The Hox-4.8 gene is localized at the 5′ extremity of the Hox-4 complex and is expressed in the most posterior parts of the body during development

Mechanisms of Development, 36 (1991) 3-13

3

© 1991 Elsevier ScientificPublishers Ireland, Ltd. 0925-4773/91/$03.50 MOD 00050

The Hox-4.8 gene is localized at the 5' extremity of the Hox-4 complex and is expressed in the most posterior parts of the body during development Pascal Doll6

1, Juan-Carlos

Izpisfia-Belmonte and Denis Duboule 1

1, Edoardo

Boncinelli 2

1European Molecular Biology Laboratory, Postfach 10.2209, D-6900 Heidelberg, Germany and 2 International Institute of Genetics and Biophysics, CNR, V. Marconi 10, 80125 Naples, Italy

(Received 3 December 1990; accepted 15 May 1991)

We report the isolation and expression pattern of a novel mouse homeobox gene, Hox-4.8. Hox-4.8 is the most 5'-located homeobox gene in the HOX-4 complex. Sequence analysis confirmed that Hox-4.8 is a member of the subfamily of AbdominalB-related Hox-4 genes and revealed strong interspecies conservation. As for the human locus, Hox-4.8 is probably the last Hox gene in this part of the HOX-4 complex. During development, Hox-4.8 transcripts are restricted to the extremities of the embryonic anteroposterior axis and limbs as well as in the developing tail bud and to the most posterior segment of the gut (the rectum). Within the limb mesenchyme, Hox-4.8 is expressed in more posterodistal regions than those of its neighbour Hox°4.7. Hence, Hox-4.8 expression appears to be related to the last significant phenotypic changes towards the extremities of the embryonic body and limb axes. Homeobox; HOX-4 complex; Mouse embryology; Morphogenesis; Pattern formation

Introduction The murine genes harbouring a homeobox related to that of the Drosophila homeotics, the Hox genes, form a multi-gene family. The 38 Hox genes characterized to date are clustered in four complexes (the HOX complexes) probably generated during evolution by large scale duplications of an ancestral homeogenecontaining cluster. Thus, a given Hox gene can have evolutionary linked counterparts or paralogs in one or several other HOX complexes. Consequently, such paralogous Hox genes are always found in the same order within their complexes (e.g. Kappen et al., 1989; Duboule and Doll6, 1989; Graham et al., 1989). This peculiar organization reflects some functional significance, since Hox genes are transcribed in a coordinate manner during mouse development. They are generally expressed in a set of identical tissues, but the extent of their expression domains along the anterior-posterior

Correspondence to: D. Duboule, EMBL,6900 Heidelberg, Germany.

(AP) axis of the embryo is related to the position of the genes along the chromosomes: genes located at more 5' positions within a HOX complex will have more posteriorly restricted expression domains (Gaunt et al., 1988; reviewed in Akam, 1989). 3'-Located genes are widely expressed in embryonic tissues up to quite sharp anterior expression boundaries in the developing hindbrain and cervical prevertebrae (pv) (e.g. Gaunt et al., 1989; Wilkinson et al., 1989), whereas 5'-positioned genes display expression bpundaries in posterior thoracic or lumbar spinal cord and prevertebrae (reviewed in Kessel and Gruss, 1990). Hence, in each Hox complex there is a colinearity between the arrangement of the genes along the chromosome and the spatial extent of their expression domains. This concept, originally proposed for the homeotic genes of the Drosophila Bithorax complex (Lewis, 1978) appears therefore to be highly applicable throughout evolution. In addition, several homeogene members of the HOX-4 complex are expressed in a coordinated manner during limb development (Doll6 et al., 1989). In that case, 5'-located genes display a more distally and posteriorly restricted expression pattern in the limb mesenchyme. Alto-

