Sox2, Tlx, Gli3, and Her9 converge on Rx2 to define retinal stem cells in vivo

This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 License.-- et al.Transcriptional networks defining stemness in adult neural stem cells (NSCs) are largely unknown. We used the proximal cis-regulatory element (pCRE) of the retina-specific homeobox gene 2 (rx2) to address such a network. Lineage analysis in the fish retina identified rx2 as marker for multipotent NSCs. rx2-positive cells located in the peripheral ciliary marginal zone behave as stem cells for the neuroretina, or the retinal pigmented epithelium. We identified upstream regulators of rx2 interrogating the rx2 pCRE in a trans-regulation screen and focused on four TFs (Sox2, Tlx, Gli3, and Her9) activating or repressing rx2 expression. We demonstrated direct interaction of the rx2 pCRE with the four factors in vitro and in vivo. By conditional mosaic gain- and loss-of-function analyses, we validated the activity of those factors on regulating rx2 transcription and consequently modulating neuroretinal and RPE stem cell features. This becomes obvious by the rx2-mutant phenotypes that together with the data presented above identify rx2 as a transcriptional hub balancing stemness of neuroretinal and RPE stem cells in the adult fish retina.DI received fellowships from the Human Frontier Science Program (HFSP) and the Japan Society for the Promotion of Science (JSPS); TT received a fellowship of the Hartmut Hoffmann Berling International Graduate School (HBIGS) in Heidelberg. The project was supported by the Collaborative Research Center SFB 873 (J.W.) of the German Research Foundation (DFG).Peer Reviewe

A global survey identifies novel upstream components of the Ath5 neurogenic network

Regulators of vertebrate Ath5 expression were identified by high-throughput screening; extending the current gene regulatory model network controlling retinal neurogenesis

Rapid identification of PAX2/5/8 direct downstream targets in the otic vesicle by combinatorial use of bioinformatics tools

A novel bioinformatics pipeline is used to discover PAX2/5/8 direct downstream targets involved in inner ear development

A trans-Regulatory code for the forebrain expression of Six3.2 in the Medaka fish

A well integrated and hierarchically organized gene regulatory network is responsible for the progressive specification of the forebrain. The transcription factor Six3 is one of the central components of this network. As such, Six3 regulates several components of the network, but its upstream regulators are still poorly characterized. Here we have systematically identified such regulators, taking advantage of the detailed functional characterization of the regulatory region of the medaka fish Six3.2 ortholog and of a time/cost-effective trans-regulatory screening, which complemented and overcame the limitations of in silico prediction approaches. The candidates resulting from this search were validated with dose-response luciferase assays and expression pattern criteria. Reconfirmed candidates with a matching expression pattern were also tested with chromatin immunoprecipitation and functional studies. Our results confirm the previously proposed direct regulation of Pax6 and further demonstrate that Msx2 and Pbx1 are bona fide direct regulators of early Six3.2 distribution in distinct domains of the medaka fish forebrain. They also point to other transcription factors, including Tcf3, as additional regulators of different spatial-temporal domains of Six3.2 expression. The activity of these regulators is discussed in the context of the gene regulatory network proposed for the specification of the forebrain.Spanish Ministerio de Economía y Competitividad (MINECO) Grants BFU2010-16031 and BFU2013-43213-P, cofounded by FEDER Funds; Comunidad Autónoma de Madrid (CAM) Grant CELL-DD S2010/BMD-2315; Fundaluce; Fundación ONCE; the Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER) del Instituto Carlos III (ISCIII); and an Institutional Grant from the Fundación Ramón Areces.Peer Reviewe

In vivo analysis of neural crest behaviour in ojoplano

Resumen del trabajo presentado al IX Meeting of the Spanish Society for Developmental Biology celebrado en Granada del 12 al 14 de noviembre de 2012.During embryogenesis, genetically programmed morphogenetics events occur through modifications in cellular architecture. These modifications may trigger behaviours such as the contraction of epithelial layers or cell migration events. In vertebrates, a key morphogenetic event takes place when neural crest cells delaminate from the dorsal neural tube and experiment a massive migration to finally contribute to a variety of tissues throughout the body. Our laboratory recently described the ojoplano (opo) mutant, named after its eyes phenotype, which is caused by optic cup folding defects (Martínez-Morales etal., 2009). Interetingly, opo also presents a neural crest phenotype, with many derivatives starkly reduced in number, albeit correctly differentiated. To understand what causes this phenotype, we are currently using an in vivo time-lapse imaging approach to study cell polarization and early migration in the neural crest, and specifically the role of opo during these events.Peer Reviewe

DOI: 10.1093/nar/gkg017 MEPD: a Medaka gene expression pattern database

The Medaka Expression Pattern Database (MEPD) stores and integrates information of gene expression during embryonic development of the small freshwater fish Medaka (Oryzias latipes). Expression patterns of genes identified by ESTs are documented by images and by descriptions through parameter

A Novel Mammal-Specific Three Partite Enhancer Element Regulates Node and Notochord-Specific <em>Noto</em> Expression

<div><p>The vertebrate organizer and notochord have conserved, essential functions for embryonic development and patterning. The restricted expression of developmental regulators in these tissues is directed by specific cis-regulatory modules (CRMs) whose sequence conservation varies considerably. Some CRMs have been conserved throughout vertebrates and likely represent ancestral regulatory networks, while others have diverged beyond recognition but still function over a wide evolutionary range. Here we identify and characterize a mammalian-specific CRM required for node and notochord specific (NNC) expression of NOTO, a transcription factor essential for node morphogenesis, nodal cilia movement and establishment of laterality in mouse. A 523 bp enhancer region (NOCE) upstream the <em>Noto</em> promoter was necessary and sufficient for NNC expression from the endogenous <em>Noto</em> locus. Three subregions in NOCE together mediated full activity in vivo. Binding sites for known transcription factors in NOCE were functional in vitro but dispensable for NOCE activity in vivo. A FOXA2 site in combination with a novel motif was necessary for NOCE activity in vivo. Strikingly, syntenic regions in non-mammalian vertebrates showed no recognizable sequence similarities. In contrast to its activity in mouse NOCE did not drive NNC expression in transgenic fish. NOCE represents a novel, mammal-specific CRM required for the highly restricted <em>Noto</em> expression in the node and nascent notochord and thus regulates normal node development and function.</p> </div