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Fig. 1. Homez structure and gene expression during embryogenesis. (A) Shematic representation of the Homez protein. HD, homeodomain; L, leucine repeats, A, acidic
domain. (B) Temporal expression of Homez by RT-PCR. RNA was extracted from embryos at the indicated stages. (C) RT-PCR analysis of Homez expression in dissected explants
of stage 10.5 embryos. The endodermal VegT, mesodermal Xbra and ectodermal Xema markers were used as dissection controls. In B and C, Histone H4 was used as a loading
control. -RT, control RT-PCR without reverse transcriptase. (D-I) Spatial expression of Homez compared to that of Ngnr1, Myt1 and N-tubulin analyzed by whole-mount in situ
hybridization. Nieuwkoop-Faber stages are indicated. (D–H) Dorsal views. (I) Lateral view. Stripes of Homez expression in the domains of primary neurogenesis in the
posterior neural plate are indicated (l, lateral; i intermediate; m, medial). Note that Homez is activated in the domains of primary neurogenesis at E12.5 when N-tubulin is not
yet detectable. (J–M) Homez expression compared to that of Ngnr1, Myt1 and N-tubulin in the neural tube of stage 32 embryos. Sections at the level of the otic vesicle are
shown. Note that XHomez+ cells are detected more medially than N-tubulin in the marginal zone. Abbreviations: AC, animal caps; ba, branchial arches; cg, cranial ganglia; e,
egg; CE, control embryo; MZ, marginal zone; VP, vegetal pole. |
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Fig. 4. Effect of Homez overexpression on the neuronal differentiation. (A) RT-PCR
analysis of the expression of the indicated genes in animal caps from embryos
overexpressing Homez (500 pg of Homez mRNA/blastomere) or from uninjected
control embryos. Note that Homez blocks Ep. Keratin expression but is not sufficient
to induce neural or neuronal markers in animal cap explants. CC, control caps; CE,
control embryo. -RT, reverse transcriptase minus as a negative control. (B–G) Dorsal
views of neurula embryos injected with 500 pg of Homez mRNA analyzed by in situ
hybridization for the indicated markers. Injected sides (IS) are indicated. Note that
overxexpression of Homez interferes with N-tubulin and CRMP4 expression but does
not affect Ngnr1 and Myt1. |
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CRMPFig.
3. Knockdown of Homez reduces the expression of late but not early neuronal markers. (A) In vivo translation of Homez-eGFP is specifically inhibited by the Homez MO.
Embryos were injected with 500 pg of Homez-eGFP or, as a control, a Dmrt5-eGFP construct, alone or in combination with 10 ng of the Homez MO, as indicated. (B–F)
Expression of the indicated neuronal genes in neurula embryos (dorsal views) injected unilaterally with 10 ng of the Homez MO and LacZ mRNA as a tracer. (G) Section of the
neural tube at the level of the hindbrain of a MO Homez injected tadpole embryo stained with N-tubulin. (H–K) Dorsal views of embryos coinjected with 500 pg Su(H)DBM
mRNA, with or without 10 ng of the Homez MO and probed as indicated. Injected sides (IS) are indicated. (L) RT-PCR analysis of the expression of the terminal neuronal
markers CRMP4, N-tubulin and BTBD6, the early postmitotic marker Myt1 and of the pan-neural markers Sox3 in animal caps derived from embryos injected with 50 pg Ngnr1
mRNA/blastomere alone or together with 2, 5, 5 or 10 ng of Homez MO or 10 ng of a standard control MO. Histone H4, internal positive control. -RT, control RT-PCR without
reverse transcriptase. A control western blot shows that similar levels of Myc-Ngnr1 have been produced under each condition. GAPH, Glyceraldehyde 3-phosphate
dehydrogenase. |
