October 24, 1997;
Anf: a novel class of vertebrate homeobox genes expressed at the anterior end of the main embryonic axis.
Five novel genes homologous to the homeobox-containing genes Xanf
-1 and Xanf
-2 of Xenopus and Hesx-1/Rpx of mouse have been identified as a result of a PCR survey of cDNA in sturgeon, zebrafish, newt, chicken and human. Comparative analysis of the homeodomain primary structure of these genes revealed that they belong to a novel class of homeobox genes, which we name Anf
. All genes of this class investigated so far have similar patterns of expression during early embryogenesis, characterized by maximal transcript levels being present at the anterior
extremity of the main embryonic body axis. The data obtained also suggest that, despite considerable high structural divergence between their homeodomains, all known Anf
genes may be orthologues, and thus represent one of the most quickly evolving classes of vertebrate homeobox genes.
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Fig. 1. (A) Comparison of amino acid sequences of known Anf homeodomains. All sequences are compared to the amino acid sequence of the
Xanf-1 homeodomain (top). Dashes indicate sequence identities to Xanf-1. The residues that are absolutely conserved within each class are shown
below as the consensus sequence (section 3.2.). (B) Table representing numbers of amino acid mismatches between different Anf homeodomains is
shown in comparison with a similar table for Otx2 homeodomains. Note much higher mutual divergence of Anf homeodomains (section 3.3).
Fig. 2. Whole-mount in-situ hybridization with digoxigenin-labelled probes to Danf (in zebrafish), Xanf-1 and Xotx2 (in Xenopus) and Ganf (in
chick) (section 3.4). (A–D). Danf expression at successive stages of zebrafish development. (A) At 70% of epiboly, Danf is expressed dorsal to the
embryonic shield. The embryo is shown from the left side, animal pole up. (B) At the 80% epiboly, the expression domain in the prospective
neuroectoderm has a trapezium-like shape. The embryo is shown from the animal pole, dorsal side up. (C) At the end of epiboly, the expression
of Danf is restricted to the crescent-shape domain at the anterior margin of the neural plate (black arrow) to a more weak, ‘M’-shaped, posterior
domain (white arrow). (D) By the eight-somites stage, the expression of Danf is restricted to dorsal telencephalon.
(E–L) The expression of Xanf-
1 and Xotx2 in Xenopus. (E) At the early midgastrula stage (stage 11), transcripts of Xanf-1 are still faintly present in cells of the leading edge of
the gastrulating mesoendoderm (cells of the presumptive prechordal plate and foregut endoderm)—black arrow. At the same time, a more
pronounced expression (white arrow) is seen in adjacent cells of the deep layer of the anterior neurectoderm. (F) At the beginning of neurulation
(stage 13), the Xanf-1 expression domain in the anterior neurectoderm has a trapezium-like shape. The anterior limit of the presumptive neural
plate is marked by triangles. (G) At the mid-neurula (stage 15), intensive expression of Xanf-1 is restricted to two domains at the anterior and
posterior borders of the initial expression territory. The anterior, more pronounced expression domain coincides with the anterior margin of the
neural plate and has a stripe of higher intensity bordering the medial anterior ridge from the anterior side (white triangle). The posterior, weaker
domain appears to surround the anterior tip of the prospective floorplate (black arrow), and comes into contact with the anterior domain by
lateral strips of weaker expression (black triangles). (H) During the second half of neurulation, in parallel to neural tube closure, the expression
of Xanf-1 is progressively down-regulated in its posterior domain (triangles). (I ) By the end of neurulation (stage 21), Xanf-1 expression appears
to be restricted exclusively to the medial part of the anterior domain corresponding to the anterior pituitary anlage (triangle). (J) At the early
neurula stage (stage 13), the homeobox-containing gene Xotx2 is expressed in a wide area, which entirely includes the Xanf-1 expression domain.
The posterior limit of this territory corresponds to the presumptive midbrain–hindbrain boundary (triangles), and the anterior limit (arrowheads)
is in the non-neural ectoderm, surrounding the anterior margin of the neural plate. (K) At the midneurula (stage 15), a transverse domain of
intensive expression is segregated in the posterior part of the Xotx2 expression territory. The anterior limit of this domain (triangles) corresponds
to the prospective zona limitans intrathalamica (zli). (L) Double-labelling in-situ hybrydization with Xanf-1 (blue) and Xotx2 ( light brown) probes
demonstrates that at stage 15, the posterior domain of Xanf-1 expression appears to be located just anterior to the domain of intensive Xotx2
expression [compare with (G) and (K)]. Triangles mark the border of the whole Xotx2 expression territory.
(M–P) Expression of Ganf in chick.
In all pictures, except (P), embryos are shown from the dorsal side, anterior end to the top. (M) In chick, Ganf expression can be first detected
in the anterior neurectoderm at HH5 stage. Note that at this stage, the expression is present in the prechordal plate, which is seen as a spot through
the neurectoderm (arrow). Triangle indicates Hensen’s node. (N) At the HH5–6 stage, Ganf is intensively expressed in a broad territory of neural
ectoderm just anterior to the rostral tip of the floor plate. (O) At the head fold stage (stage HH8-, three somites) the expression of Ganf is localized
in cells of the anterior neural fold with a local maximum of intensity in its medial part. Two symmetrical weaker local spots of expression are seen
in the lateral folds (triangles). (P) At the eight-somite stage, very weak Ganf expression is seen as two symmetrical strips in dorsal telencephalon
(arrow). The embryo is shown from the ventral side. Scale bar is 100 mm for A–L and 500 mm for M–P.