Green YS, Vetter ML.

EY012274 NEI NIH HHS , P30 EY014800-07 NEI NIH HHS , R01 EY012274-13 NEI NIH HHS , R01 EY012274-14 NEI NIH HHS , P30 EY014800 NEI NIH HHS , R01 EY012274 NEI NIH HHS

GSE27084: NCBI

	Figure 1. Candidate target genes upregulated by overexpression of EBF2 or EBF3. hGR-XEBF2 or hGR-XEBF3 mRNA was injected into one cell of two-cell stage embryos, followed by DEX treatment from the late gastrula stage (stage 11/11.5) to the neurula stage (stage 14/15). β-Galactosidase (β-gal) mRNA was co-injected as a marker of the injected side. In all panels the right side is the injected side, showing the light blue color of X-gal staining. (A-T) The expression levels of pcdh8 (A,B), peripherin (C,D), greb1 (E,F), nf-m (G,H), kcnk5 (I,J), nscl-1 (K,L), neurod (M,N), aml1 (O,P), activin beta b (Q,R), and emx1 (S,T) are strongly upregulated by EBF2 and EBF3 (brackets). Panels (A-R) show dorsal views, while (S,T) show anterior views.
	Figure 2. Downregulation of transcription factor candidate target genes after knockdown of EBF2 and EBF3. One cell of two-cell stage embryos was injected with either control MO, both EBF2 MO and EBF3 MO (2MO + 3MO), or dominant negative Xenopus EBF3 (DN-XEBF) mRNA. β-Galactosidase (β-gal) mRNA was co-injected as a marker of the injected side. In all panels the right side is the injected side, showing the light blue color of X-gal staining. (A-C) The expression of sox2 was not changed in all three conditions. (D-O) The expression of nscl-1 (E,F), neurod (H,I), aml1 (K,L), and emx1 (N,O) is downregulated by EBF2 MO and EBF3 MO, and by DN-XEBF (brackets), while control MO does not change their expression levels (D,G,J,M). Panels (A-L) show dorsal views of neurula stage embryos (stage 15/16), and (M-O) are anterior views of tail bud stage embryos (stages 25 to 28).
	Figure 3. Downregulation of non-transcription factor candidate target genes after knockdown of EBF2 and EBF3. One cell of two-cell stage embryos was injected with either control MO, both EBF2 MO and EBF3 MO (2MO + 3MO), or dominant negative Xenopus EBF3 (DN-XEBF) mRNA. β-Galactosidase (β-gal) mRNA was co-injected as a marker of the injected side. In all panels the right side is the injected side, showing the light blue color of X-gal staining. (A-R) The expression of pcdh8 (B,C), peripherin (E,F), greb1 (H,I), nf-m (K,L), kcnk5 (N,O), and activin beta b (Q,R) is downregulated by EBF2 MO and EBF3 MO, and by DN-XEBF (brackets), while control MO does not change their expression levels (A,D,G,J,M,P). Panels (A-I) and (P-R) are neurula stage embryos (stage 15/16), and (J-O) are tail bud stage embryos (stages 25 to 28). All panels show dorsal views.
	Figure 4. Neuronal expression for ebf genes and transcription factor candidate target genes. (A-F) ebf2 (A-C) and ebf3 (D-F) are expressed in multiple regions of the developing nervous system, including the trigeminal placodes (yellow arrows), olfactory placodes (black arrows), some domains in the brain, the spinal cord (white arrows), and neural crest derivatives like the branchial arches. (G-L) nscl-1 (G-I) and neurod (J-L) are expressed in the trigeminal placodes and three stripes of primary neurons in the neural plate (black arrowheads) at stage 15, and are strongly expressed in the trigeminal placodes, olfactory placodes, and spinal cord at stage 23. At stage 28, nscl-1 is expressed in the olfactory placodes, some domains in the midbrain/hindbrain, spinal cord and cranial ganglia IX and X. At stage 28, neurod is expressed in the olfactory placodes, retina, otic placodes, cranial ganglia, spinal cord and some domains in the brain. (M-O) aml1 is expressed in the lateral primary neuron stripe at stage 15, sensory neurons of the spinal cord (white arrowheads) at stage 23, and the olfactory placodes and otic placodes at stage 28. (P-R) emx1 is weakly expressed in the prospective forebrain region at stage 15. At stages 23 and 28, this gene is expressed in the dorsal forebrain region. Stage 15 embryos show dorsal views except (P) (anterior view). Stage 23 and 28 embryos show lateral views.
