Mouse Alx3: an Aristaless-Like Homeobox Gene Expressed During Embryogenesis in Ectomesenchyme and Lateral Plate Mesoderm

DEVELOPMENTAL BIOLOGY 199, 11–25 (1998) ARTICLE NO. DB988921 View metadata, citation and similar papers at core.ac.uk brought to you by CORE

provided by Elsevier - Publisher Connector Mouse Alx3: An aristaless-like Homeobox Gene Expressed during Embryogenesis in Ectomesenchyme and Lateral Plate Mesoderm

Derk ten Berge, Antje Brouwer, Sophia El Bahi,* Jean-Louis Gueńet,† Benoıˆt Robert,* and Frits Meijlink Hubrecht Laboratory, Netherlands Institute for Developmental Biology, Uppsalalaan 8, 3584CT Utrecht, The Netherlands; *Institut Pasteur, De´partement de Biologie MolećulaireGeńe´tique Molećulaire du De´veloppement, 28, rue du Dr. Roux, 75724 Paris Cedex 15, France; and †Institut Pasteur, Geńe´tique des Mammife`res, De´partement d’ Immunologie, 28, rue du Dr. Roux, 75724 Paris Cedex 15, France

Mouse Alx3 is a homeobox gene that is related to the Drosophila aristaless gene and to a group of vertebrate genes including Prx1, Prx2, Cart1, and Alx4. The protein encoded contains a diverged variant of a conserved peptide sequence present near the carboxyl terminus of at least 15 different paired-class-homeodomain proteins. Alx3 is expressed in mouse embryos from 8 days of gestation onward in a characteristic pattern, predominantly in neural crest-derived mesenchyme and in lateral plate mesoderm. We detected prominent expression in frontonasal head mesenchyme and in the ﬁrst and second pharyngeal arches and some of their derivatives. High expression was also seen in the tail and in many derivatives of the lateral plate mesoderm including the limbs, the body wall, and the genital tubercle. aristaless-related genes like Alx3, Cart1, and Prx2 are expressed in overlapping proximodistal patterns in the pharyngeal arches. Similar, but more lateral patterns have been described for the Distal-less-related (Dlx) genes. Intriguingly, expression and to some extent function of aristaless and Distal-less in Drosophila also have overlapping as well as complementary aspects. Alx3 was localized to chromosome 3, near the droopy-ear (de) mutation. © 1998 Academic Press Key Words: homeobox gene; pharyngeal arches; skeletogenesis; neural-crest-derived mesenchyme; lateral plate mesoderm; limb development; in situ hybridization; mouse embryology; proximodistal patterning; aristaless; Cart1.

INTRODUCTION mutation analysis using gene targeting or by the analysis of natural mutants. While many (and most likely all) Hox genes contribute to morphogenesis of the skeleton as an The vertebrate skeleton is derived from three different implication of their general role in rostrocaudal patterning embryological sources: the neural-crest-derived ectomesen- (see for a review Maconochie et al., 1996), mutations in chyme, the somitic (paraxial) mesoderm, and the lateral plate mesoderm. In spite of this heterogeneous ancestry, it members of many other subfamilies (for instance Msx1 and has emerged in recent years that many genes that are -2, Dlx-1 and -2, goosecoid, Prx1, Cart1, and Alx4) lead to a involved in patterning of the mesoderm and in skeleton variety of different skeletal defects (Satokata et al., 1994; formation are expressed in more than one of these types of Jabs et al., 1993; Rivera-Perez et al., 1995; Yamada et al., mesenchyme. Factors involved in skeletogenesis include 1995; Martin et al., 1995; Qiu et al., 1995, 1997; Zhao et al., growth factors and their receptors, collagens, and different 1996; Qu et al., 1997b). types of transcription factors (Erlebacher et al., 1995; Hall We are interested in the morphogenetic function of a and Miyake, 1996). Among these transcription factors, subclass of paired-(prd-) related, non-Pax homeobox genes homeodomain proteins take a prominent place. Several of that are expressed in speciﬁc patterns in embryonic mesen- them have been linked to functions in skeletogenesis by chyme. This group of genes includes Prx1 and Prx2 (previously