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gether, these features have led to the proposal that the Hox gene network could be involved in positional signalling in the embryo, via multiple combinations of homeogene products along the AP body axis or the limb axis (e.g. Holland and Hogan, 1988; Gaunt et al., 1988; Kessel and Gruss, 1990). The most 3' genes isolated so far, Hox-l.6 and Hox-2.9, are expressed up to posterior regions of the head, in the hindbrain, a region of the CNS that is transiently segmented, and in some pharyngeal arches (reviewed in Lumsden, 1990; Wilkinson and Krumlauf, 1990). This suggests that positional information within the anterior part of the head and brain may be achieved by a distinct system. In contrast, the characterization of the 5' regions of the Hox loci suggests that the Hox genes could be functional up to extreme posterior regions, and it is therefore of interest to see whether the expression patterns of extreme 5' genes in a HOX complex correlate with the posterior end of the developing vertebrate embryos. As a model system, we have studied the 5' part of the HOX-4 complex and could identify four adjacent genes, Hox-4.4, -4.5, -4.6 and -4.7, which appear to be all related to the Drosophila Abdominal B (AbdB) gene (Izpisfia-Belmonte et al., 1991a). These four genes are expressed only in limbs and in posterior areas of the embryonic trunk, where their expression boundaries span the prevertebral column from pv 21 to pv 29. Here we report the isolation of Hox-4.8, a novel homeogene located 5' to Hox-4.7. Since no other homeobox sequence could be detected between Hox-4.8 and another transcription unit lying upstream in the genome, the Evx-2 gene (Bastian and Gruss, 1990; Bastian et al., in preparation), we believe that Hox-4.8 may be the last Hox gene in this part of the HOX-4 complex. From day 9.5 postcoitum (p.c.) and later, Hox-4.8 transcripts could be detected only in very posterior regions of the embryo giving rise to the tail, and in the

most posterior segment of the gut and urogenital tract. Hox-4.8 transcripts are also specific to the developing limbs, where they are the most distally and posteriorly restricted among Hox-4 transcripts. Hox-4.8 is the first of these genes not to be expressed in kidneys.

Results

Cloning of the Hox-4.8 homeobox A genomic cosmid clone containing sequences 5' to the Hox-4.7 homeogene (cosB, Fig. 1A) was analyzed by Southern blotting under reduced stringency conditions with a probe containing the human HOX41 homeobox. This homeobox is located in the human HOX-4 locus at about 6 kb upstream of the human Hox-4.7 homologue (D'Esposito et al., 1991). A crosshybridizing sequence was identified and mapped 5 kb 5' to the Hox-4.7 homeobox. DNA sequencing of a 1.5 kb HindlII-HindlII subclone (Fig. 1B) confirmed the presence of a homeobox. Fig. 1A shows a map of 80 kb of the mouse HOX-4 complex. The nucleotide sequence and predicted amino-acid (aa) sequence of the Hox-4.8 homeodomain reveals that it is more similar to the AbdB homeodomain (31 out of 60 identical aa) than to any other Drosophila homeodomains (e.g. 26/60 with Antp). Thus, Hox-4.8 belongs to the group of AbdB-like genes of the HOX-4 complex (Izpisfa-Belmonte et al., 1991a). When compared to the other AbdB-like Hox-4 sequences, the Hox-4.8 homeodomain is more similar to those of its neighbours Hox-4.7 and -4.6 (36/60 and 35/60, respectively) than those of the Hox-4.5 and -4.4 genes (32/60 for both). Furthermore, several residues are identical or conserved between Hox-4.8 and Hox-4.6 but different in other homeodomains (Fig. 2C). Such common aa changes are found to a lesser extent between Hox-4.8 and Hox-4.7. Hox-4.8 and Hox-4.6 also lack a trypto-

