Naef V , De Sarlo M , Testa G , Corsinovi D , Azzarelli R , Borello U , Ori M .

             neuroblast fate specification
             embryonic brain development

	Figure 1. The spatial expression pattern of Xenopus mex3A. Whole mount in situ hybridization approaches show that mex3A is expressed in the central nervous system. (A) Mex3A expression at blastula stage (stage 3). (B,C) At neurula (stage 20) and at tadpole stages (stage 27), mex3A is expressed in: neural tube, developing eye, neural crest cells (white arrowhead) and otic vesicle (white arrow). At stage 37, an accumulation of mex3A transcript persisted in the most anterior region of the central nervous system (D). (E,F) In situ hybridization on frozen tissue of transverse sections on WT embryos at stage 41. ISH signal was revealed using fluorescent Fast Red and is visualized in red. Nuclei were revealed in blue with Hoechst. (E,F) Mex3A is expressed in CMZ of the retina (red arrow), in ventricular zone of the midbrain and in subventricular zone of the hindbrain.
	Figure 2. (A) Embryos injected with gfp (250 pg) and mex3A (500 pg) mRNA in one dorsal blastomere at the four-cells stage showing fluorescence only in the neural plate at neurula stage were cultured till different stages of development for WISH analysis. In each panel the asterisk (*) indicates the injected side of the embryo. (B,C) mRNA distribution of sox2 and msi-1 (sox2 phenotype 54%, n = 116; msi-1 phenotype 50%, n = 80) in mex3A overexpressing embryos. The arrow in (D) showed the lack of elrC expression in the anterior neural plate. (D–E′) mRNA distribution of elrC at 18 (phenotype 54%, n = 114, D) and at 23 (phenotype 57%, n = 70, E,E′) stages in mex3A overexpressing embryos. The arrow in (E′) shows the lack of neurons in the anterior neural tube. (F,G) pH3 positive cells were counted in the areas defined by the black rectangles. Statistical evaluation of the data is shown (n = 40). Abbreviations: n total number of processed embryos; error bars indicate standard error of the means (SEM); ***p ≤ 0.001.
	Figure 3. Loss of mex3A alters neuronal specification and differentiation (A) Structure of mex3A-morpholino oligonucleotide. The MO targets the second translation start site. (B) mRNA distribution of sox2 and pcna in mex3A morphants and controls. (C) Quantification of the data in B. (D) qRT-PCR analysis. Relative expression levels of each gene are normalized to gapdh expression. (E) mRNA distribution of N-tubulin and elrC in mex3A morphants and controls. (F) Quantification of the data in E. (G) qRT PCR analysis. (Abbreviations: n number of evaluated embryos in total; error bars indicate standard error of the means (SEM); *p ≤ 0,05, ***p ≤ 0.001, ****p ≤ 0.0001.
	Figure 4. The inhibition of mex3A function causes defects in anterior neuronal development. In each panel, the asterisk indicates the injected side of the embryo (A) Representative images and (B) quantification of the effect of injection of mex3A-MO and co-injection of mex3A-MO with zebrafish mex3A mRNA on the eye phenotype. (C) Image showing the anatomy of Xenopus brain. Diagrams showing a dorsal view of isolated brains. (D) Bright field images of Xenopus brains at stage 41, anterior to the top after unilateral injection of mex3A-MO or CoMO. (E) Statistical evaluation of the brain size in injected embryos. (F–I) pH3 staining in mex3A-deficient embryos at stage 24. Mex3A depletion leads to a significant reduction of proliferating cells compared to the un-injected side, whereas the CoMO injection does not influence on proliferation. pH3 positive cells were counted in the areas defined by the black rectangles. Statistical evaluations of the data are shown. Statistical quantifications of the data are given. Abbreviations: n, total number of evaluated embryos in total; SEM, error bars indicate standard error of the means; ns, not significant. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001.
	Figure S2. Gene expression of mex3A in E18 mouse embryo. Coronal cryostat sections of mouse embryo (embryonic day 18) processed by in situ hybridization to visualize mex3a expression pattern. Ob, olfactory bulbs; oe, olfactory epithelium; r, retina; c telencephalic cortex; vz, ventricular cortical zone; svz subventricular zone of basal ganglia; ncc, cartilagineous derivatives of neural crest cells; hi, developing hippocampal formation.