known as MHox and S8, respectively), Alx3, Alx4, and Cart1 ration and sectioning of embryos and subsequent treatment and (Opstelten et al., 1991; Rudnick et al., 1994; Cserjesi et al., hybridization of sections with radioactive probes were essentially 1992; Zhao et al., 1993; Qu et al., 1997a,b). Mice in which as described by Wilkinson et al. (1987) using modifications de- Prx1 has been inactivated have defects in a subset of cranial scribed previously (Vogels et al., 1990; Opstelten et al., 1991). and appendicular cartilages and bones and sometimes have Sections hybridized with radioactive probes were photographed by vertebral malformations (Martin et al., 1995); disruption of double exposure of dark-field and bright-field images; a red filter Cart1 leads to acrania and meroanencephaly due to a defect in was used during the dark-field exposure. closure of the neural tube at the level of the midbrain (Zhao et al., 1996); inactivation of Alx4 was recently shown to result in polydactyly but also body wall defects (Qu et al., 1997a). The Probes proteins encoded by these genes carry moderately similar, prd-class, homeodomains with a glutamine at the critical Alx3. The DNA template used to generate Alx3 probes was the position 50 (known as prd/Q50 homeodomains; Wilson et al., same for radioactive and digoxigenin labeling. A plasmid consisting of a 1.856-kb Alx3 cDNA cloned in pEXLox (Novagen) was cut with 1993) in contrast with the genuine prd and some of the Pax NcoI and transcribed with Sp6 polymerase. This results in an proteins that carry S50 homeodomains. Furthermore, rather antisense RNA complementary to the 3Ј-untranslated region of the than a paired domain that defines the Pax proteins, they mRNA and the part encoding the C-terminal 66 amino acids. contain a conserved C-terminal peptide sequence of unknown hXBP. A probe for murine hXBP was generated by using T7 function that we call the aristaless-domain after the Drosoph- polymerase on EcoRI-digested plasmid muhXBP-1EX (obtained ila gene aristaless (al) (Schneitz et al., 1993; Campbell et al., from Drs. A. Reimold and L. Glimcher, Boston). This probe 1993). This domain is present in at least 15 different aristaless- contains 478 bp from the 3Ј untranslated region of murine hXBP related genes with diverse biological roles. Other aristaless- (Clauss et al., 1993). related genes have been linked for instance to functions in eye Cart1. Although rat and human Cart1 cDNAs have been development (Burmeister et al., 1996; Mathers et al., 1997). characterized (Zhao et al., 1993, 1994; Gordon et al., 1996), no Here we describe cloning of the mouse Alx3 gene, its mouse clone has been described in the literature. Based on its genetic localization, and its expression pattern during em- sequence present in the EMBL/Genbank database, an EST clone bryogenesis. A previous report on hamster Alx3 dealt with (Accession No. AA388194) represents a mouse Cart1 cDNA. We expression in cell lines and adult tissues (Rudnick et al., purchased this cDNA, cloned in the vector pSport1, from Genome 1994). Alx3’s highly localized and characteristic expression Systems (St. Louis, MO; clone ID 790077) and reconfirmed by pattern suggests a function in patterning of neural-crest- sequencing its extreme high similarity at the level of the inferred translation product (65 identical amino acids in a stretch of 66, in derived mesenchyme and lateral plate mesoderm. comparison to human Cart1; data not shown). Single-stranded Since Alx3 displays considerable structural similarity to antisense probes were generated by restriction with EcoRI and the Cart1 gene (Zhao et al., 1993), we directly compared the using SP6 polymerase. expression patterns of these genes. Interestingly, aristaless- related genes like Alx3, Cart1, and Prx2 are expressed in proximodistal overlapping patterns in the mandibular arch that appear to be more medial and complementary to those Nomenclature of the Distal-less-related Dlx genes. We strive to follow the recommendations of the Committee on Standardized Genetic Nomenclature for Mice (1994). MATERIALS AND METHODS

