Zebrafish gbx1 refines the Midbrain-Hindbrain Boundary border and mediates the Wnt8 posteriorization signal

International audienceBACKGROUND: Studies in mouse, Xenopus and chicken have shown that Otx2 and Gbx2 expression domains are fundamental for positioning the midbrain-hindbrain boundary (MHB) organizer. Of the two zebrafish gbx genes, gbx1 is a likely candidate to participate in this event because its early expression is similar to that reported for Gbx2 in other species. Zebrafish gbx2, on the other hand, acts relatively late at the MHB. To investigate the function of zebrafish gbx1 within the early neural plate, we used a combination of gain- and loss-of-function experiments. RESULTS: We found that ectopic gbx1 expression in the anterior neural plate reduces forebrain and midbrain, represses otx2 expression and repositions the MHB to a more anterior position at the new gbx1/otx2 border. In the case of gbx1 loss-of-function, the initially robust otx2 domain shifts slightly posterior at a given stage (70% epiboly), as does MHB marker expression. We further found that ectopic juxtaposition of otx2 and gbx1 leads to ectopic activation of MHB markers fgf8, pax2.1 and eng2. This indicates that, in zebrafish, an interaction between otx2 and gbx1 determines the site of MHB development. Our work also highlights a novel requirement for gbx1 in hindbrain development. Using cell-tracing experiments, gbx1 was found to cell-autonomously transform anterior neural tissue into posterior. Previous studies have shown that gbx1 is a target of Wnt8 graded activity in the early neural plate. Consistent with this, we show that gbx1 can partially restore hindbrain patterning in cases of Wnt8 loss-of-function. We propose that in addition to its role at the MHB, gbx1 acts at the transcriptional level to mediate Wnt8 posteriorizing signals that pattern the developing hindbrain. CONCLUSION: Our results provide evidence that zebrafish gbx1 is involved in positioning the MHB in the early neural plate by refining the otx2 expression domain. In addition to its role in MHB formation, we have shown that gbx1 is a novel mediator of Wnt8 signaling during hindbrain patterning

Subdivisions of the adult zebrafish pallium based on molecular marker analysis [v1; ref status: indexed, http://f1000r.es/4m2]

Background: The telencephalon shows a remarkable structural diversity among vertebrates. In particular, the everted telencephalon of ray-finned fishes has a markedly different morphology compared to the evaginated telencephalon of all other vertebrates. This difference in development has hampered the comparison between different areas of the pallium of ray-finned fishes and the pallial nuclei of all other vertebrates. Various models of homology between pallial subdivisions in ray-finned fishes and the pallial nuclei in tetrapods have been proposed based on connectional, neurochemical, gene expression and functional data. However, no consensus has been reached so far. In recent years, the analysis of conserved developmental marker genes has assisted the identification of homologies for different parts of the telencephalon among several tetrapod species. Results: We have investigated the gene expression pattern of conserved marker genes in the adult zebrafish (Danio rerio) pallium to identify pallial subdivisions and their homology to pallial nuclei in tetrapods. Combinatorial expression analysis of ascl1a, eomesa, emx1, emx2, emx3, and Prox1 identifies four main divisions in the adult zebrafish pallium. Within these subdivisions, we propose that Dm is homologous to the pallial amygdala in tetrapods and that the dorsal subdivision of Dl is homologous to part of the hippocampal formation in mouse. We have complemented this analysis be examining the gene expression of emx1, emx2 and emx3 in the zebrafish larval brain. Conclusions: Based on our gene expression data, we propose a new model of subdivisions in the adult zebrafish pallium and their putative homologies to pallial nuclei in tetrapods. Pallial nuclei control sensory, motor, and cognitive functions, like memory, learning and emotion. The identification of pallial subdivisions in the adult zebrafish and their homologies to pallial nuclei in tetrapods will contribute to the use of the zebrafish system as a model for neurobiological research and human neurodegenerative diseases

Subdivisions of the adult zebrafish pallium based on molecular marker analysis [version 2; referees: 2 approved, 1 approved with reservations]

Background: The telencephalon shows a remarkable structural diversity among vertebrates. In particular, the everted telencephalon of ray-finned fishes has a markedly different morphology compared to the evaginated telencephalon of all other vertebrates. This difference in development has hampered the comparison between different areas of the pallium of ray-finned fishes and the pallial nuclei of all other vertebrates. Various models of homology between pallial subdivisions in ray-finned fishes and the pallial nuclei in tetrapods have been proposed based on connectional, neurochemical, gene expression and functional data. However, no consensus has been reached so far. In recent years, the analysis of conserved developmental marker genes has assisted the identification of homologies for different parts of the telencephalon among several tetrapod species. Results: We have investigated the gene expression pattern of conserved marker genes in the adult zebrafish (Danio rerio) pallium to identify pallial subdivisions and their homology to pallial nuclei in tetrapods. Combinatorial expression analysis of ascl1a, eomesa, emx1, emx2, emx3, and Prox1 identifies four main divisions in the adult zebrafish pallium. Within these subdivisions, we propose that Dm is homologous to the pallial amygdala in tetrapods and that the dorsal subdivision of Dl is homologous to part of the hippocampal formation in mouse. We have complemented this analysis be examining the gene expression of emx1, emx2 and emx3 in the zebrafish larval brain. Conclusions: Based on our gene expression data, we propose a new model of subdivisions in the adult zebrafish pallium and their putative homologies to pallial nuclei in tetrapods. Pallial nuclei control sensory, motor, and cognitive functions, like memory, learning and emotion. The identification of pallial subdivisions in the adult zebrafish and their homologies to pallial nuclei in tetrapods will contribute to the use of the zebrafish system as a model for neurobiological research and human neurodegenerative diseases

