Gene 200 (1997) 25–34
Anf: a novel class of vertebrate homeobox genes expressed at the
anterior end of the main embryonic axis1
Olga V. Kazanskaya a, Elena A. Severtzova a, K. Anukampa Barth b, Galina V. Ermakova a,
Sergey A. Lukyanov a, Alex O. Benyumov c, Maria Pannese d, Edoardo Boncinelli d,e,
Stephen W. Wilson b, Andrey G. Zaraisky a,*
a Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaja 16/10,
V-437 Moscow, 117871, Russia
b Development Biology Research Centre, Randall Institute, King’s College, Drury Lane, London, WC2B 5RL, UK
c Biological Faculty, Moscow State University, 117234 Vorobievi gori, Moscow, Russia
d Istituto Scientifico HS Raffaele, Via Olgettina 60, 20132 Milan, Italy
e Centro Infrastrutture Cellulari, CNR, Via Vanvitelli 32, 20129 Milano, Italy
Received 26 January 1997; accepted 16 May 1997
Abstract
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. © 1997 Elsevier Science B.V.
Keywords: Homeobox genes; Embryo; Forebrain patterning
1. 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 homeodo* Corresponding author: Tel: +7 (095) 3363622;
Fax: +7 (095) 3306538; e-mail: zar@humgen.siobc.ras.ru
1 GeneBank accession numbers: U65433, U65436 and U82811.
Abbreviations: aa, amino acid; Anf, class of homeobox-containing
genes; Aanf, Danf, Ganf, Hanf, Panf, Xanf-1, Xanf-2, members of Anf
class of genes in different species ; bp, base pair; en, engrailed; eve,
even-skipped; msh, muscle segment homeobox gene; ftz, fushi tarazu; flh,
floating head; Not, class of genes encoding negative transcription regulators; Otx, class of orthodenticle-related genes; PAX, class of pairedrelated genes; PCR, polymerase chain reaction; Prop.1, Prophet of Pit1 gene; Xotx2, Xenopus homologue of Otx2; zli, zona limitans
intrathalamica.
0378-1119/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved.
PII S 03 7 8 -1 1 1 9 ( 9 7 ) 0 0 3 26 - 0
main [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 Xanf1 in zebrafish (Danio rerio—Danf ), sturgeon (Acipenser
26
O.V. Kazanskaya et al. / Gene 200 (1997) 25–34
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.
2. Materials and methods
2.1. 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 extirpated from embryos in appropriate
physiological solutions using an eye-surgery microknife
and a fused glass capillary.
2.2. Preparation of amplified cDNA samples
Total RNA was purified using homogenization of
tissue explants with guanidine isothiocyanate and
phenol/chloroform extraction (Chomczynski and Sacchi,
1987). First cDNA strand synthesis and preparation of
amplified cDNA, involving oligo dA-tailing of the first
cDNA strand and subsequent PCR with T(13)-stretch
containing primer (5∞CGCCAGTCGACCG( T ) ), were
13
performed exactly as described earlier (Lukyanov et al.,
1995). First cDNA strand and amplified cDNA were
purified from unincorporated dNTPs and the excess of
primers using Wizard PCR Prep DNA Purification
System (Promega).
2.3. Amplification of homeobox fragments from cDNA
by PCR
The following degenerated oligonucleotides were
designed to amplify Anf homeobox fragments. For the
RELSWYR motif: 5∞-AGAGAGARCTNAGYTGGTA; for the NS/CYPGID motif: 5∞-AAYTCA-
TAYCCNGGTATWGAT; for the ESQFLI/M motif:
5∞-TATYAGRAAYTGQGAYTC; for the WFQNRR
motif: 5∞-GNCGRTTYTGRAACCA; where N=A, G,
C or T; Q=T or G; R=A or G; W=A or T; Y=C or T.