Embryos. Mouse embryos were F2 from crosses between C57Bl/6 Chromosomal Mapping and CBA mice. Gestation was assumed to have begun at midnight A polymorphic microsatellite was identified between the Mus before the plug was observed. spretus-derived mouse strains SPR and SEG on one side and the Cloning procedures. cDNA libraries used were a 10.5-day- C57BL/6 strain on the other side, using the oligonucleotides embryo cDNA library in ␭gt10 obtained from B. Galliot (Gene`ve) and 5Ј-CCAGAACTGACAGCCATCC-3Ј and 5Ј-GGAAGAAGTGAT- a 16.5-day-fetal mouse library in ␭ExLox (Novagen). Nonstringent TTGCCCAC-3Ј as forward and reverse primers, respectively, in screening was done by hybridizing at 50°C in 5ϫ SSC. DNA was sequenced using an ABI 373 automatic sequencer. Other nucleic acid PCR amplifications of DNA. Amplification was performed with 20 procedures were according to standard procedures. The GenBank ng of DNA for 30 cycles (94°C for 30 s, 58°C for 1 min, and 72°C for accession number of the mouse Alx3 cDNA sequence is U96109. 12 s) and resulted in a 158-nucleotide fragment with C57BL/6 DNA RNA blot hybridization. Twenty micrograms of total RNA was and a significantly shorter fragment with spretus-derived strain loaded on 1.2% agarose gels and blotted to Qiabrane (Qiagen). This DNA. DNA fragments were analyzed on 3.5% Seakem:0.5% blot was hybridized with oligo-labeled Alx3 cDNA probes in 0.5 M NuSieve GTG agarose (FMC) gels. DNA samples were prepared from a set of consomic mice or from offsprings of the EUCIB sodium phosphate buffer containing 1% BSA, 1 mM Na2EDTA, and 7% SDS at 65°C; the most stringent wash was in 40 mM sodium (http://www.HGMP.MRC.AC.UK/MBx/MBxHomepage.html) phosphate buffer containing 1 mM Na2EDTA and 1% SDS. (Breen et al., 1994). The segregation profile of Alx3 was compared In situ hybridizations. Whole-mount, non-radioactive hybrid- to those of markers previously typed in the EUCIB using Excel 5 for izations were essentially as described by Wilkinson (1992). Prepa- the Macintosh.