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Fig. 2. (A) Nucleotide sequence of the mouse Hox-4.8 homeobox and flanking regions, and alignment to the sequences of the h u m a n and chicken cognate genes (the identical nucleotides are dashed). T h e open rectangle shows the homeobox; the grey rectangle shows an in-frame stop codon present in all sequences. (B) Conceptual amino-acid sequence of Hox-4.8 in m o u s e (upper line), h u m a n and chicken (lower lines, the identical aminoacids are dashed). The rectangle shows the homeodomain. (C) T h e sequence of the Hox-4.8 h o m e o d o m a i n is aligned to those of its neighbouring genes (Hox-4.7 to -4.4) and to the AbdB sequence. Only the aa differing from the AbdB reference sequence are indicated. T h e aa positions are n u m b e r e d according to Scott et al., 1989. T h e position of the alpha-helices is shown (hi, h2, h3). O n the right are indicated the percentages of identity between Hox-4.8 and the other h o m e o d o m a i n s (first column), and the percentages of similarity (when conservative aa changes are taken into account - second column).

phan residue at position - 6 to - 7 from the homeodomain, this residue being present in the other AbdBlike metazoan Hox genes (Izpisfia-Belmonte et al., 1991a). Sequence comparison with the human and chicken Hox-4.8 homologues reveals a complete interspecies conservation since the mouse Hox-4.8 homeodomain is 100% identical to the human and chicken

homologues (Fig. 2A, B). This sequence identity extends further N-terminally to the homeodomain along 14 residues (Fig. 2B) but is interrupted in more upstream regions. At the point of divergence, the corresponding nucleotide sequence is in good agreement with the splice acceptor consensus sequence (Shapiro and Senapathy, 1987). At their C-termini, the mouse,

Fig. 3. Hox-4.8 expression pattern during early stages of mouse development. (A) Selected histological section of a 8.5 day old embryo hybridized to a Hox-4.8 riboprobe, photographed under bright-field (left panel) and dark-field (middle panel) illumination to show the hybridization signal grains. No significant labelling was detected with the Hox-4.8 probe. However, specificlabeling is detected in the posterior regions of the embryo, on a neighbouring section hybridized with a control Hox-4.4 riboprobe (right panel) A, anterior, P, posterior. (B) Selected section of a 9.5 day old embryo. Hox-4.8 transcripts are restricted to posterior areas of the embryo, adjacent to the allantois, hd, head; ht, heart; al, allantois, br, branchial arch; nt, neural tube; m, posterior mesoderm.

human and chicken proteins are almost identical and terminate at the same positions. The regular spacing of Hox-4 homeogenes in the 5' part of the complex is shown in Fig. 1A. Six genes (Hox-4.3, -4.4, -4.5, -4.6, -4.7 and -4.8) are clustered within 60 kb of DNA, and the distance between two homeoboxes is 5 - 1 0 kb. Several lines of evidence suggest that there is no additional homeogene at a comparable distance 5' to Hox-4.8. No homeobox-cross-hybridizing sequence can be detected by Southern blot analysis among 12 kb of mouse genomic D N A upstream of Hox-4.8 probed with different Hox probes (Hox-4.8, -4.7, -4.4; not shown). Accordingly, no homeobox was detected in 12 kb of 5' flanking sequences from the human Hox-4.8 homologue. In addition, the Evx-2 gene, a murine homologue of the Drosophila

even-skipped (eve) gene (Bastian and Gruss, 1990) lies 13 kb upstream of the Hox-4.8 gene in both human and mice (D'Esposito et al., 1991; Bastian et al., in preparation). Evx-2 however is transcribed from the opposite D N A strand.

