Dirksen ML and Jamrich M (1992), A novel, activin-inducible, blastopore lip-spec...

XB-ART-23863

Genes Dev 1992 Apr 01;64:599-608. doi: 10.1101/gad.6.4.599.

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A novel, activin-inducible, blastopore lip-specific gene of Xenopus laevis contains a fork head DNA-binding domain.

Dirksen ML , Jamrich M .

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The organizer region, or dorsal blastopore lip, plays a central role in the initiation of gastrulation and the formation of the body axis during Xenopus development. A similar process can also be induced in ectodermal explants by activin or by injection of activin mRNA into embryos. We have searched early embryo-specific cDNA libraries for genes containing the fork head box sequence that encodes a DNA-binding domain similar to that of the Drosophila homeotic gene fork head and rat hepatocyte nuclear factor HFN3 beta. These genes were subsequently tested for expression in the organizer region of blastula/gastrula-stage embryos as well as inducibility by activin. Our effort resulted in the isolation of a gene, XFKH1, that is primarily expressed in the dorsal blastopore lip of early gastrulae and is inducible by activin. At later stages it is expressed in the notochord and neural floor plate. Because of its spatial and temporal expression pattern, as well as its inducibility by activin, this gene is a good candidate to have a regulatory function in the initial processes of axis formation in Xenopus laevis embryos.

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Species referenced: Xenopus laevis
Genes referenced: foxa4 tbx2

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	Figure 1. The nucleotide sequence of XFKHl and its corresponding amino acid sequence. [A] Amino acids are in the single-letter IP AC code. The fkh domain is boxed in. The termination codon is indicated by an asterisk (*). (B) Comparison of the fkh domain of XFKHl to the similar domains in the rat transcription factor HNF-3P (Lai et al. 1991) and Drosophila homeotic gene fkh (Weigel et al. 1989). Asterisks indicate identities.
	Figure 2. Developmental expression of XFKHl. {A) Northern blot analysis of 10 txg of total RNA from different stages of embryos. [Bottom] Ethidium bromide staining of the gel; [top] autoradiography. (Stage E) Egg; (stage 8) blastula; (stages 10,12) gastrula; (stages 15,20) neurula; (stages 30,40) tadpoles. The positions of 18S and 28S mRNAs are indicated by arrows. [B] Northern blot analysis of RNA from dissected halves of embryos. Equal amounts of RNA (10 \|xg) were loaded per lane. (A) Animal; (V) vegetal; (D) dorsal; (Ve) ventral.
	Figure 3. Whole-mount in situ hybridization analysis of XFKHl expression in Xenopus blastula and gastrula. {A) In situ hybridization of XFKHl to stage 9'A Xenopus blastula. This view from the vegetal pole of the embryo shows that the XFKHl RNA is expressed in the dorsal marginal zone of the embryo, (d) Dorsal; (v) ventral. [B] In situ hybridization of XFKHl to stage 11 Xenopus gastrula. Notice intense hybridization in the dorsal marginal zone in this vegetal view of the embryo, (d) Dorsal; (v) ventral. (C) Lateral view of a stage lO'/z embryo hybridized with XFKHl cRNA. Most of the signal is in the organizer region (arrow). [D] Lateral view of stage 11 Vi embryo. Hybridization is primarily in the invaginating marginal zone of the dorsal blastopore lip (arrow).
	Figure 4. Whole-mount in situ hybridization of XFKHl to Xenopus neurulae. [A] Dorsal view of stage 12 embryo showing the extent of hybridization of XFKHl in the dorsal region of the embryo. Anterior is left; posterior is right. (B) Posterior view of stage 12'/2 embryo hybridized with XFKHl showing the dorsal labeling of XFKHl RNA. Hybridization is to a narrow strip of chordal mesoderm and overlying neural tissue. (C) Dorsal view of stage 18 embryo showing the XFKHl-labeling pattern. At this point of development, labeling extends into the brain area. [D] Lateral view of stage 20 embryo showing the inactivation of XFKHl gene in the posterior region of the embryo. Forebrain area appears to be negative as well (arrow). (£) In situ hybridization of XFKHl and engrailed 2 to a stage 18 embryo. Arrow indicates the transverse band of engrailed 2 hybridization. The hybridization resulting from the XFKHl probe extends anteriorly beyond that of engrailed 2, which marks the boundary between midbrain and hindbrain. (f) Whole-mount in situ hybridization of XFKHl to Xenopus exogastrula. Lateral view of a Xenopus exogastrula. Hybridization signal is visible in the notochord. No signal appears in the ectoderm (arrow). The ectodermal part broke off during the examination of the embryo.
	Figure 5. Sections of embryos hybridized with XFKHl. [A] Transverse section through the trunk region of a stage 15 embryo. Hybridization of XFKHl is to the notochord and putative neural floor plate. [B] In situ hybridization of XFKHl to a stage 16 embryo. Section is through the hindbrain region. The upper arrow shows the hybridization in the neural floor plate; the lower arrow shows the hybridization to the notochord. This transverse section is slightly oblique. The dorsal area is more anterior than ventral. (C) Higher magnification of the XFKHl hybridization to the stage 16 embryo. Hybridization is to the notochord (n) and neural floor plate (arrow). Somites( s) and other areas of the embryo are negative.
	Figure 6. Effects of growth factors on the expression of XFKHl. Northern blot analysis of RNA (10 fjig) from induced and uninduced animal caps. [Left] Ethidium bromide staining; [upper right) control hybridization with clone 1 Al 1 that is known to be induced by activin and FGF; {lower right) same filter hybridized by XFKHl (induction by activin only). (Lane 1) FGF; (lane 2) control; (lane 3) activin.
	Figure 7. Northern analysis of XFKHl induction in the presence and absence of protein synthesis. Equal amounts of RNA (10 \|jLg) from induced and uninduced animal caps were loaded per lane. (Lane 1] Control; (lane 2) activin (no cycloheximide); (lane 3] activin and cycloheximide.