RESULTS referred to here as the aristaless-domain, is only present in homeodomains belonging to the prd- class, either of the Cloning and Structural Characteristics Q50 or the K50 type. Figure 1B shows that proteins con- of Mouse Alx3 taining an ‘‘aristaless-like’’ homeodomain may lack an aristaless-domain, as for instance in the case of Phox2 A random-primed, day 10.5 mouse embryo cDNA library (Valarche´ et al., 1993). It is also interesting that splice in ␭gt10 (obtained from B. Galliot, Gene`ve) was screened variants of Prx1 have been described that encode versions of under low-stringency conditions with a mixture of probes the Prx1 transcription factor with and without the for Prx2 and Prx1. Among several positive clones, one aristaless-domain (Kern et al., 1992). contained a partial cDNA that was identified through A remarkable feature of the Alx3 protein is an abundance sequence analysis as the mouse homologue of the Alx3 in prolines, on both sides of the homeodomain. In the 148 homeobox gene. Hamster Alx3 was previously cloned from amino acids N-terminal from the homeodomain, 30 (20%) a golden hamster insulinoma cDNA library (Rudnick et al., are prolines, and C-terminally there are 22 prolines of 119 1994). Screening of a 16.5-day embryo library (Novagen) amino acids (18%). The homeodomain itself contains one under high-stringency conditions with a probe derived from conserved proline. Even in the highly related Cart1 protein this partial cDNA clone resulted in cloning of a complete (see below), only 8 prolines are present N-terminal and 7 1856-bp cDNA, the sequence of which confirmed its iden- C-terminal from the homeodomain. The N-terminal part of tity as Alx3. Figure 1A shows the mouse Alx3 sequence and Alx4 is also rich in prolines (26 of 152 ϭ 17%) but in this its inferred translation product. Sequence similarity with case 11 prolines are concentrated in a 16-amino-acid the hamster protein is very high, the homeodomains being stretch, similar to domains present in many different pro- identical and only 14 (5%), mostly conservative, changes teins; these are associated in a few cases with functions as being present in the remaining 283 amino acids (Fig. 1A). In activation domain and other roles (Mitchell and Tjian, the hamster Alx3 sequence published by Rudnick et al. 1989; Williamson, 1995). Abundance of prolines throughout (1994), however, the ATG triplet proposed to encode the most of the protein was reported for at least one other translation initiation codon is followed six nucleotides homeodomain protein, Hesx1, originally known as Prh downstream by a second ATG. Only this second triplet has (proline-rich homeodomain protein; Crompton et al., 1992), been conserved in the mouse sequence, the first correspond- but its significance remains unclear in both cases. The ing to CGA in the mouse sequence, an extremely unlikely abundance of prolines is even evident in the aristaless- initiation codon. This opens the possibility that in the domain of Alx3, making it a very unusual version: four hamster too, only the second ATG is used, the first being in prolines are present in the 16-amino-acid sequence, a rather poor context according to the ‘‘Kozak-rules’’ (a whereas all other known aristaless-domains do not contain pyrimidine at position Ϫ3; for a discussion, see Kozak, any prolines. As prolines have a major impact on the 1989, and references therein). three-dimensional structure of a protein domain (see for Alx3 belongs to the large class of genes that encode a instance Williamson, 1994), insertion of four of them in a prd/Q50 homeodomain, denoting moderate similarity to short peptide would be expected to strongly modify any the prd homeodomain, but the presence of a glutamine on structural characteristics previously present. position 50 (Wilson et al., 1996). Figure 1B gives a compari- As noted by others, Alx3, Cart1, and Alx4 encode pro- son of the Alx3 homeodomain with a series of selected teins sharing very high sequence similarity inside the similar homeodomains including the prd/K50 (Ptx) home- homeodomain (90% identity), as well as considerable simi- odomains, as well as for comparison the genuine prd- (S50) larity in the stretch between the homeodomain and the homeodomain. carboxyl terminus (Zhao et al., 1993; Rudnick et al., 1994; Only limited sequence similarity outside the homeodo- Qu et al., 1997b). Figure 1C compares Alx3, Alx4, and Cart1 main is usually found between different homeodomain sequences in the region between the homeo- and aristaless- proteins, except in homologues from different species. The domains, confirming the high degree of relatedness of these resemblances that one does find are an important criterion three proteins, especially between Alx3 and Cart1. for classification of the genes, since they are indicative of a N-terminal from the homeodomain, the similarity was phylogenetic relationship and suggest involvement in com- barely detectable (not shown). mon molecular interactions. As indicated in Fig. 1B, a subset of the proteins whose homeodomains are listed contains close to the C-terminus a conserved 16-amino-acid Genetic Localization sequence with the consensus sequence RXSSIAAL R- A polymorphism was identified for the Alx3 locus be- LK AKEHS (underlining indicates strongest conservation). tween the C57BL/6 and M. spretus-derived (SPR and SEG) Currently 15 different genes have been reported that encode mouse strains, within the first 150 nucleotides of intron 3 a protein carrying a variant of this sequence, disregarding (our unpublished data). The distribution of this polymor- homologous genes of different species. The variant present phism was analyzed in a panel of consomic mice. These are in Orthopedia (Otp) was already noticed by Simeone et al. inbred strains in which a complete chromosome from a (1994) to be conserved in three homologous Otp proteins given strain has been substituted for the homologous chro- from Drosophila, sea urchin, and mouse. This sequence, mosome of another strain or species. The panel we used was

FIG. 1—Continued

generated by raising an F1 between C57BL/6 (M. musculus) the EUCIB which defines breakpoints every 10 cM or less in and STF/Pas (M. spretus) parental strains and then by Chr3 (Fig. 2A). This showed that Alx3 is located between systematically backcrossing selected offsprings of this F1, the D3Nds20 and D3Mit42 markers. We further analyzed checking at each generation for the presence of a specific animals which define breakpoints every centiMorgan (Fig. chromosome (J. L. Gueńet, unpublished data). In some 2B); Alx3 was localized between Cd2 and D3Mit102, which strains, only a fraction of a given STF/Pas chromosome was are 46.84 and 47.28 cM from the centromere on the EUCIB retained in a C57BL/6 background, thus allowing for sub- map, respectively. Thus, Alx3 maps in the close vicinity of chromosomal localization. This analysis permitted to as- the mutation droopy ear (de), which has been located on sign the gene to mouse chromosome 3, telomeric to the mouse Chr3 by reference to the mutation osteopetrosis D3Mit9 microsatellite marker, which is located 42 cM from (Curry, 1959), now known to be allelic to the macrophage- the centromere on the EUCIB map. To refine localization, a stimulating factor gene (Csfm) (Yoshida et al., 1990) and to pedigree analysis was performed, in a panel of animals from the polymorphic marker D3Mit104 (Dietrich et al., 1994).