Hox-4.8 expression during development We analyzed the Hox-4.8 developmental expression by in situ hybridization on sections of mouse embryos at various developmental stages, from day 8.5 p.c. to day 15.5 p.c. The Hox-4.8 gene is transcribed in essentially three broad domains: along the embryonic AP axis, in the developing limbs and during the development of the genital tubercle. The latter will be described independently in relation to the expression

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Fig. 4. Posterior specificity of Hox-4.8 transcripts. (A) Section through the tail bud of a 11.5 day old fetus, showing Hox-4.8 boundary of expression m the tail somites (s, white arrows). In this posterior level, the neural tube (nt) is also labelled. (B) Sagittal section of a 12.5 day old fetus. Hox-4.8 transcripts are restricted anteroposteriorly to the tail (tl), and are also strongly expressed in the genital tubercle (gt). The apparent labelling in the liver (li) is due to a combination of slightly higher background grains and optical refringence caused by red blood cells, hd, head. (C) Schematic diagram of the restricted transcript domains of Hox-4 genes along the AP axis, in the CNS (left) and prevertebral column (right). The anterior expression boundaries of various genes are indicated, and the transcript domains of AbdB-related genes (Hox-4.4 to -4.8) are indicated by increasing intensities of grey. The colinearity between the expression domains and the order of the genes is illustrated as well as the extreme posterior specificity of the Hox-4.8 transcripts. HB, hindbrain; SC, spinal cord; PV, prevertebrae.

patterns of other Hox-4 genes in developing genitalia (in preparation). Hox-4.8 is expressed in the most posterior areas

A time course of the Hox-4.8 gene expression was performed by in situ hybridization. No specific signal could be detected in day 8.5 p.c. embryos although serial sections encompassing very posterior areas were analyzed (Fig. 3A). Hox-4.8 transcripts were detected earliest in 9.5 day old embryos and were restricted along the AP embryo axis to very posterior areas (Fig. 3B). In these domains, the transcripts are widely dis-

tributed in the undifferentiated presomitic and lateral mesoderm, the open neural plate and the hindgut. Hox-4.8 transcripts were detected up to very posterior embryonic cells contacting the allantois (Fig. 3B). Hox-4.8 transcripts remain restricted to posterior levels during subsequent differentiation of the corresponding germ layers. By day 11.5 p.c., Hox-4.8 is only expressed in the posterior neural tube and the tail somites. Fig. 4A shows the boundary of expression along the very posterior somites which are still epithelialized in these areas at this developmental stage. By day 12.5 p.c., weak hybridization signals are still detected in the tail (Fig. 4B). Labelling was comparatively

far more intense in the genital tubercle (Fig. 4B) and in the limbs (compare, for example, with Fig. 7D, which shows the limbs of the same embryo). Due to its weakness, Hox-4.8 expression boundary along the pv was difficult to ascertain, but was mapped within caudal pv, between pv 32 and 33. Labeling equally extends until the posterior extremity of the tail where somites have not yet differentiated. In subsequent developmental stages, no clear signal could be detected in the tail region (not shown). Thus, Hox-4.8 displays the most posterior domain of expression among all HOX homeogenes described so far (Fig. 4C). Altogether, the murine AdbB-related Hox-4 genes (Hox-4.4, -4.5, -4.6, -4.7 and -4.8) are expressed at distinct AP levels in regions posterior to the low thoracic spinal cord and first lumbar prevertebra. The Hox-4.8 gene is also expressed in the fetal gut at all stages studied. The transcript distribution is also restricted posteriorly along the digestive tract since transcripts are detected only in the rectum (Fig. 5A). Interestingly, Hox-4.8 transcripts are localized in both the rectum mesenchyme and epithelium. In later developmental stages (days 15.5 to 17.5 p.c.), labeling becomes restricted to certain areas of the rectum: preferential labeling is observed within the epithelium that is invaginating to form the anlagen of the crypts (glands) of Lieberkiihn (Fig. 5B). In the mesenchymal wall, transcripts are found in a concentric layer which may correspond to the muscularis primordium (Fig. 5B). The neighbour gene Hox-4.7 is expressed in the same regions but exclusively in the mesenchymal wall, as is generally the case for other Hox genes (Fig. 5A). Hox-4.8 transcripts display a very sharp expression boundary which corresponds to the limit between rectum and anus (Fig. 5A). This separation is identified morphologically by a change in the type of epithelium, from a glandular-type of epithelium in the rectum (labeled by the Hox-4.8 probe) to the malpighian anal epithelium (non-labeled, Fig. 5C). Similarly, Hox-4.8 expression is also posteriorly restricted along the mesenchyme of the urogenital apparatus. It is expressed in the urinary bladder wall, and in the posterior part of the uterus mesenchyme, where transcripts show a well-defined boundary of expression (Fig. 5A). In males, Hox-4.8 is also expressed in the mesenchyme of the urinary bladder and in a limited part of the epididymis (not shown). Hox-4.8 transcripts are not detected at any stage either in the male or female genital ridges or gonads, or in the metanephros or definitive kidney anlagen (Fig. 6). Thus, the gene members of the HOX-4 complex are differentially expressed in the genito-urinary apparatus. Only 3'-located genes (Hox-4.2, -4.3 and -4.4) are expressed in somatic cells of the genital ridges and gonads (see also IzpisfiaBelmonte et al., 1990) whereas all but the most 5' gene Hox-4.8, are expressed in the metanephros (Fig. 6).

