Mead PE et al. (1998), Cloning of Mix-related homeodomain proteins usi...

XB-ART-14283

Proc Natl Acad Sci U S A 1998 Sep 15;9519:11251-6. doi: 10.1073/pnas.95.19.11251.

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Cloning of Mix-related homeodomain proteins using fast retrieval of gel shift activities, (FROGS), a technique for the isolation of DNA-binding proteins.

Mead PE , Zhou Y , Lustig KD , Huber TL , Kirschner MW , Zon LI .

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We have developed a technique, fast retrieval of gel shift activities (FROGS), that allows for the rapid isolation of proteins that interact with DNA. Using this technique, we have isolated two proteins that are structurally similar to Mix.1, a PAX class homeodomain protein with ventralizing activity in Xenopus. The Mix family of proteins are expressed during late blastula and gastrula stages of Xenopus development. During gastrulation, these genes are expressed at high levels in distinct, yet overlapping regions in mesoderm and endoderm. The members of the Mix family heterodimerize with each other and overexpression of each results in severe axial abnormalities. Mix.3 and Mix.4 can directly induce primitive ectoderm to become endoderm whereas Mix.1 cannot. Injection of Mix.3 or Mix.4 RNA in the whole embryo results in extensive ectopic endodermin mRNA expression. The expression of the Mix family homeoproteins is differentially regulated by activin, Vg1, BMP-4, and fibroblast growth factor, supporting a model in which the Mix homeoproteins are downstream effectors of growth factor signaling during endoderm and ventral mesoderm formation.

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Species referenced: Xenopus
Genes referenced: a2m bmp4 fabp2 fgf2 gdf1 mix1 pdx1 smad1 sox17a

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	FIG. 1. (A) FROGS. Protein from pools of 100 cDNA plasmids was synthesized in vitro by coupled transcription/translation. A gel mobility shift assay on radiolabeled P3 site was performed in the presence or absence of HA, a truncated mutant of Mix.1 that encodes just the homeodomain. Pools with a specific gel mobility shift were sib-selected to isolate pure clones. (B) Sib selection of a sample pool. The gel mobility shift is detected in the primary pool (pool 22). The activity becomes more prominent with sib selection to pools of 12 cDNA clones (22B) and finally with the pure cDNA clone (22B8) when homodimers are evident. The HA heterodimer is marked. Note: the unprogrammed lysate (labeled H2O) contains no added plasmid and characteristically gives high background on gel mobility shift analysis.
	FIG. 2. Sequence analysis of the Mix family of homeoproteins. Conserved residues are shaded and the homeodomain is indicated by the solid bar.
	FIG. 3. (A) Mix family members can heterodimerize with each other. The full-length clones were tested for their ability to heterodimerize on a candidate P3 site with the truncated Mix.1 homeodomain construct HA. Each combination yields an extra band demonstrating dimerization (arrowheads). (B) Expression patterns of the Mix family of homeoproteins. (Top, i–iii) Vegetal view of blastula (stage 9). The embryos are slightly tilted to show a portion of the marginal zone. (Middle, iv–vi) Transverse section of early gastrula (stage 101). (Bottom, vii–ix) Vegetal view of mid-late gastrula (stage 10.5–11). White arrows indicate the dorsal axis. (C) Growth factor signaling upstream of the Mix homeoproteins. Embryos were injected with growth factor mRNA at the one-cell stage, animal pole explants were dissected at stage 8, and the expression of each Mix family member was analyzed at stage 11 by RT-PCR. The TGF-b family members activin, AVg1, and BMP-4 induce expression of each of the Mix family of homeoproteins. Basic FGF induced expression of Mix.4 alone. Expression of the Mix genes was not stimulated by Xwnt-8.
	FIG. 4. Ectopic expression of Mix.1, Mix.2, Mix.3, and Mix.4 leads to axial abnormalities. Embryos were injected at the one-cell stage with 1 ng of each synthetic mRNA. b-galactosidase RNA was included as a negative control.
	FIG. 5. Mix genes and hematopoiesis. (A) Mix genes pattern mesoderm to a ventral (hematopoietic) fate. Embryos were injected with mRNA at the one-cell stage in the animal pole (1 ng of each RNA), animal caps were explanted at stage 8 and cultured to sibling stage 36 in the presence of bFGF, and expression of globin RNA was examined by RT-PCR. M11 is a dominant negative mutant of Mix.1. (B) Mix genes induce erythroid cells in animal pole explants. Embryos were injected with plasmid DNA (300 pg, pcDNA3.0) at the one-cell stage in the animal pole and animal caps were explanted at stage 8 and cultured in the presence of basic FGF (20 ng/ml) to sibling stage 36. Disaggregated animal caps were stained with O-dianisidine (T.L.H., Y.Z., P.E.M., and L.I.Z., manuscript submitted), cytocentrifuged onto glass slides, and photographed at 3200.
	FIG. 6. Expression of Mix family proteins and markers of endoderm differentiation in animal pole explants and whole embryos. (A) Expression of Mix family members leads to the induction of endoderm. Embryos were injected at the one-cell stage with 1 ng of each RNA. Smad1 and M11 are included as negative controls. Animal pole explants were dissected at stage 8 and cultured to sibling stage 36. RT-PCR analysis demonstrated that Mix.3 and Mix.4 induce endodermal markers edd, IFABP, GATA-5, XSox17a, and XlHbox8. Overexpression of Mix family members, without added growth factors, failed to induce mesoderm markers such as globin. (B) Whole embryo in situ hybridization for edd. Embryos were injected with Mix RNA (500 pg/blastomere) at the two-cell stage, cultured to sibling control stage 38, and then fixed and stained for edd expression by in situ hybridization. Mix.3 led to ectopic edd expression (54/55 injected embryos) whereas Mix.1 did not (0/37 injected embryos).
	FIG. 7. Mix.1 antagonizes Mix.3 induction of endoderm markers. Embryos were injected at the one-cell stage with a constant dose of Mix.3 RNA (200 pg) and an increasing amount of Mix.1 RNA (200–1000 pg). Animal poles were explanted at stage 8 and cultured for 2 days (stage 36). Endoderm marker expression was determined by RT-PCR analysis.

References [+] :

Ang, A targeted mouse Otx2 mutation leads to severe defects in gastrulation and formation of axial mesoderm and to deletion of rostral brain. 1996, Pubmed