	Figure 5. Neuronal expression for non-transcription factor candidate target genes. (A-C) pcdh8 is expressed in medial and intermediate stripes (arrowheads) of primary neurons, in one posterior stripe between the two stripes of primary neurons, and in the anterior domain of the neural plate at stage 15, and in the spinal cord (white arrows) and some domains in the brain at stages 23 and 28. (D-F) peripherin is expressed in the trigeminal placodes (yellow arrows) and three stripes of primary neurons (arrowheads) at stage 15, and in the trigeminal placodes, olfactory placodes (black arrow), spinal cord, retina and many domains in the brain at stages 23 and 28. (G-I) greb1 is expressed as a band in the prospective midbrain/hindbrain region at stage 15, and in the midbrain/hindbrain region and spinal cord at stage 23, and in the olfactory placodes, spinal cord, and many domains in the brain at stage 28. (J,K) nf-m is not expressed at stage 15, but at stages 23 and 28 it is expressed in the trigeminal placodes and spinal cord. (L-N) kcnk5 is weakly expressed from anterior to posterior along the dorsal midline, with stronger expression in the anterior end of the neural fold at stage 15. It is expressed in retina, otic placode, and several domains in the brain at stages 23 and 28, and in spinal cord at stage 28. (O-Q) activin beta b is expressed in two bands in the prospective midbrain/hindbrain region and diffusely throughout the anterior neural plate at stage 15, and it is expressed in the retina and some brain domains at stages 23 and 28. Stage 15 embryos show dorsal views, and stages 23 and 28 embryos show lateral views.
	ebf2 (early B-cell factor 2) gene expression in Xenopus laevis embryos, NF stage 15, as assayed by in situ hybridization, dorsal view, anterior left. Key: trigeminal placodes (yellow arrows), olfactory placodes (black arrows), three stripes of primary neurons in the neural plate (black arrowheads).
	ebf2 (early B-cell factor 2) gene expression in Xenopus laevis embryos, NF stage 23, as assayed by in situ hybridization, lateral view: dorsal up, anterior left. Key: trigeminal placode (yellow arrow); olfactory placode (black arrow), spinal cord (white arrows).
	ebf2 (early B-cell factor 2) gene expression in Xenopus laevis embryos, NF stage 28, as assayed by in situ hybridization, lateral view, dorsal up, anterior left. Key: olfactory placode (black arrow); spinal cord (white arrows)
	ebf3 (early B-cell factor 3) gene expression in Xenopus laevis embryos, NF stage 15, as assayed by in situ hybridization, dorsal view, anterior left. Key: trigeminal placode (yellow arrows); developing neurons of the neural plate and neural plate border (black arrowheads).
	ebf3 (early B-cell factor 3) gene expression in Xenopus laevis embryos, NF stage 23, as assayed by in situ hybridization. Lateral view: dorsal up, anterior left. Ley, trigeminal placode (yellow arrow); olfactory placode ( black arrow); neurons of the spinal cord ( white arrow).
	ebf3 (early B-cell factor 3) gene expression in Xenopus laevis embryos, NF stage 28, as assayed by in situ hybridization. Lateral view: dorsal up, anterior left.
	nhlh1 (nescient helix loop helix 1) gene expression in Xenopus laevis embryos, NF stage 15, as assayed by in situ hybridization. Dorsal view, anterior left.
	nhlh1 (nescient helix loop helix 1) gene expression in Xenopus laevis embryos, NF stage 23, as assayed by in situ hybridization,lateral view: dorsal up, anterior left. Note trigeminal placode (yellow arrow), olfactory placode (black arrow), domains of the spinal cord (white arrows)
	nhlh1 (nescient helix loop helix 1) gene expression in Xenopus laevis embryos, NF stage 28, as assayed by in situ hybridization. Lateral view: dorsal up, anterior left.