PCR with previously amplified cDNA samples (see
above) was performed using a thermostable DNA polymerase mixture of KlenTaq (AB Peptides, USA) and
pfu (Stratagene) at a ratio of 150:1 U/U under the
following conditions: two cycles at 94°C for 1 min, 46°C
for 30 s, 72°C for 2 min, then 25–28 cycles at 94°C for
30 s, 56°C for 30 s, and 72°C for 2 min. In the case of
human, the template for PCR reactions was phage DNA
prepared from human undifferentiated teratocarcinoma
NT2/D1 cDNA library (provided by Telethon Institute
of Genetics and Medicine). In this case, an additional
round of PCR (with one initial and one novel, ‘internal’,
degenerate primer) was performed after a 1000-fold
dilution of the PCR product that had been obtained in
the first round. Purified PCR products were subcloned
into pBluescribe KS vector (Stratagene, USA) and
20–30 colonies in each case were tested by PCR using
‘internal’ degenerate primers (that were not used in
initial PCR) in combination with a primer specific for
the pBluescribe KS vector. All positive clones were then
sequenced using Promega ‘fmol’ sequencing kit. In some
cases, positive clones were identified using hybridization
of the filter replicas with a Xanf-1 derived probe in low
stringency conditions.
2.4. Amplification of 5∞- and 3∞-ends of Anf cDNAs by
RACE
To obtain the remaining regions of Anf cDNAs, a
technique based upon the theory of suppression-PCR
was used (Lukyanov et al., 1995; Chenchik et al., 1996).
In contrast with the previously published method,
inverted terminal repeates were introduced into amplified cDNA samples (see above) not by a ligation procedure, but using 10 cycles of PCR with elongated
T(13)-stretch containing primer ( ETP): 5∞-AGCACTCTCCAGCCTCTCACCGCAGTCGACCG ( T ) .
13
When this PCR was completed, the reaction mixture
was diluted 1000 times (to avoid amplification with ETP
in further rounds of PCR), and two rounds of PCR 25
cycles each (also interrupted by the 1000 times dilution)
were performed with a pair of nested specific primers to
the homeobox. These primers were successively combined in the first round of PCR with a primer representing the outer part of ETP (5∞-AGCACTCTCCAGCCTCTCACCGCA), and in the second round with
( T ) -stretch containing primer (see Section 2.2).
13
The following pairs of nested specific primers were
used to amplify 3∞-ends of Anf cDNAs (for each species,
the first primer is the one used at the first round of
PCR); for sturgeon: 5∞-CCCAGGACGGCTTTCAGTG
and 5∞-GCTCTAGAGTGGAGCCCAGATCGAGGT;
O.V. Kazanskaya et al. / Gene 200 (1997) 25–34
for zebrafish: 5∞-AACAGCCTTCTCCAGTGT and
5∞-TCTAGATCAAGATATTAGAGAGTGTT; for newt:
5∞-AGCTGGTACCGAGGTCGGA and 5∞-TCTAGAGGCCGAGGACAGCCTTCAG; for chick: 5∞-AGGGGTAGAAGACCGAGAACT and 5∞-TCTAGACTGCTTTCACTAGAAACCAG.
Pairs of nested specific primers used to obtain 5∞-ends
of Anfs cDNAs were as follows; for sturgeon: 5∞-CATCCAGCTCCAGTTTGCAC and 5∞-AGAAGCTTAGGGTAGGGGTTCACTCT; for zebrafish: 5∞-ATAGTTCACTTGGAAAACAC and 5∞-GAAAGCTTCTAATATCTTGATCTGAAC; for newt: 5∞-CTCTCGAATGTCAATGCCG and 5∞-AGAAGCTTAATGTCAATGCCGGGATAC; for chick: 5∞-GGGATCTCTTCAGTTTTGC and 5∞-AGAAGCTTCGGTTCTGGAACCAGAT.
The longest PCR bands (presumably representing the
full-length 3∞ and 5∞-cDNA ends) were excized from the
agarose gel and cloned into pBluescribe KS vector
(Stratagene, USA) for further sequencing. In the case
of Hanf, 3∞- and 5∞-cDNA ends were isolated by PCR
from the phage DNA of teratocarcinoma cDNA library
using primers specific to phage in combination with the
following pairs of nested specific primers; for the 3∞ end:
5∞-CAAGAACTGCTTTTACTCAAAA and 5∞-GTGTTAGAAAATGTCTTTAG, for the 5∞ end: 5∞-CCTTTTCAGTTTTGCACG and 5∞-CTGGATTCTRTCTTCCTCTAG.