FIG. 1. Sequence of mouse Alx3 and comparison of structural features with related genes. (A) DNA sequence and deduced translation product of mouse Alx3 cDNA. Amino acids shown in lowercase show differences with the hamster cDNA. As explained in the text, we believe that in both genes the ATG encoded by nucleotides 149–151 (this sequence) is the authentic translation start. Homeodomain and aristaless-domain are shaded. Note abundance of prolines in all portions of the protein except the homeodomain. (B) Listing of homeodomains with high similarity to that of Alx3 and aristaless-domains if present in the same protein. (Third column) Percentage of identical amino acids in the homeodomain, compared to that of Alx3. Where possible, mouse sequences are shown, and homologues from other species are not included. CER08B4 is the name of a Caenorhabditis elegans locus containing several putative genes, one of which encodes the homeodomain shown (Accession No. Z68008). Since this sequence is from an incomplete genomic fragment, the absence of the aristaless-domain is uncertain [References: Alx3, this paper; Alx4, Qiu et al., 1997; aristaless, Schneitz et al., 1993; Cart1, Zhao et al., 1993; Chx 10, Liu et al., 1994; CER08B4, Wilson et al., 1994; DRG11, Saito et al., 1995; OG-12 and Uncx-4, Rovescalli et al., 1996; Otp, Simeone et al., 1994; Phox2, Valarche´ et al., 1993; Prx1, Kern et al., 1992, and Cserjesi et al., 1992; Prx2, Opstelten et al., 1991; Ptx1, Lamonerie et al., 1996; Ptx2, Semina et al., 1996; Ptx3, Smidt et al., 1997; Rax, Furukawa et al., 1997; SpPrx1, Martinez and Davidson, 1995, unpublished entry from GenBank (Accession No. D85080)]. (C). Comparison of amino acid sequences of mouse Alx3 and Alx4 and rat Cart1 between the homeodomain and the carboxyl-terminus. Numbering is as above (A) for Alx3, as in Qiu et al. (1997) for Alx4, and as in Zhao et al. (1993) for Cart1. * indicates identity and ϩ a conservative change. Alx3 and Cart1 are more similar to each other (52% identical amino acids) than Alx3 and Alx4 (34% identities in this region).

Early expression. In embryos of about 7.5 days p.c. (mid to late streak), no expression was observed (not shown). We did not analyze earlier stages, but using blot analysis of total RNA, we did not detect expression in E14 embryonic stem cells, a model for the inner cell mass cells of blastocyst-stage embryos (not shown). In embryos of 8.0 (not shown) to 8.5 and 9.0 (Figs. 4A, 4B, and 4D) days, expression becomes first visible in the allantois and in a region near the tail bud. The latter expression is especially high in ventral surface ectoderm between the allantois and tail bud and less in the underlying mesoderm (Fig. 4D) in contrast with expression at later stages, which is predominantly located in mesenchyme. Alx3 expression in the tail bud region overlaps with an epithelial ridge ventral from the tail bud region, termed the ventral ectodermal ridge or groove (VER), a structure proposed to have a role in tail mesoderm formation (Gru¨neberg, 1956; Gajovic and Kostovic-Knezevic, 1995). Between 9.0 (Fig. 4C) and 9.5 (Fig. 4E) days p.c., the expression pattern changes markedly: strong expression areas appear in the anterior mesenchyme of the head, at multiple paired areas in the flank mesoderm, and in the body wall. Two of these pairs of patches of expression correspond to the sites of outgrowth of the limbs and the FIG. 2. Segregation of the Alx3 genes in offsprings of the EUCIB. precursors of the genital tubercle (Fig. 4E). The first column represents the microsatellite markers used as references in this study and the second column (d) their distance Head and Pharyngeal Arches from the centromere on the EUCIB map. Anchor markers are in bold type, and the Alx3 row is in italics. Each column then The time of appearance and also the location of the represents the status of a specific offspring from the back-cross early (day 9.0–10.5; Figs. 4C, 4E, and 4G) expression of (identified by its number on top), either homozygous for a C57BL/6 Alx3 in the head suggest that the expressing cells are or spretus allele (1) or heterozygous (2), for each marker analyzed. postmigratory and migrating neural-crest-derived mesen- The limit between the shaded and the white areas defines the position of the chromosome breakpoint for each animal analyzed.

The latter is located at position 51.9 cM from the centromere on the EUCIB map.