	greb1 (growth regulation by estrogen in breast cancer 1) gene expression in Xenopus laevis embryos, NF stage 15, as assayed by in situ hybridization. Dorsal view, anterior left.
	greb1 (growth regulation by estrogen in breast cancer 1) gene expression in Xenopus laevis embryos, NF stage 23, as assayed by in situ hybridization. Lateral view: dorsal up, anterior left.
	inhbb (inhibin, beta B) gene expression in Xenopus laevis embryos, NF stage 15, as assayed by in situ hybridization. Dorsal view, anterior left.
	inhbb (inhibin, beta B) gene expression in Xenopus laevis embryos, NF stage 23, as assayed by in situ hybridization. Lateral view: dorsal up, anterior left.
	inhbb (inhibin, beta B) gene expression in Xenopus laevis embryos, NF stage 28, as assayed by in situ hybridization. Lateral view: dorsal up, anterior left.
	kcnk5 (potassium channel, subfamily K, member 5) gene expression in Xenopus laevis embryos, NF stage 15, as assayed by in situ hybridization. Dorsal view, anterior left.
	kcnk5 (potassium channel, subfamily K, member 5) gene expression in Xenopus laevis embryos, NF stage 28, as assayed by in situ hybridization. Lateral view: dorsal up, anterior left.
	nefm (neurofilament medium chain) gene expression in Xenopus laevis embryos, NF stage 23, as assayed by in situ hybridization. Lateral view: dorsal up, anterior left.
	nefm (neurofilament medium chain) gene expression in Xenopus laevis embryos, NF stage 28, as assayed by in situ hybridization. Lateral view: dorsal up, anterior left.
	pcdh8 (protocadherin 8) gene expression in Xenopus laevis embryos, NF stage 15, as assayed by in situ hybridization. Dorsal view, anterior left.
	pcdh8 (protocadherin 8) gene expression in Xenopus laevis embryos, NF stage 28, as assayed by in situ hybridization. Lateral view: dorsal up, anterior left.
	prph (peripherin) gene expression in Xenopus laevis embryos, NF stage 15, as assayed by in situ hybridization. Dorsal view, anterior left.
	prph (peripherin) gene expression in Xenopus laevis embryos, NF stage 23, as assayed by in situ hybridization. Lateral view: dorsal up, anterior left.
	prph (peripherin) gene expression in Xenopus laevis embryos, NF stage 28, as assayed by in situ hybridization. Lateral view: dorsal up, anterior left.
	Figure 6. The identification of direct and indirect candidate targets of EBF3 by RT-QPCR. hGR-XEBF3 mRNA and Noggin mRNA were injected into one-cell stage embryos, and animal caps were collected at the blastula stage (stage 9). The animal caps were divided into four groups, based on CHX and DEX treatment: -C-D, -C+D, +C-D, and +C+D. After a 3.5-hour incubation with CHX and/or a 3-hour incubation with DEX, total RNA was isolated from each animal cap group. RT-QPCR was conducted with the isolated total RNA. The expression level was normalized with the expression level of histone h4 and then normalized to the expression level of -C+D, for each gene, at 100 arbitrary units. (A-C) The expression levels of all candidate target genes in controls (-C-D and +C-D) are very low compared to the DEX-treated condition of -C+D. (A) The expression level of peripherin in +C+D (61%) is only partially reduced compared to -C+D, indicating that the majority of its expression is controlled by EBF3 directly. (B) The expression level of pcdh8 in +C+D (5%) is much lower than in -C+D and is similar to the levels of the control conditions, indicating that it is an indirect candidate target. (C) The expression level of aml1 in +C+D (37%) is lower than the expression level in -C+D but higher than levels of the control conditions, indicating that its expression is partially controlled by EBF3 directly. Error bars represent standard error of the mean. The results for the remaining candidate target genes are shown in Additional file 6. N = 3 replicates, 20 to 30 animal caps per condition.

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