To minimize the possibility of PCR mistakes, Anf
cDNAs were finally sequenced using templates obtained
by 30 cycles of PCR directly from the first cDNA stands
or, in the case of human, from phage DNA, with the
KlenTaq (AB Peptides, USA)/pfu (Stratagene) mixture
(Barnes, 1994) and the following specific primers to the
most 5∞ and 3∞-terminal regions of cDNAs (these primers
were designed on the base of sequencing information
obtained at previous steps); for sturgeon: 5∞-CAATATTTATTAAGCAATAAC and 5∞-AAATGCCAGGCTCGCAG; for zebrafish: 5∞-TCAGTTGGAGTTAAATTAAAGG and 5∞-ATTATTTATTTATATTTTGGCC; for newt: 5∞-GCTTCCGCCACGCGATC and
5∞-ACCATTAGAAAATGTTTTTATTC; for chick:
5∞-GGGTACCATCCATCAGCA and 5∞-GGAAAAGCTTCACTTTCTCCAC, for human: 5∞-GCTCTGTGCAGACCACGAGA and 5∞-TCTGTGTCTAGTACCCTGGT.
The following conditions of PCR were used in all the
experiments described above; denaturation: 94°C for
30 s; annealing at 56°C for 30 s; extension: 72°C for
2 min.
Final sequences were deposited into GeneBank under
accession numbers: U65433–U65436 and U82811.
2.5. Whole-mount in-situ hybridization
Whole-mount in-situ hybridization on zebrafish was
performed as described in Xu et al. (1994) and for other
27
species as described in (Harland, 1991), with digoxigenin-labelled probes and, in the case of Otx-2, with
fluorescein-labelled probes.
3. Results and discussion
3.1. 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 suppressionPCR 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 intensive expression of Xanf-1 homologues
should be roughly the same in different species. Taking
into account that in every case, the extirpated tissue
piece (which presumably contained the majority of Anf
expressing cells) was at least 20 times smaller than the
whole embryo, one may suppose that cDNA samples
obtained by this technique should be enriched with Anf
templates by about 20 times in comparison with the
total cDNA routinely prepared from the whole embryos.
This strategy had an additional advantage because as
total cDNA samples were obtained by PCR, it was not
necessary to collect a large amount of embryonic polyA
RNA for the first strand synthesis.
In the next step, the homeobox fragments of Anf
genes were obtained from the enriched cDNA samples
by PCR with a set of degenerate primers corresponding
to Anf specific conservative amino acid motifs (see
Materials and Methods). This set allowed us to perform
a PCR survey with four different pairs of primers and,
in addition, easily to test the homeobox specificity of
the PCR products. Interestingly, only one Anf class gene
was identified for each of the species investigated, despite
the fact that, in each case, several positive clones were
analysed by sequencing.
To obtain the remaining fragments of Anf cDNAs,
we used the initial samples of total cDNA and a
technique based on the principle of suppression-PCR
(Siebert et al., 1995; Chenchik et al., 1996). The utility
of this strategy allowed us quickly to isolate overlapping
fragments representing Anf cDNA sequences of the
following lengths; for Anf: 1091 bp; for Danf: 717 bp;
for Panf: 929 bp. The sequences with the longest ORFs
starting from the methionine codon were 174, 161 and
185 aa, respectively. For Ganf, overlapping fragments
representing a sequence of 865 bp with the longest ORF
of 184 aa were isolated. The obtained fragments of Ganf
cDNA, however, do not contain the entire ORF. In the
case of human, overlapping fragments representing a
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O.V. Kazanskaya et al. / Gene 200 (1997) 25–34
Hanf cDNA sequence of 754 bp were isolated by the
same PCR strategy, but using phage DNA of teratocarcinoma cDNA library as the template. The longest ORF
of Hanf starting from methionine was 185 aa.
3.2. Primary protein structure
Anf proteins vary in length from 161 (in zebrafish) to
187 amino acids (in Xenopus). The main feature of these
proteins which permitted all Anf genes to be assigned
to a distinct class is the much higher degree of identity
revealed when their homeodomains are compared with
each other (more than 75%), than with any homeodomain of other known classes ( less than 55%). Moreover,
amino acid sequences of three regions that form secondary structures directly involved in protein–DNA interactions and thus determine functional specificity of the
homeodomain (the N-terminal arm, the region near the
start of a-helices 2 and a-helix 3/4), demonstrate the
highest identity within the Anf class (Fig. 1A) and clear
differences with analogous sequences of other
homeodomains.
