Biologists Ltd.Biology Open 03/2015; 4(4). DOI:10.1242/bio.20149977 · 2.42 Impact Factor
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- "The topographic location of the eye field corresponds to an anterior region where BMP, Wnt, and Nodal signals are repressed and IGF and FGF signaling is active (reviewed in Andreazzoli, 2009). The specific combination of these signals allows the concomitant expression of the Eye Field Transcription Factors (EFTFs), which are responsible for determining the features of early retinal progenitors and to trigger the following phases of eye development (Zuber et al., 2003). In fact, co-injection of EFTFs, or their activator Noggin, is sufficient to induce a retinal fate in pluripotent ectodermal cells, which, following transplantation, are able to generate a complete retina (Viczian et al., 2009; Lan et al., 2009). "
ABSTRACT: Background: The transcription factor Rx1, also known as Rax, controls key properties of retinal precursors including migration behavior, proliferation and maintenance of multipotency. However, Rx1 effector genes are largely unknown. Results: To identify genes controlled by Rx1 in early retinal precursors, we compared the transcriptome of Xenopus embryos overexpressing Rx1 to that of embryos in which Rx1 was knocked-down. In particular, we selected 52 genes coherently regulated, i.e., actived in Rx1 gain of function and repressed in Rx1 loss of function experiments, or vice versa. RT-qPCR and in situ hybridization confirmed the trend of regulation predicted by microarray data for the selected genes. Most of the genes upregulated by Rx1 are coexpressed with this transcription factor, while downregulated genes are either not expressed or expressed at very low levels in the early developing retina. Putative direct Rx1 target genes, activated by GR-Rx1 in the absence of protein synthesis, include Ephrin B1 and Sh2d3c, an interactor of ephrinB1 receptor, which represent candidate novel effectors for the migration promoting activity of Rx1. Conclusions: This study identifies previously undescribed Rx1 regulated genes mainly involved in transcription regulation, cell migration/adhesion and cell proliferation that contribute to delineate the molecular mechanisms underlying Rx1 activities. Developmental Dynamics, 2014. © 2014 Wiley Periodicals, Inc.Developmental Dynamics 10/2014; 243(10). DOI:10.1002/dvdy.24145 · 2.67 Impact Factor
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- "ecursors have been reported to be between 30 and 36 hr ( Young , 1985 ; Alexiades and Cepko , 1996 ) . The fact that cells plated on polyLM dis - played a faster doubling time implies that the substrate can accelerate cell cycling . This could be mediated through the activation of early eye field network genes that most often drive proliferation ( Zuber et al . , 2003 ; Agathocleous and Harris , 2009 ) . An interesting candi - date is Sonic hedgehog ( Shh ) , because hedgehog signal - ing acts through a double - pronged action of increasing proliferation and differentiation in the early retina and is involved in retinal ganglion cell generation ( Locker et al . , 2006 ; Agathocleous et al . , 2007 ) "
ABSTRACT: Polylaminin (polyLM) is a flat biomimetic polymer of laminin capable of promoting axonal growth both in vitro and in vivo. It is assembled in a cell-free system when laminin 111 is incubated in acidic pH, whereas incubation in neutral buffer leads to the formation of bulky and irregular polymers (LM). In the present work, we compared the behaviors of cells isolated from the P1 rat retina on polyLM and LM. PolyLM induced cellular spreading and the outgrowth of neurites in contact with the substrate, whereas LM led to the formation of large clusters of cells, with neurites growing only inward. After 24 hr in culture, the number of cells on polyLM increased threefold, and this increase was inhibited by 60% in the presence of the PKA inhibitor H89 and by 41% in the presence of the PKC inhibitor chelerythrine chloride, whereas both inhibitors abolished neuritogenesis. Neither the cell number nor the outgrowth of neurites was affected by the ERK1/2 inhibitor PD98059 on polyLM. On the other hand, PD98059 was able to reduce the cell number on LM, whereas the other inhibitors were not. Immunostaining of P1 retina with an antilaminin antibody revealed that the protein was expressed not only at its inner surface but also within the neuroblast layer in close contact with individual cells. Our results indicate that, when provided in its active polymerized form, laminin can influence both neuritogenesis and proliferation of retinal cells. © 2013 Wiley Periodicals, Inc.Journal of Neuroscience Research 01/2014; 92(1):24-34. DOI:10.1002/jnr.23298 · 2.73 Impact Factor