Sölter M et al. (1999), Characterization of a subfamily of related wing...

XB-ART-11981

Mech Dev 1999 Dec 01;891-2:161-5. doi: 10.1016/s0925-4773(99)00195-1.

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Characterization of a subfamily of related winged helix genes, XFD-12/12'/12" (XFLIP), during Xenopus embryogenesis.

Sölter M , Köster M , Hollemann T , Brey A , Pieler T , Knöchel W .

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The fork head domain family of genes defines a growing group of proteins that serve important regulatory functions in pattern-forming events of both invertebrates and vertebrates. Here we add three closely related, novel members to this family in Xenopus laevis, termed XFD-12, XFD-12' and XFD-12". All three genes reveal indistinguishable expression patterns during Xenopus embryogenesis. During gastrulation, XFD-12 type transcripts are detected exclusively in the superficial layer of cells within the Spemann organizer territory. In the open neural plate, XFD-12 type expression defines a row of cells located along the dorsal midline and destined to become the floor plate of the neural tube. After closure of the neural tube, XFD-12 type encoding mRNAs are only detected in the tailtip and a small area located at the midbrain/hindbrain boundary. Within the Spemann organizer and in the floor plate area, expression of XFD-12 type genes is only partially overlapping with XFD-1 expression.

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Species referenced: Xenopus laevis
Genes referenced: chrd egr2 en2 foxa4 foxd4l1.1 tbx2

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	Fig. 3. Spatial distribution of XFD-12' transcripts analyzed by whole-mount in situ hybridization. XFD-12' expression in different embryonic stages (as indicated) is visualized either in purple (A-H,K,L) or in brown (I,J,M,N), staining for XFD-1(Knoèchel et al., 1992) (I,J), En-2 (Hemmati-Brivanlou et al., 1991) (M) and Krox-20 (Bradley et al., 1993) (N) is shown in red. Expression of XFD-1 (I',J') is indicated in purple. Stained embryos were gelatin-embedded and vibratome-sectioned at 30 mm. Magnified regions are boxed and demarcated by the corresponding letter. (A) Lateral view (bottom, vegetal pole; right, dorsal), (B-E) dorsal view (blastopore/posterior, bottom), (K-N) lateral view (posterior, right), (F,F') sagittal sections (orientated as in (a), (G,I,I') sagittal sections (top, dorsal; blastopore, right), (L',L'',M',N') sagittal sections (top, dorsal; anterior, left), (H,J,J') transverse sections within the posterior third of the embryo (top, dorsal).
	Fig. 4. Effects of UV and LiCl treatment, as well as of dorsal BMP-4 and ventral chordin injection on XFD-12' expression. 400 pg BMP-4 RNA were injected into each dorsal blastomere and 200 pg chordin RNA were injected into each ventral blastomere of four-cell stage embryos. These, as well as UV and LiCl treated embryos were fixed at stage 11 and analyzed by whole-mount in situ hybridization.
	Fig. 2. Temporal expression of XFD-12′ during Xenopus embryogenesis. Stages of RNA preparations (Nieuwkoop and Faber, 1975) are indicated above each column. RT-PCR was performed using the following primers: XFD-12′ (F, 5′-GAACCCACCATAATTCCC TG-3′ and R, 5′-CTGAATGTGTAGGCTGTAGG-3′; 122 bp; 28 cycles) and histone H4 (F, 5′-CGGGATAACATTCAGGGTATCACT-3′ and R, 5′-ATCCATGGCGGTAACTGTCTTCCT-3′; 188 bp; 21 cycles).
	Fig. 1. A novel subfamily of closely related Xenopus winged helix factors. (A) Amino acid sequences of XFD-12, XFD-12′ and XFD-12″. Sequences were deduced from the corresponding cDNAs (EMBL database accession AJ242676 (XFD-12), AJ242677 (XFD-12′) and AJ242678 (XFD-12″)). Residues in XFD-12′ and 12″ being identical to those in XFD-12 are indicated by dots, deletions are shown by dashes. The winged helix domain is boxed. (B) Winged helix domain alignments. Members of the fork head/HNF-3 subfamily 5b (Kaufmann and Knöchel, 1996) being characterized by the GICE/D motif (amino acid positions 35–38) have been aligned for their winged helix domains. The partial XFLIP sequence (King and Moore, 1994; XFLIP update: accession number U04872) is identical to the corresponding positions of XFD-12, XFD-12′ as well as of XFD-12″ (see also Fig. 1A), however, the 95 bp nucleotide sequence of XFLIP yields only a perfect match to XFD-12′ (one silent mismatch to XFD-12′, three silent mismatches to XFD-12; data not shown). Positions of helices H1, H2 and H3 (involved in DNA binding) as found in HNF-3γ structure (Clark et al., 1993) are indicated. Note that Xenopus XFD-6 (Scheucher et al., 1995), rodent HFH-2/genesis (Clevidence et al., 1993, Sutton et al., 1996), chicken CWH-3 (Freyaldenhoven et al., 1997) and zebrafish fkd6 (Odenthal and Nüsslein-Volhard, 1998) are most closely related, but differ from XFD-12 and Drosophila FD3 (Häcker et al., 1992), as well as from zebrafish fkd8 (Odenthal and Nüsslein-Volhard, 1998).
	foxd4l1.1 (forkhead box D4 like 1, gene 1) gene expression in bisected Xenopus laevis embryo, assayed via in situ hybridization, NF stage 10, dorsal up.
	foxd4l1.1 (forkhead box D4 like 1, gene 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 11, dorsal view, blastopore to bottom.
	foxd4l1.1 (forkhead box D4 like 1, gene 1) gene expression in bisected Xenopus laevis embryo, assayed via in situ hybridization, NF stage 12.5, dorsal up.
	foxd4l1.1 (forkhead box D4 like 1, gene 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 31, lateral view, anterior left, dorsal up (L) and higher magnification of brain region (L') and tail tip (L").
	foxd4l1.1 (forkhead box D4 like 1, gene 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 22, lateral view, anterior left, dorsal up