The primary structure of helix 3/4 (the so-called
recognition helix) plays the most important role in DNA
sequence recognition (Gehring et al., 1994). Amino acid
residues in the more variable positions of this helix
(positions 1, 2, 5, 6 and 9) are expected to be more
significant for control specificity of DNA binding than
the conservative frame work amino acids (Scott et al.,
1989; Gehring et al., 1994). The residue in the ninth
position is the most critical among all variable amino
acids for the formation of the homeodomain–DNA
complex. Thus, in the bicoid protein, changing a lysine
residue at the ninth position to glutamine or serine
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).
O.V. Kazanskaya et al. / Gene 200 (1997) 25–34
allows the homeodomain to bind to DNA consensus
sequences, which are normally recognized respectively
by the Antennapedia or paired class homeodomains
(Hanes and Brent, 1989). All known homeodomains of
the Anf class have a glutamine residue at position 9 of
the recognition helix (Fig. 1A). Therefore, theoretically,
they should not bind DNA sequences recognized by the
products of such homeobox genes as PAX, Otx and
goosecoid. However, Anf proteins may recognize the
same DNA motifs as Antennapedia, engrailed, msh and
some other homeodomains.
In contrast with members of the other homeobox
families, Anf proteins demonstrate a specific set of
amino acid residues in other variable positions of the
recognition helix. Despite these amino acids being less
critical for DNA binding, they are also suspected to be
important for the affinity of the homeodomain to specific
nucleotide sequences (Desplan et al., 1988; Scott et al.,
1989; Gehring et al., 1994). In the case of Anf proteins,
asparagine residues at the second position seem to be
the most intriguing. As far as we know, this amino acid
is present at this position in only two homeodomains
from more than 300 described so far: in ceh-10 (Hawkins
and McGhee, 1990) and Oct-4 (Scholer et al., 1990).
Moreover, these two homeodomains belong to different
classes ( Kappen et al., 1993), and it seems likely,
therefore, that an asparagine residue at the second
position represents an arbitrary variation only in these
representatives of the two classes. By contrast, asparagine occupies the second position of the recognition
helix in all known Anf homeodomains. Such a high
conservation indicates an important role of asparagine
at this position for Anf homeodomain functioning.
Interestingly, a critical role for the amino acid residue
at the second position in DNA binding has been demonstrated in Drosophila for the fushi tarazu (ftz) homeodomain (Furukubo-Tokunaga et al., 1992).
Amino acid residues 1–7 of the N-terminal arm of
the homeodomain interact with the minor groove of the
DNA and also may influence the homeodomain binding
specificity (Gibson et al., 1990; Lin and McGinnis,
1992). These residues form an entirely conserved
sequence, GRRPRTA, in all known homeodomains of
the Anf class ( Fig. 1a). This sequence is most similar to
the N-terminal arm consensus sequences of the en, evenskipped (eve) and prd homeodomains, differing from en
and eve by the first and the second and from the prd
by the first, second and third amino acid residues.
Amino acids located in the third putative DNAinteracting region (the loop between a-helices 1 and 2
and in the N-terminal part of helix 2) also form a
common sequence, PGID (Fig. 1A), which appears to
be specific exclusively for Anf class homeodomains. As
has been shown in experiments with the ftz homeodomain, a residue at position 28 is the most important for
DNA binding among all other variable residues of this
29
region ( Furukubo-Tokunaga et al., 1992). This is an
isoleucine residue in Anf proteins. This amino acid in
this position is quite rare and, besides the Anf class, is
only present in some homeodomains of the paired class.
All Anf homeodomains are flanked by specific conservative sequences: K/RRE/A,D,TL/QN/SWY from the
aminoterminus and RESQFLM/IV/AK/R from the carboxyterminus. These sequences have no homologues
among other known flanking sequences that have been
previously described for classes of homeodomains.