Hox-4.8 expression in developing limbs

Within the forelimb buds, Hox-4.8 transcripts are detected earliest in a 9.5 day old embryo where a very weak signal is detected in some sections of the posterior areas (Fig. 7A). By days 10.5 and 11.5 p.c., a strong hybridization signal appears in the mesenchyme of both forelimbs and hindlimbs (Fig. 7B,C). Hox-4.8 expression in the limbs follows the rules reported for other Hox-4 genes (Doll6 et al., 1989). The Hox-4.8 transcripts are indeed spatially restricted to a more distal and posterior area than that containing the Hox4.7 transcripts, in both fore- and hindlimb buds (Fig. 7B,C). Interestingly, the Hox-4.8 domain does not extend up to the most proximal cells in the post-axial side of the limb, in contrast to other Hox-4 genes (e.g. Fig. 7B and data not shown). By day 12.5 p.c., Hox-4.8 transcripts are still extremely distally restricted to mesenchymal cells of the footplates (Fig. 7D) and remain quite strongly expressed in the same areas during all subsequent developmental stages. As described for other Hox-4 transcripts, the expression becomes progressively localized to cells surrounding the phalangeal bone anlagen in the digits (Fig. 7E). In late stages (days 14.5 to 17.5 p.c.), Hox-4.8 expression is maximal when compared to that of other Hox-4 genes (not shown).

Discussion

As a paradigm to understand the functional organization of the murine HOX complexes, we are studying the 5' region of the mouse HOX-4 complex. In this genomic locus, five Hox genes have been characterized (Hox-4.4 to Hox-4.8) which are related to the Drosophila AbdB homeotic gene (Duboule and Doll6, 1989; IzpisOa-Belmonte et al., 1991a and this work). These AbdB-like genes are probably involved in the specification of posterior areas along the AP axis of the developing mouse embryo, from the thoraco-lumbar junction posteriorwards. The same genes are used to give positional landmarks within the developing limbs (Doll6 et al., 1989, Izpisfia-Belmonte et al., 1991b). Since the expression domains of 5'-located genes are always more posterior (in the trunk) or more posterodistal (in the limbs) than those of the 3'-located neighbour genes it was of interest to define the extreme 5' member and to look at its expression along the corresponding axes (i.e. within the tail and the distal extremities of the limbs). The Hox-4. 8 homeobox is related to that of the Drosophila AbdB homeotic gene

The genomic location and sequence of the Hox-4.8 homeobox suggest that it belongs to the group of