Expression of Mouse Alx3 during Embryogenesis General. We studied the expression pattern of the Alx3 gene by a combination of RNA blot analysis, non- radioactive whole-mount in situ hybridizations on 7.5–11.5 postconception (p.c.) embryos, and radioactive in situ hybridizations on sections of parafﬁn-embedded embryos of 10.5–14.5 days p.c. RNA blot analysis showed that Alx3 is expressed as an approximately 2.2-kb mRNA in embryos of 10.5–14.5 days p.c. (Fig. 3). Expression in organs of adult animals has been reported by Rudnick et al. (1994). The in situ hybridization experiments revealed highly characteristic expression domains in the head, the limbs, the tail region, and the body wall. Expression was found mostly in FIG. 3. Blot analysis of RNA from total embryos. Twenty micro- loose mesenchymal cells and occasionally in adjacent epi- grams of RNA from embryos of 10.5–14.5 days (as indicated) was thelia. Expressing mesenchyme was either neural-crest- analyzed. Twelve micrograms of adult pancreas RNA was also derived craniofacial ectomesenchyme or lateral plate- included in this blot. The same blot was hybridized with both derived mesoderm. No expression was observed in axial or human ␤-actin and rat GAPDH probes as a control for quantity and paraxial mesoderm nor in neural tissues or endoderm. quality of mRNA present in the RNA preparations.

FIG. 4. Whole-mount in situ hybridization analysis of Alx3 expression in 8.5- to 11.5-day mouse embryos and of Cart1 expression at 10.5 days. The stage (days p.c.) is indicated in the upper right corner of each panel. B shows at higher magnification of the expression area near the tail bud in a similar embryo as shown in A. D shows a cross-section of a slightly more advanced embryo, demonstrating expression in surface ectoderm and amnion. (E) Short white arrow points at fore limb, black arrow at hind limb; long white arrow indicates progenitor of genital tubercle. (F) Cross-section of embryo shown in E. Expression inside the brain vesicle is an artifact. The signal seen in E and G in the hind brain region was also found in negative controls, but not in sections of similar embryos hybridized with the same, but radioactively labeled probe. (H) 10.5-day embryo hybridized with mouse Cart1 probe. Arrow points at mesonephric and paramesonephric ducts that hybridize to this probe and not to Alx3. (I) 11.5-day embryo hybridized with Alx3 probe; (inset) genital tubercle at higher magnification. Abbreviations used: Al, allantois; Am, amnion; ba, first branchial arch; di, diencephalic vesicle; ec, ventral surface ectoderm; g, hind gut; gt, genital tubercle; tel, telencephalic vesicle; hl, hind limb.

chyme (Le Douarin et al., 1993; Osumi-Yamashita et al., craniofacial structures. Expression at 9.0–12.5 days was 1994; Trainor and Tam, 1995). In subsequent stages, particularly high in the lower frontal part of the head expression in the skull was found predominantly in (Figs. 4C, 4E, 4G, and 4I; Figs. 5B and 5C), with a rostral

FIG. 5. Radioactive in situ hybridization analysis of Alx3 expression on sections of 10.5- to 14.5-day mouse embryos. Expression related with head and branchial arches. Age (days p.c.) is indicated in right upper corner. The signal seen in the retina is an artifact. (A, B, D, and F, transversal sections; C, E, and G, sagittal sections). Abbreviations used: 3v, third ventricle; a1, first branchial arch; bo, basioccipital; bs, basisphenoid; bw, body wall; d, diencephalon; f, frontonasal process; fl, fore limb; h, heart; ib, incisor bud; lp, lateral nasal process; mb, molar bud; mc, Meckel’s cartilage; mp, medial nasal process; nc, nasal cavity; os, ossification of mandible progenitor; pal, secondary palate; pm, premaxilla; t, telencephalon; to, tongue; v, ventricle.