Along with the described homologous regions, there
are two further conservative motifs: P/HH/YRPW and
FT/SID/EH/SILGL near the N-terminuses of Anf proteins. The last sequence has some similarity with the
octapeptide of paired-type proteins: HSIAGILG (Burri
et al., 1989). Despite this similarity, Anf genes seem not
to be tightly related with paired class genes. Indeed, Anf
homeodomains do not demonstrate any exceptional
homology with paired class homeodomains (not more
than 54%). In addition none of the Anf proteins contains
other important feature of many paired-type proteins—
the paired domain.
Outside the regions mentioned above, Anf proteins
have a low homology with each other. The only exception is the products of two genes identified in Xenopus
laevis, Xanf-1 and Xanf-2, demonstrating an unusually
high identity of about 90% over the whole sequence.
Interestingly, the same degree of identity (90%) is
revealed when the translated regions of the cDNAs of
these two genes are compared. This fact clearly indicates
that the duplication that generated Xanf-1 and Xanf-2
was a quite recent event, which probably happened only
in the evolutionary branch leading to Xenopus. Indeed,
genes that diverged so long ago that now they are
present in different classes of Vertebrates (for example,
homeobox genes otx-1 and otx-2, en-1 and en-2) usually
demonstrate a considerably higher degree of identity at
the protein level than at the nucleotide level. Bearing in
mind the well-known phenomenon of duplication of the
Xenopus genome, one can hypothesize that these two
genes represent a pair of pseudo-allelic genes present
only in this, or maybe also in other species of this
Amphibian genus (Richter et al., 1990). Indeed, analogues pairs of pseudo alleles have been revealed for
some other Xenopus homeobox genes (Fritz et al., 1989).
3.3. Unusual evolutionary non-stability of the Anf
homeodomain primary structure
As the Anf homeodomains from different species
show a considerable divergence from each other and fail
to obviously subdivide into different sub-classes
( Fig. 1B), it is possible that the genes coding for these
homeodomains are all non-orthologous homologues,
and in each species, there may exist as-yet unknown Anf
30
O.V. Kazanskaya et al. / Gene 200 (1997) 25–34
O.V. Kazanskaya et al. / Gene 200 (1997) 25–34
genes, encoding homeoproteins with homeodomains less
diverged from known Anf genes in other species.
Although we cannot completely discount this possibility, it seems unlikely that we isolated five non-orthologous genes (Xanf-1, Danf, Panf, Ganf and Aanf ) from
cDNA samples derived from similar embryonic stages
and tissues. If all five genes are non-orthologous, then
we would expect at least four unknown Anf genes in
each of the species studied, all of them expressed at the
same time and in the same tissues. If we assume that in
each of the species, transcripts of all five genes are
present in equal concentrations in the tissues studied,
then we can make a rough calculation as to the likelihood that we isolated non-orthologous genes in each
species.
The probability of finding only non-orthologous genes
during screening of these cDNA samples is:
P=p · p · p · p · p , where p is the probability of find1 2 3 4 5
1
ing only one type of sequence in the first species;
p —the probability of finding another, but also only
2
one, type of sequence in the second species, etc. If, for
instance, only two independent clones were analysed for
each of the species studied (in reality, we analysed more
than two), then: p =5/52; p =4/52; p =3/52;
1
2
3
p =2/52; p =1/52. Thus, P=5!/510, or approximately
4
5
0.00001. Clearly, it appears to be very unlikely that we
would isolate different non-orthologous Anf genes in
31
each species, and so we can conclude that at least some
of these five genes are likely to be true orthologues.
If only some of the genes that we isolated are
orthologues, then we would expect to see a sharp
difference in the number of mismatches when comparing
homeodomains of the orthologous group with nonorthologous homologous. Instead, we find that all five
homeodomains considered above show approximately
similar degrees of divergence from each other ( Fig. 1B).
Given these considerations, and the similarities in
expression described below, we feel that it is probable
that all five genes are indeed orthologues.
However, we should caution that our analysis cannot
exclude the possibility that several very homologous Anf
genes may present in the same genome (for example,
Xanf-1 and Xanf-2). In such cases, it is probably not
feasible to assign orthology to one of the pair and not
the other. Also, we cannot exclude that there may still
be unknown Anf genes in the species studied, which are
expressed at other times and in other tissues, or that
there are other more divergent Anf genes that would
not have been amplified using our primer sets.