-4.8

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Fig. 5. Expression of Hox-4.8 in the posterior gut and genito-urinary organs. (A) Sagittal section through the rectum (re), uterus (ut) and urinary bladder (bl) of a 16.5 day old female fetus, hybridized to Hox-4.8 and Hox-4.7 probes. The white arrow points to the sharp boundary of cells expressing Hox-4.8, which corresponds to the rectum-anus boundary. Note the anterior boundary of Hox-4.8 transcripts in the uterus mesenchyme, while Hox-4.7 transcripts extend more anteriorly in the uterus. A longer part of the gut is seen in the -4.8 panel (towards the upper part of the figure) which explains why the -4.8 signal seems to extend more anteriorly than the -4.7 signal. (B) High-power magnification of the rectum wall of a 17.5 day old fetus (as an example, a corresponding area is boxed in A). Within the epithelium (ep), Hox-4.~8 labelling is more intense in the deep invaginations (crypts of Lieberkiihn, wide arrow) than in the apical cells. Within the mesenchyme (me), labeling is found in a concentric cell layer (small arrow). (C) High-power magnification of the recto-anal junction in a 16.5 day old fetus, showing the sharp boundary of Hox-4.8 transcripts in the epithelial and mesenchymal layers (arrows). A corresponding area is boxed in A.

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Fig. 6. Comparison of Hox.4 transcript domains in the developing kidney (metanephros) and gonadal anlage (genital ridge). Adjacent sagittal sections of a 12.5 day old fetus were hybridized to probes specific for Hox-4.2 to -4.8 as indicated. The metanephros (m) and genital ridge (g) region is enlarged (boxed in the scheme). Note that only Hox-4.2 and -4.4 are expressed in genital ridge cells and that Hox-4.8 is the only gene whose transcripts are not detected in the metanephros.

homology 1 within the Hox gene superfamily (Acampora et al., 1989). Paralogs have been characterized in the human HOX1 and HOX3 complexes (D'Esposito et al., 1991) and in the mouse HOX-1 (P. Gruss, personal communication). The Hox-4.8 homeodomain is more similar to the Drosophila AbdB homeodomain than to any other Antp-like (class I) homeodomain. When compared to other members of the HOX-4 complex, the Hox-4.8 homeodomain can clearly be classified among the AbdB-related genes together with Hox-4.4, -4.5, -4.6 and -4.7. Within this subfamily, the Hox-4.8 homeodomain sequence is significantly closer to those of Hox-4.7 and Hox-4.6 than from those of Hox-4.5 and Hox-4.4. Furthermore, a number of amino acids different from the AbdB homeodomain are shared by Hox-4.8, Hox-4.7 a n d / o r Hox4.6. Such common amino acid substitutions are not found between Hox-4.8 and Hox-4.5 or Hox-4.4. This suggests that Hox-4.8, -4.7 and -4.6 are phylogenetically closely related. The absence in Hox-4.8 and Hox-4.6 of the consensus Trp residue usually found at positions - 6 or - 7 from the AbdB-like homeodomains (Izpisfia-Belmonte et al., 1991a) reinforces the possibility that Hox-4.8 and Hox-4.6 are the result of a late duplication. A few amino acid substitutions are specific for the Hox-4.8 homeodomain. Such amino acid changes are found essentially in the first and second alpha helices. Interestingly, one of these substitutions involves the 'consensus' residue Phe 21 which is replaced by a Tyr 21. Among all homeodomains reported to date, only the Drosophila caudal gene shows the same replacement (Mlodzick et al., 1985). As already observed for the Hox-4.7 gene (Izpisfia-Belmonte et al., 1991a), the consensus residue Tyr 25 is exchanged for a Phe 2~. Finally, Hox-4.8 displays one amino acid substitution within the highly conserved DNA recognition

helix 3. Such a difference in specific amino acid could slightly alter the Hox-4.8 DNA binding a n d / o r recognition specificities. A comparison of the mouse, human and chicken Hox-4.8 sequences reveals high interspecies conservation which reflects their functional equivalence.