Characterization of light lesion paradigms and optical coherence tomography as tools to study adult retina regeneration in zebrafish

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Protein expression pattern of Fgf receptors.

<p><b>A</b>) Fgfr1a protein is detected in the photoreceptor layer colocalizing with UV cones (green) (white arrow), INL and GCL (white arrowhead). <b>B</b>) Expression of Fgfr3 is detected in the outer part of the INL next to the UV cone synaptic terminals (white arrowhead). <b>C, D</b>) Fgfr3 is colocalized with the synaptic terminals of UV cones and rods (white arrows). Scale bars = 20 µm.</p

Gene expression analysis in the adult zebrafish pallium

<p>Dataset 1 Expression of eomesb in the embryonic brain and the adult pallium in zebrafish.<br>Raw data of Figure S2 and additional image files of eomesb expression in the embryo and the adult pallium.</p> <p>Dataset 2 Images of negative control.<br>No signal was detected in the absence of the riboprobe, demonstrating that the antibody reacts specifically with the synthetic RNA.</p> <p>Dataset 3 Expression of eomesa in the zebrafish pallium.<br>Raw data of Figure 1 and additional image files of eomesa expression in the adult pallium.</p> <p>Dataset 4 Expression of emx1, emx2 and emx3 in the zebrafish larval brain. Raw data of Figure S3 and additional image files of emx gene expression in the zebrafish larvae.</p> <p>Dataset 5 Expression of emx1, emx2 and emx3 in the zebrafish pallium.<br>Raw data of Figure 2 and additional image files of emx gene expression in the adult pallium.</p> <p>Dataset 6 Expression of Prox1 in the zebrafish pallium.<br>Raw data of Figure 3 and additional image files of Prox1 expression in the adult pallium.</p> <p>Dataset 7 Expression of ascl1a in the zebrafish pallium.<br>Raw data of Figure 4 and additional image files of ascl1a expression in the adult pallium.</p> <p> </p

No influence of Fgf inhibition on neurogenesis in the CMZ.

<p>The numbers represent the average of BrdU+ cells in the CMZ (± standard deviation). For this experiment, transgenic and control siblings were heatshocked for one- or seven days, respectively. BrdU-positive cells in the CMZ of both eyes of at least three individuals were counted for each time point.</p

Double labeling of Caspase-3 positive cells.

<p><b>A</b>) Casp3 (red) and HuC/D (green), marker for mature neurons such as amacrine and ganglion cells, do not colocalize in the INL (white arrow) and GCL (white arrowhead). <b>B</b>) Glutamine synthetase (GS, green), a marker for MGC, colocalizes with Casp3+ (red) cells in the INL (white arrows). Scale bar = 20 µm.</p

Fgf receptors, ligands and downstream target expression in specific layers of the adult zebrafish retina.

<p><b>A</b>) <i>fgfr1a</i> expression in the INL and GCL. <b>B</b>) <i>fgfr2</i> signal in the INL <b>C</b>) <i>fgfr3</i> expression in the outer part of the INL <b>D</b>) <i>fgfr4</i> expression in the INL next to the CMZ (black arrow). <b>E</b>) <i>fgf8a</i> expression in the INL and weakly in the GCL. <b>F</b>) <i>fgf20a</i> expression in the ONL, INL and GCL. <b>G</b>) <i>fgf24</i> is detectable in the INL and GCL. <b>H–M</b>) Fgf pathway target gene expression. <b>H</b>) <i>spry1</i> expression in the INL and GCL. <b>I</b>) <i>spry2</i> signal in POS, INL and GCL. <b>J</b>) <i>spry4</i> expression in the INL and weakly in the GCL. <b>K</b>) <i>dusp6</i> expression is strong in the POS, and in the INL and GCL. <b>L</b>) Strong <i>etv5a</i> expression is found in the POS, INL and GCL. <b>M</b>) <i>etv5b</i> expression is widespread in the ONL, INL and GCL and enriched in the POS. <b>N</b>) Summary of ISH expression data: + expression, − no detectable expression. GCL, ganglion cell layer: white arrowhead; INL, inner nuclear layer: black arrowhead; ONL, outer nuclear layer; POS, photoreceptor layer: black arrow. Scale bar = 20 µm.</p