Giudetti G et al. (2014), Characterization of the Rx1-dependent transcrip...

XB-ART-48953

Dev Dyn 2014 Oct 01;24310:1352-61. doi: 10.1002/dvdy.24145.

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Characterization of the Rx1-dependent transcriptome during early retinal development.

Giudetti G , Giannaccini M , Biasci D , Mariotti S , Degl'innocenti A , Perrotta M , Barsacchi G , Andreazzoli M .

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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.

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Species referenced: Xenopus laevis
Genes referenced: atrx bahcc1 bzw2 ccdc12 chpf crx ddr1 dgcr2 efnb1 eif4g1 enox1 fat1 fli1 gpatch8 gtpbp1 hey1 kdm7a kif26a med12 mef2d mrps5 myod1 myos ncoa6 nek2 notch1 p4ha1 p4ha2 pfdn6 polr2a ppfia1 psme1 rab11fip4 rab6a rax rbfox2 rbm24 rnf19a rpl9 scaf4 sh2d3c slc25a17 strn3 tcf3 tcf7l1 tcp1 thg1l tle2 tob2 trip12
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	Figure 1. A: Venn diagram identifying the overlap of genes regulated in gain- and loss-of-function experiments. B: Pie chart of genes coherently regulated by Rx1. Percentages of each group classified according to their putative molecular function are indicated.Download figure to PowerPoint
	Figure 2. Expression pattern of genes regulated by Rx1. In situ hybridizations performed on whole mount (stage 13, early neurula; stage 16, mid-neurula; stage 19, late neurula; stage 27, early tailbud; stage 33, late tailbud), and on cryo-sections (stage 42, early tadpole, detail of the retina). Stage 13–19 embryos are frontal views with dorsal to the top. Stage 27 and 33 embryos are lateral views with dorsal to the top and anterior to the left.Download figure to PowerPoint
	Figure 3. Validation of Rx1 regulated genes by RT-qPCR. Relative quantification of selected genes in Rx1-injected embryos as compared to their expression levels in control embryos (baseline). Error bars indicate SEM. P < 0.05; *P < 0.01(Student's t-test).Download figure to PowerPoint
	Figure 4. Alteration of the expression of genes controlling migration/adhesion in Rx1 gain- and loss-of function experiments. A–I: Embryos injected unilaterally with control standard morpholino (CoMo; A, D, G), MoRx1 (B, E, H), or Rx1 (C, F, I) and analyzed at early neurula stage. Frontal views, dorsal to the top. The probes used are indicated in the top right corner of each panel. J: Relative quantification by RT-qPCR of Sh2d3c, Efnb1, and Fat1 in embryos injected with GFP alone (GFP), or coinjected with either control standard morpholino and GFP (CoMo) or MoRx1 and GFP (MoRx1). Error bars indicate SEM. P < 0.05; *P < 0.01 (Student's t-test). No significant difference is observed between GFP and CoMo samples.Download figure to PowerPoint
	Figure 5. Identification of direct candidate targets of Rx1 by RT-qPCR. Relative quantification of candidate gene expression in animal caps injected with Rx1GR in the presence or absence of CHX (C) or DEX (D). For each condition, the expression level was normalized with the expression level of GAPDH and then normalized to the expression level of the −C+D condition, which was set at 100 arbitrary units. Statistical significance is indicated for the comparison of +C−D vs. +C+D and −C−D vs −C+D conditions. Error bars indicate SEM. P < 0.05; *P < 0.01 (Student's t-test).Download figure to PowerPoint
	bzw2 (basic leucine zipper and W2 domains 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 19, anterior view, dorsal up.
	bzw2 (basic leucine zipper and W2 domains 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 27, lateral view, anterior left, dorsal up.
	bzw2 (basic leucine zipper and W2 domains 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 33, lateral view, anterior left, dorsal up.
	fat1 (FAT atypical cadherin 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 16, anterior view, dorsal up.
	fat1 (FAT atypical cadherin 1) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 27, lateral view, anterior left, dorsal up.
	rab11fip4 (RAB11 family interacting protein 4 (class II)) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 19, anterior view, dorsal up.
	rab11fip4 (RAB11 family interacting protein 4 (class II)) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 33, lateral view, anterior left, dorsal up.
	rbfox2 (RNA binding fox-1 homolog 2 ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 19, anterior view, dorsal up.
	rbfox2 (RNA binding fox-1 homolog 2) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 27, lateral view, anterior left, dorsal up.
	sh2d3c (SH2 domain containing 3C) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 19, anterior view, dorsal up.
	sh2d3c (SH2 domain containing 3C) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 33, lateral view, anterior left, dorsal up.
	slc25a17 (solute carrier family 25 (mitochondrial carrier; peroxisomal membrane protein, 34kDa), member 17) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 19, anterior view, dorsal up.
	slc25a17 (solute carrier family 25 (mitochondrial carrier; peroxisomal membrane protein, 34kDa), member 17) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 33, lateral view, anterior left, dorsal up.