Anf: a novel class of vertebrate homeobox genes expressed at the anterior end of the main embryonic axis1
Introduction
Homeobox-containing genes play crucial roles in regional patterning and cell differentiation during development of multicellular organisms. Protein products of these genes act as specific transcription factors, binding with regulatory elements of target genes by means of a common conserved 60-aa motif, called the homeodomain [reviewed by Scott et al. (1989)and McGinnis and Krumlauf (1992)]. It is the primary structure of this motif that primarily determines DNA binding specificity of homeodomain-containing proteins both in vitro and in vivo [reviewed by Gehring et al. (1994)]. The homeobox gene family can be subdivided into about 30 classes of genes, each characterized by its own specific consensus sequence of the homeodomain (Kappen et al., 1993).
In the present study, we characterize a novel class of homeobox genes encoding a homeodomain consensus sharply different from those of other classes. The first member of this class, Xanf-1, was cloned in Xenopus laevis (Zaraisky et al., 1992). Xanf-1 homologues have since been identified in mouse, Hesx-1 (Thomas et al., 1995) [this gene was also described as Rpx (Hermesz et al., 1996)], and in Xenopus, Xanf-2 (Mathers et al., 1995). Xanf-2 appears to be very similar to Xanf-1 and probably is a pseudo-allelic copy of it. Besides these genes, we now describe five novel homologues of Xanf-1 in zebrafish (Danio rerio—Danf), sturgeon (Acipenser baeri—Aanf), newt (Pleurodeles waltlii—Panf), chick (Gallus gallus—Ganf) and human (Homo sapiens—Hanf).
In all species investigated, these genes are expressed at the most anterior extremity of embryo in a very restricted time interval during gastrulation and neurulation; moreover, in the anterior neurectoderm (in Xenopus laevis, this region corresponds to the anterior neural fold), they are expressed most intensively. Based upon the name of the first member of this class of genes (Xanf-1) and upon their expression patterns, we propose to name the family Anf genes. All data currently obtained indicate that Anfs could be involved in the early patterning of the most anterior region of the main embryonic body axis.
Section snippets
Embryo manipulations
Sturgeon (Acipenser baeri) and zebrafish (Danio rerio) embryos were obtained by natural spawning or by artificial fertilization, and staged according to Detlaff and Ginsburg (1954)and Kimmel et al. (1995), respectively. Newt (Pleurodelis waltlii) embryos were collected by natural spawning and staged according to Gallien and Durocher (1957). Fertile white leghorn chicken (Gallus gallus) eggs were incubated at 38°C and staged according to Hamburger and Hamilton (1951). Tissue pieces were
Isolation of Anf nucleotide sequences
To isolate cDNAs of Xanf-1 homologues in sturgeon, zebrafish, newt and chick, we used a PCR-based strategy. To enrich initial samples of total cDNA with the required templates, they were obtained by a suppression-PCR technique, directly from individual pieces of anterior neurectoderm extirpated from embryos just after the end of gastrulation (Lukyanov et al., 1995; see also Materials and Methods). Anterior tissue at this stage was selected with the assumption that the place and the time of
Conclusions
- 1.
We cloned cDNA of five novel genes homologous to the homeobox-containing genes Xanf-1 and Xanf-2 of Xenopus and Hesx1/Rpx of mouse in sturgeon (Aanf), zebrafish (Danf), newt (Panf), chicken (Ganf) and human (Hanf). 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.
- 2.
Our data suggest that Anf class may be one of the most quickly evolving classes of vertebrate homeobox genes.
- 3.
Early Anf expression
Acknowledgements
We would like to thank Oleg Vasiliev for technical assistance and fruitful discussions. We are also grateful to Anna Stornaiouolo, Antonello Mallamaci and Giovanni Lavorgna and Dominic Delaney for providing expert advice. We thank the Telethon Institute of Genetics and Medicine (TIGEM) for supplying the NT2/D1 library. This work was supported by grants from the Russian Human Genome Project, the Russian Foundation for Fundamental Investigations (95-04-11320a and 97-04-49883), and INTAS
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GeneBank accession numbers: U65433, U65436 and U82811.