Belyaeva OV , Lee SA , Adams MK , Chang C , Kedishvili NY .

AA12153 NIAAA NIH HHS , GM83029 NIGMS NIH HHS , R01 AA012153 NIAAA NIH HHS , R01 GM083029 NIGMS NIH HHS

Xla Wt + aldh1a2 + retinol (fig.5.a, b)
Xla Wt + aldh1a2 + retinol (fig.5.a, b)
Xla Wt + aldh1a2 + sdr16c5 + retinol (fig.5.a, b)
Xla Wt + aldh1a2 + sdr16c5 + retinol (fig.5.a, b)
Xla Wt + rdh10 MO (fig.6.c)
Xla Wt + retinol (fig.5.a, b)
Xla Wt + retinol (fig.5.a, b)
Xla Wt + sdr16c5 (fig.5.c)
Xla Wt + sdr16c5 + retinol (fig.5.a, b)
Xla Wt + sdr16c5 + retinol (fig.5.a, b)
Xla Wt + sdr16c5 + retinol (fig.5.b)
Xla Wt + sdr16c5 MO (fig.6.c)
Xla Wt + sdr16c5 MO (fig.6.c)
Xla Wt + sdr16c5 MO (fig.7.a)
Xla Wt + sdr16c5 MO (fig.7.b)
Xla Wt + sdr16c5 MO (fig.7.d)
Xla Wt + sdr16c5 MO (fig.7.e)
Xla Wt + sdr16c5 MO (fig.7.f)
Xla Wt + sdr16c5 MO (fig.7.g)

	FIGURE 4. Expression pattern of endogenous rdhe2 in frog embryo. A, Western blot analysis of extracts (50 μg of total protein) of neurula stage wild-type embryos or embryos injected with in vitro synthesized rdhe2 mRNA revealing a single major band of the expected size (∼34 kDa), which is increased in injected embryos, thus confirming that antibody is specific. B and C, Western blot analysis of dissected gastrula embryos (B) and stage 39 tadpoles (C), 30 μg of total protein per lane. D, whole mount in situ hybridization with antisense rdhe2 probe. E, in situ hybridization of dissected embryos with rdhe2 probe. Dashed lines indicate where embryos were cut in F. F, embryos stained with rdhe2 probe further dissected to better reveal staining of notochord (left, cross-section through the trunk; center, sagittal cut through the upper portion of the trunk), or paraffin-embedded and sectioned at 50 μm (right). G, 50-μm sections of prestained paraffin-embedded specimen revealing staining in the brain, eye, and otic vesicle. NC, notochord; NT, neural tube; FB, forebrain; MB, midbrain; HB, hindbrain, OV, otic vesicle; OB, olfactory bulb.
	FIGURE 5. Overexpression of frog rdhe2 in Xenopus embryos. A, wild-type or mRNA-injected embryos treated with all-trans-retinol and incubated until early tadpole stages, fixed, and divided into severe, moderate, mild, and normal groups according to the severity of manifested defects. The figure shows representative embryos for each phenotypic class. B, proportion of embryos manifesting defects of different severity after injections of rdhe2 mRNA (4 ng/embryo), raldh2 mRNA (1 ng), or a mixture of both. Embryos were treated with 5 or 8 μM all-trans-retinol from stage 8 to stage 14. C, expression of anterior marker genes otx2, krox20, en2, and rx2a in injected (arrow) versus uninjected side of the embryo analyzed by whole mount in situ hybridization.
	FIGURE 6. MO-mediated knockdown of endogenous rdhe2. A, Western blot analysis of protein extracts (30 μg/lane) from neurula embryos injected with 4 ng of rdhe2 mRNA alone or mixed with 50 ng of rdhe2 MO. MO essentially prevents an increase in the newly synthesized rdhe2 protein levels over those preexisting in wild-type embryos (bottom). Top, staining for β-actin as a loading control. B, Western blot analysis of protein extracts prepared from an equal number of rdhe2 and control morphants (st. 32) using rdhe2 (bottom) or actin (top) antibodies shows a decrease in endogenous rdhe2 levels. C, array of phenotypes displayed by rdhe2 and rdh10 morphants compared with control morphants. Phenotypes were divided into severe, strong, moderate, and mild groups with the representative embryos shown for each group. An arrow shows an incompletely closed neural tube typical for severely affected rdhe2 morphants. Dramatic phenotypic changes in morphants compared with the moderate decrease in protein levels in B may reflect a strong localized effect of MO at sites where newly synthesized rdhe2 protein is critical for embryogenesis. D, proportion of different phenotypic groups in rdhe2 morphants compared with control morphants and wild-type embryos. E, co-injection of rdhe2 rescue mRNA (4 ng) and rdhe2 MO (30 ng) decreasing the frequency and severity of defects displayed by morphants. F, proportion of different phenotypic groups in embryos injected with 25 ng of rdhe2 MO or rdh10 MO, or a mixture of both (50 ng total). Control MO (25 ng) was added to individual rdhe2- and rdh10-MO injections to equalize the total oligonucleotide amount.
	FIGURE 7. Decreased retinoid signaling in rdhe2 morphants. A, rdhe2- or control-MO (155 ng) mixed with the lineage tracer were injected into one dorsal blastomere at four-cell stage. The injected side is indicated by an arrow. D, rdhe2- or control-MO (50 ng/embryo) was injected into two dorsal blastomers. D, embryos were treated with 0.01 or 0.1 μM RA where specified. A, whole mount in situ hybridization with antisense RNA probes was performed for the following markers (stages and frequencies of observed phenotypes in parentheses): A, otx2 and krox20 (st. 20, 31/77); B, en2 (st. 189, 42/74); C and D, hoxd4 (st. 189, 30/46); E, sox3 (st. 134, 10/12); F, shh (st.14, 23/28); G, myod (st. 302, 15/42). Arrows in G indicate irregular somites as revealed by myod staining. H, tissues of rdhe2 and control morphants were incubated over the monolayer of F9-RARE-lacZ cells; β-galactosidase activity was revealed with X-gal substrate. The results are representative of three independent experiments.
	sdr16c5 ( short chain dehydrogenase/reductase family 16C, member 5) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 36, lateral view, anterior left, dorsal up.
	sdr16c5 ( short chain dehydrogenase/reductase family 16C, member 5) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 39, lateral view, anterior left, dorsal up.
	sdr16c5 ( short chain dehydrogenase/reductase family 16C, member 5) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 26, lateral view, anterior left, dorsal up.

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