Given the possibility that all known Anf genes are
orthologues, the unusually high degree of divergence
between their homeodomains is noteworthy. Indeed,
homeodomains of all other known homeobox-containing orthologues in Vertebrates listed in Stein et al. (1996)
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 Xanf1 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.
32
O.V. Kazanskaya et al. / Gene 200 (1997) 25–34
appear to diverge less than 5% (with the only exception
of NOT2 class, in which divergence achieves 10%), even
if such distant organisms as fishes and mammals are
compared (see, as an example, the table of the numbers
of mismatches between different Otx2 homeodomains
in Fig. 1B). Conversely, in the case of Anf proteins, the
divergence is already 5% between mammalian species
(mouse and man) and more than 20% if mammals and
fishes are compared.
Interestingly, all the changes in Anf homeodomains
appear to be restricted exclusively to regions that presumably do not contact DNA. In this respect, one can
speculate that in different species, Anf may bind the
same DNA motifs, but if the Anf class of homeoproteins
has less protein–protein interactions than other classes
of homeodomains, they could escape the strong stabilizing pressure of natural selection in regions not involved
in DNA binding. Alternatively, the amino acid substitutions in more divergent regions may not be of a neutral
character, but could be stipulated by some type of
co-evolution of Anf proteins and their co-factors.
3.4. Embryonic expression and possible functions
A specific feature of all known Anf genes is their
expression within the most anterior region of the embryonic body axis during gastrulation and neurulation. The
expression has a transient character, starting early
during gastrulation, reaching maximal intensity around
gastrulation–neurulation transition and then gradually
decreasing (Fig. 2). In mice and frogs, early Anf expression has previously been shown to occur both in ectoderm and in mesendodermal cells, primarily of the
prechordal plate [Fig. 2E; see also Mathers et al. (1995),
Zaraisky et al. (1995), Hermesz et al. (1996) and
Thomas and Beddington (1996)]. It is also possible that
comparable mesendodermal tissues weakly express Anf
in chicks ( Fig. 2M ) and in fish, although the strong
ectodermal expression and very thin hypoblast layer in
fish makes this difficult to ascertain.
In ectoderm, Anfs are initially (from the midgastrula
stage) expressed within a broad territory of the anterior
neural plate (Fig. 2B, F, N ). This territory appears to
be localized entirely within the expression domain of
another early expressing head marker, the homeobox
gene Xotx2 (Fig. 2G). The latter gene is expressed at
this stage in a wide area, which includes the neural plate
anterior to the presumptive midbrain–hindbrain boundary, along with a fragment of non-neural ectoderm,
surrounding the anterior margin of the neural plate.
As neurulation proceeds, the posterior margin of the
initial trapezium-shaped territory of the Anf expression
domain becomes increasingly curved, possibly because
of morphogenetic movements associated with floorplate
extension in the midline of the neural plate (Fig. 2C, G,
N, O). At the same time, at least in zebrafish and
Xenopus, intense expression of Anf becomes restricted
to two domains just 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, whereas the posterior,
weaker domain appears to surround the anterior tip of
the prospective floorplate (Fig. 2C, G).
According to neural plate fate maps in Xenopus and
chick, the anterior domain of Anf expression coincides
in its medial part with the location of pituitary anlage
and in its lateral parts with the presumptive dorsal
telencephalon and anterior part of dorsal diencephalon
(Couly and LeDouarin, 1987; Eagleson and Harris,
1990). This is supported by a later analysis of Danf
expression, which remains present in dorsal regions of
the anterior forebrain (Fig. 2D).
