XB-ART-44151Biochem Biophys Res Commun November 11, 2011; 415 (1): 11-6.
The homeobox leucine zipper gene Homez plays a role in Xenopus laevis neurogenesis.
The Homez gene encodes a protein with three atypical homeodomains and two leucine zipper motifs of unknown function. Here we show that during neurula stages, Xenopus Homez is broadly expressed throughout the neural plate, the strongest expression being detected in the domains where primary neurons arise. At later stages, Homez is maintained throughout the central nervous system in differentiating progenitors. In accordance with this expression, Homez is positively regulated by neural inducers and by Ngnr1 and negatively by Notch signaling. Interference with Homez function in embryos by injection of an antisense morpholino oligonucleotide results in the specific disruption of the expression of late neuronal markers, without affecting the expression of earlier neuronal and early neurectodermal markers. Consistent with this finding, Homez inhibition also interferes with the expression of late neuronal markers in Ngnr1 overexpressing animal cap explants and in Notch inhibited embryos. In gain of function experiments, Homez inhibits the expression of late neuronal markers but has no effect on earlier ones. These data suggest a role for Homez in neuronal development downstream of proneural/neurogenic genes.
PubMed ID: 22001922
Article link: Biochem Biophys Res Commun
Species referenced: Xenopus laevis
Genes referenced: btbd6 dmrta2 dpysl3 foxi1 homez krt12.4 myc myt1 neurog1 neurog2 nog nos1 notch1 sox3 tbxt tcf3 tubb2b vegt
Morpholinos: homez MO1
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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.|
|Fig. 2. Homez is regulated positively by attenuation of Bmps and Ngnr1 and negatively by Notch. (A) RT-PCR analysis of Homez expression in animal cap explants isolated from embryos injected with increasing doses of noggin mRNA or from uninjected control animal caps (CC). CE, control embryos. Ep. Keratin and the pan-neural Sox3 gene were used as controls for neural induction. Histone H4 was used as a loading control. -RT, control RT-PCR without reverse transcriptase. (B–D) In situ analysis of Homez expression in neurula embryos (dorsal views) injected unilaterally with the indicated mRNA and LacZ mRNA. Injected sides (IS) are indicated.|
|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.|
|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.|
|Supplementary Fig. 1. (A) Sequence alignment of full length predicted amino-acid sequences from Xenopus laevis, mouse and human Homez proteins. Genbank Accession Nos.: Xenopus, BC071005; mouse, NM001177705; human, NM020834. Dashes represent gaps introduced into the amino acid sequence in order to obtain optimal sequence homology. Identical amino-acids are boxed. The homeodomains, leucine repeats and acidic domain are indicated, respectively in red, blue and green rectangles. Xl, Xenopus laevis; Hs, Homo sapiens; Mm, Mus musculus. (B) Subcellular localization of a Flag-tagged Homez protein in animal pole cells of early gastrula embryos. Note that immunoreactivity is only detected in the nucleus.|
|homez (homeobox and leucine zipper encoding) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anterior right, dorsal up.|