	Fig. S3 Functional rescue experiments. Zebrafish mex3A sequence and the mex3A-Mo used in the functional analysis. The morpholino is not able to bind efficiently the zebrafish sequence. The pairing between mex3A-Mo and the zebrafish mex3A sequence if affected by 18 mismatches (B) The overexpression of 800pg of zebrafish mex3A reproduced the overexpression phenotype observed with the Xenopus mex3A mRNA (sox2 phenotype 52%, n=82; tubb phenotype 47%, n=78). In the rescue experiments we injected 12 ng of mex3A-MO and 500 pg of zebrafish mex3A. (C, D) The rescue has been calculated on the percentage of embryos in which the correct expression of Sox2 and tubb was restored. (Sox2 n= 122; tubb n=116); error bars indicate standard error of the means (s.e.m); ** p≤0,01.
	FIGURE 1. The spatial expression pattern of Xenopus mex3A. Whole mount in situ hybridization approaches show that mex3A is expressed in the central nervous system. (A) Mex3A expression at blastula stage (stage 3). (B,C) At neurula (stage 20) and at tadpole stages (stage 27), mex3A is expressed in: neural tube, developing eye, neural crest cells (white arrowhead) and otic vesicle (white arrow). At stage 37, an accumulation of mex3A transcript persisted in the most anterior region of the central nervous system (D). (E,F) In situ hybridization on frozen tissue of transverse sections on WT embryos at stage 41. ISH signal was revealed using fluorescent Fast Red and is visualized in red. Nuclei were revealed in blue with Hoechst. (E,F) Mex3A is expressed in CMZ of the retina (red arrow), in ventricular zone of the midbrain and in subventricular zone of the hindbrain.
	FIGURE 2. (A) Embryos injected with gfp (250 pg) and mex3A (500 pg) mRNA in one dorsal blastomere at the four-cells stage showing fluorescence only in the neural plate at neurula stage were cultured till different stages of development for WISH analysis. In each panel the asterisk (*) indicates the injected side of the embryo. (B,C) mRNA distribution of sox2 and msi-1 (sox2 phenotype 54%, n = 116; msi-1 phenotype 50%, n = 80) in mex3A overexpressing embryos. The arrow in (D) showed the lack of elrC expression in the anterior neural plate. (D–E′) mRNA distribution of elrC at 18 (phenotype 54%, n = 114, D) and at 23 (phenotype 57%, n = 70, E,E′) stages in mex3A overexpressing embryos. The arrow in (E′) shows the lack of neurons in the anterior neural tube. (F,G) pH3 positive cells were counted in the areas defined by the black rectangles. Statistical evaluation of the data is shown (n = 40). Abbreviations: n total number of processed embryos; error bars indicate standard error of the means (SEM); ***p ≤ 0.001.
	FIGURE 3. Loss of mex3A alters neuronal specification and differentiation (A) Structure of mex3A-morpholino oligonucleotide. The MO targets the second translation start site. (B) mRNA distribution of sox2 and pcna in mex3A morphants and controls. (C) Quantification of the data in B. (D) qRT-PCR analysis. Relative expression levels of each gene are normalized to gapdh expression. (E) mRNA distribution of N-tubulin and elrC in mex3A morphants and controls. (F) Quantification of the data in E. (G) qRT PCR analysis. (Abbreviations: n number of evaluated embryos in total; error bars indicate standard error of the means (SEM); *p ≤ 0,05, ***p ≤ 0.001, ****p ≤ 0.0001.
	FIGURE 4. The inhibition of mex3A function causes defects in anterior neuronal development. In each panel, the asterisk indicates the injected side of the embryo (A) Representative images and (B) quantification of the effect of injection of mex3A-MO and co-injection of mex3A-MO with zebrafish mex3A mRNA on the eye phenotype. (C) Image showing the anatomy of Xenopus brain. Diagrams showing a dorsal view of isolated brains. (D) Bright field images of Xenopus brains at stage 41, anterior to the top after unilateral injection of mex3A-MO or CoMO. (E) Statistical evaluation of the brain size in injected embryos. (F–I) pH3 staining in mex3A-deficient embryos at stage 24. Mex3A depletion leads to a significant reduction of proliferating cells compared to the un-injected side, whereas the CoMO injection does not influence on proliferation. pH3 positive cells were counted in the areas defined by the black rectangles. Statistical evaluations of the data are shown. Statistical quantifications of the data are given. Abbreviations: n, total number of evaluated embryos in total; SEM, error bars indicate standard error of the means; ns, not significant. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001.

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