The posterior, weaker domain of Anf expression in
zebrafish and Xenopus may coincide, in its medial part,
with the presumptive hypothalamic region and, in its
lateral parts, with the position of presumptive zona
limitans intrathalamica (zli), a boundary that divides
ventral and dorsal thalamus. This is suggested by the
location of this domain just rostral to the anterior end
of the prospective floor plate, and second, by the result
of double-labelling in-situ hybridization experiments
with probes to Xanf-1 and Xotx2. In these experiments,
we find that the posterior boundary of Xanf-1 expression
coincides with the anterior boundary of the diencephalic–mesencephalic domain of the Xotx2 expression
( Fig. 2K, L), which is known to coincide with the zli
(Boncinelli et al., 1993; Simeone et al., 1993; Mori et al.,
1994). A similar location for the posterior boundary of
Danf expression has been shown in zebrafish by comparison of Anf expression with that of flh, which is known
to be expressed just dorsal to the zli (Barth et al., in
preparation).
Thus, early Anf expression divides the neural plate
into two prospective forebrain territories corresponding
to tissue rostral to the zli, which expresses both Otx and
Anf genes and more posterior tissue that only expresses
Otx genes. This subdivision is consistent with recent
models of forebrain organization that place a fundamental subdivision of the forebrain at the zli ( Figdor and
Stern, 1993; Puelles and Rubenstain, 1993; Macdonald
et al., 1994). Anf genes are the earliest known genes to
respect this subdivision, raising the possibility that they
may be involved in its establishment.
During further development, Anf expression is progressively down-regulated in its posterior domain and,
by the end of neurulation, appears to be restricted
exclusively to the medial part of the anterior domain.
At the latest stage at which the expression can be
detected by in-situ hybridization, transcripts are localized to the dorsal telencephalon in zebrafish ( Fig. 2D),
and in all species examined to an ectodermal derivative
initially connected with the presumptive telencephalon,
O.V. Kazanskaya et al. / Gene 200 (1997) 25–34
the anterior pituitary anlage ( Fig. 2I, P; see also Hermesz
et al., 1996; Mathers et al., 1995; Zaraisky et al., 1995;
Barth et al., in preparation).
Interestingly, the medial, more prominent part of the
posterior Anf expression domain ( Fig. 2C and G) coincides with the floor of the rostral diencephalon, a region
that includes cells of the prospective neurohypophisis.
Therefore, it appears that both local high points of Anf
expression, in the anterior neural ridge and near the
rostral end of the floor plate, mark cells whose progenitors will contribute to the same definitive organ, the
pituitary.
As Anf genes are restricted in their expression to the
most anterior part of the embryonic body axis and
transcripts appear to be present in tissues derived from
all three embryonic layers (in the meso-endoderm and
in the neurectoderm), one may hypothesize that Anf
genes in the forebrain region like Hox genes in the trunk
region could be involved in specification of embryonic
subdivisions [reviewed by Scott et al. (1989) and
McGinnis and Krumlauf (1992)]. There is some experimental evidence supporting this hypothesis. When Xanf1 is ectopically expressed in cells of the ventral marginal
zone of Xenopus early gastrulae, it is able to induce in
these cells some properties normally characteristic of
the dorsal anterior meso-endoderm, i.e. attraction to
anterior locations and an ability to organize the formation of secondary embryonic axis at the ventral side of
embryo (Zaraisky et al., 1995). In addition, overexpression of Xanf-1 in the neurectoderm can, in some cases,
lead to the transformation of more posterior brain
tissues to telencephalic structures (Zaraisky et al., in
preparation).
Another possible role of Anf genes may be connected
with the regulation of pituitary differentiation. Thus, it
has been shown recently, that in mouse, Rpx/Hesx-1
could be directly involved in the repression of early
pituitary differentiation, inhibiting until stages 12.5–13.5
the expression of a pituitary-specific homeobox gene
Prop-1 (Sornson et al., 1996).
4. Conclusions
(1) We cloned cDNA of five novel genes homologous
to the homeobox-containing genes Xanf-1 and Xanf2 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 divides the neural plate into
33
two prospective forebrain territories corresponding
to tissue rostral to the zli, which expresses both Otx
and Anf genes and more posterior tissue that only
expresses Otx genes. This subdivision is consistent
with recent models of forebrain organization that
place a fundamental subdivision of the forebrain at
the zli. Thus, Anf genes are the earliest known genes
to respect this subdivision, raising the possibility
that they may be involved in its establishment.
Acknowledgement
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 (95-INRU-1152) and Wellcome Trust funding to S.W. O.V.K.
was supported by an EMBO East European fellowship.
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