Is Hox-4.8 the 5' end of the HOX-4 complex? We have unsuccessfully searched for the presence of a putative homeobox 5' of the Hox-4.8 gene. This is consistent with the structure of the human HOX-4 locus where only a divergent homeobox could be detected 13 kb upstream of the human HOX41 (the Hox-4.8 homologue; D'Esposito et al., 1991). This homeobox belongs to the Evx-2 gene, one of the two murine homologues of the Drosophila even-skipped gene (Bastian and Gruss, 1990). In both mouse and human, Evx-2 appears to be transcribed from the opposite DNA strand (D'Esposito et al., 1991; Bastian et al., in preparation). The presence of the Evx-2 transcription unit at this position suggests that Hox-4.8 could mark the 5' limit of the HOX-4 complex.

Hox-4.8 is expressed in extreme posterior domains during development The Hox-4.8 expression along the AP axis of mouse fetuses and in the limbs is in good agreement with colinearity. The spatial and temporal regulation of the Hox-4.8 expression during development fits well with its extreme 5' position in the HO'X-4 complex in that it is restricted to very posterior regions of the mouse embryo. By day 9.5 p.c., the earliest stage at which Hox-4.8 transcripts could be detected, these are restricted to very posterior regions where somites have

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Fig. 7. Expression of Hox-4.8 in the developing limbs. (A) Sections of a 9.5 day old embryo, crossing the forelimb buds (fib). The orientation of the section is indicated on the scheme, the plan of section being orthogonal to the drawing. A weak Hox-4.8 signal is detected in posterior cells of the forelimb bud in this section (arrow). (B-C) Comparison of Hox-4.8 and Hox-4.7 transcript domains in the forelimb (B) and hindlimb (C) buds of a 10.5 day old fetus. A, anterior; P, posterior. (D) Sagittal section through the limbs of a 12.5 day old fetus. Hox-4.8 labeling is intense and distally restricted to the footplates mesencbyme, fl, forelimb; hi, hindlimb; hd, head; ey, eye. (E) Section through the toes of a 17.5 day old fetus. Hox-4.8 transcripts are detected in the mesenchymal cells surrounding the bone anlage.

n o t yet c o n d e n s e d . L a t e r , Hox-4.8 t r a n s c r i p t s a p p e a r to b e l i m i t e d to t h e tail, t h e genital t u b e r c l e , a n d t h e p o s t e r i o r - m o s t s e g m e n t o f t h e gut (the r e c t u m ) . I n t e r estingly, Hox-4.8 is t h e only g e n e o f the H O X - 4 complex t h a t shows no d e t e c t a b l e e x p r e s s i o n in t h e

metanephros metanephric level since it ing f r o m t h e will t h e n b e

or definitive kidney anlagen. The b l a s t e m a o r i g i n a t e s f r o m a very p o s t e r i o r condenses above the ureteric bud expandp o s t e r i o r p a r t o f t h e m e s o n e p h r i c duct. It d i s p l a c e d a n t e r i o r l y to t h e fetal gonads.

12 The expression patterns of the Hox-4 genes in the urogenital tractus correlate well with the level of origin of the mesonephros, gonad and metanephros (see Fig. 6). Only the most 3'-located genes, Hox-4.2, -4.3 and -4.4 are expressed in the genital ridge and fetal gonads. The same genes, plus Hox-4.5, -4.6 and -4.7 are expressed in the metanephros, probably because the metanephric blastema originates from an AP level included in their respective mesodermal domains of expression. Such an expression pattern illustrates the importance of level specificity (as compared to organ specificity) in the regulation of Hox gene expression. The extreme genomic position of the Hox-4.8 gene makes it expressed too posteriorly to be transcribed in the metanephros. A number of homeogenes are expressed during development in various areas of the digestive tract. Their expression is generally confined to the mesenchymal layer of the stomach and gut, whereas the endodermderived epithelial layer is negative (e.g. Galliot et al., 1989). In contrast, Hox-4.8 transcript distribution in the rectum spans the mesenchymal and the epithelial layers. Few Hox genes were described as being expressed in the gut endodermal layer, either in the foregut (Hox-l.6; Doll6 and Duboule, 1989) or hindgut (Hox-3.1; Le Mouellic et al., 1988). It is intriguing that the endodermal expression concerns essentially the anterior and posterior extremities of the alimentary tract. The Hox-4.8 gene is also expressed in the developing limbs according to a spatial regulation which is compatible with the general logic of expression of the Hox-4 genes in these structures (Doll6 et al., 1989). During early outgrowth (days 9.5-11.5 p.c.), Hox-4.8 transcripts are restricted to areas slightly more distal and more posterior than those where the Hox-4.7 transcripts are detected. This restricted expression in the limb extremities is conserved during subsequent stages as illustrated by the limited expression of Hox-4.8 in the hindlimb and forelimb footplates. As for other Hox-4 genes, Hox-4.8 transcripts remain preferentially expressed around the differentiating skeletal elements (i.e. the digit cartilages, days 14.5-17.5 p.c.). Thus, as for the trunk, the Hox-4.8 gene could be involved in determining the most postero-distal structures of the limbs. Recent analyses of the chicken Hox-4 genes during wing morphogenesis support this hypothesis (Izpisfia-Belmonte et al., 1991b; Nohno et al., 1991). The very posterior expression domain of Hox-4.8 appears to mark the last significant morphological boundary towards the posterior extremity of the mouse fetus. The tail, for instance, has a metamerized organization which consists of about 30 caudal vertebrae of rather similar morphologies and a rudimentary neural canal and vascular system. Hox-4.8 transcripts are also specific for the last posterior segment of the gut. Such

features support the idea that Hox-4.8 might be the last homeoprotein from the Hox-4 complex required to set up properly a network of positional information in the posterior extremities of the developing body.

Materials and Methods

Isolation and mapping of Hox-4.8 A mouse genomic cosmid clone (cos B, see Fig. 1) was screened with a 195 bp PstI-HaelII fragment containing the major part of the human Hox-4I homeobox (D'Esposito et al., 1991), labeled by random priming. Southern blots of various digests of cos B were hybridized under low-stringency conditions (42% formamide, 2 x SSC, 0.1% SDS, 1 × Denhardt's, 1 mM EDTA, 0.1 mg/ml denatured salmon sperm DNA at 37°C). The 5' region of cos B was mapped by classical procedures using partial restriction enzyme digests and end-labeled DNA fragments. Various DNA fragments were subcloned into Bluescript (Stratagene) and pGEM (Promega) vectors.

DNA sequencing The nucleotide sequences were determined with the dideoxy method (Sequenase R kit). The sequence of the Hox-4.8 homeobox was read on both DNA strands.

Riboprobe preparation Two types of ass-labelled antisense RNA probes were synthesized for in situ hybridization, using a T7 polymerase reaction according to standard procedures, from two DNA templates subcloned into pGEM1. As shown in Fig. 1B, probe A spans a 1.3 kb PvulI-HindlII fragment, and probe B covers a shorter 185 bp PstIHaelII fragment containing most of the homeobox. After transcription, probe length was reduced by limited alkaline hydrolysis. Both types of probes displayed the same spatial labelling pattern on embryo sections. Since labeling intensity was weaker with probe B, we used probe A in most experiments.

In situ hybridization Mouse embryos and fetuses were obtained from natural matings between (C57 BI6 × SJL) F1 mice. Midday of the day of vaginal plug was designated as day 0.5 p.c. Embryo recovering, sectioning and in situ hybridizations were carried out as previously described (Doll6 and Duboule, 1989), except that the pre-hybridization was omitted.

13

Acknowledgements We thank T. Rijkers for helpful discussions and H. Davies, A. Cooper and the staff of the EMBL photolab for preparing the manuscript.

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