border near the level of the eye. We never saw expression pharyngeal arch was detectable at 10.5 days (Figs. 4G and in the neural crest itself nor in migrating cells close to 5B). At 12.5–14.5 days, Alx3 was strongly expressed in the neural tube. Sections of 9.5-day embryos showed high neural-crest-derived mesenchyme in the nasal process, levels of Alx3 mRNA in ectomesenchyme ﬂanking the developing jaws, and the distal part of the tongue. This is diencephalon (Fig. 4F). At 10.5 days p.c. and later, radio- shown for 12.5-day embryos in Fig. 5C and for 14.5-day active in situ hybridization of sectioned embryos showed embryos in Figs. 5E and 5F. In the upper jaw (Fig. 5E), expression in a thin layer of mesenchymal cells surround- Alx3 expression was present in the premaxilla but absent ing the telencephalic vesicles (shown for 12.5 days, Fig. from the secondary palate. Expression in the lower jaw 5A) and at 14.5 days similarly around the ventricles (Fig. was high in the area surrounding Meckel’s cartilage, that 5D). Expression in the remaining mesoderm of the cranial itself barely expressed Alx3, and was elevated near the vault was generally low but above background (Figs. 5A developing incisor and molar buds (Figs. 5C, 5E, and 5F). and 5B and data not shown). Figures 5D and 5F show high Similar to the expression near the nasal cavity, there was expression in mesenchyme around the nasal cavity in- at 14.5 days in the developing jaws a striking median cluding the nasal septum and a contrasting lack of location. Figure 5G shows expression in the mesenchy- expression at more lateral positions. mal precursors of the sphenoid bone around Rathke’s Earliest expression in the ﬁrst (mandibular) arch was pouch, the developing pituitary. At this position the seen in 9.5-day embryos and was located distally (Fig. 4E). sphenoid will form the sella turcica that serves as a Lower but similarly distal expression in the second receptacle for the hypophysis. Expression was essentially

FIG. 6. Radioactive in situ hybridization analysis of Alx3 expression on sections of 10.5- to 14.5-day mouse embryos. Expression in limbs, body wall, and tail. Age (days p.c.) is indicated in right upper or lower corner. See legends to Figs. 4 and 5. (A, B, C, and E) Cross-sections; (D, F, and G) sagittal sections. Abbreviations used: cpf/cph, cartilage primordia of bones in fore/hind limb; d, diaphragm; lu, lung; mn, mesonephros; pp, pleuro-peritoneal membrane; (p)t, (proximal) tail; uh, umbilical hernia/midgut loop; us, urogenital sinus.

absent from the basioccipital (Fig. 5G), which is meso- hind limb (Fig. 4G). At these stages, Alx3 expression was derm-derived, and present in the neural-crest-derived higher in the anterior part of the limb bud, but especially at basisphenoid (see Le Douarin et al., 1993), illustrating the later stages the expression domain extended also to distal prevalence of expression in ectomesenchyme of the head and distal-posterior regions (Fig. 4G). Furthermore, expres- rather than in mesoderm. sion extended into the flank mesoderm anteriorly from the bud. Subsequently the pattern evolved into a bipartite expression domain consisting of a larger anterior and Limbs smaller distal-posterior domain, as is visible in the 11.5-day At early stages, Alx3 was expressed in four pairs of spots whole mount shown in Fig. 4I. With increasing age, expres- in the lateral plate mesoderm, two of which overlap with sion became further confined to narrow distal and superfi- the anterior part of the areas where the limbs develop (Figs. cial areas: compare Figs. 4E, 4G, and 4I and Fig. 6A. At 14.5 4C and 4E). A pair of more anterior spots is seen at the level days expression was present in particular locally under- of the heart, and a posterior pair of spots corresponds to the neath the surface of the limbs (Fig. 6A): expression followed area where the genital tubercle will develop (Fig. 4E). This the profile of the surface epithelium rather than the outline asymmetry with respect to the limb field became evident in of the cartilage elements. This would not suggest a direct embryos in which limb outgrowth was visible, for instance correlation at this stage with cartilage differentiation or at 9.5 days for the fore limb (Fig. 4E) and at 10.5 days for the bone formation in the limb.

Cloacal Region and Tail The genital eminence is the precursor of the genitalia, but is at the stages studied here indistinguishable between male and female mice. It is formed from the fusion (at around 11 days of gestation) between two halves forming from lateral plate mesoderm. As shown in Fig. 4I, Alx3 was expressed in the caudal part of the developing genital tubercle of an 11.5-day embryo. As in the case of the limbs, expression in the genital eminence was at early stages (9.5 days) foreshad- owed by expression spots in posterior lateral plate mesoderm (Fig. 4E). Sections of embryos of 12.5–14.5 days (Figs. 6D–6F) showed that expression in the genital eminence surrounded the urogenital sinuses. This expression area was continuous with expression in the tail. Expression in the tail was prominently ventrally concentrated. Until 12.5 days, expression appeared to extend to the ectoderm (Figs. FIG. 7. Absence of Alx3 in developing pancreas. Radioactive in 6B–6D), but from 13.5 days on, it was restricted to the situ hybridization of sagittal sections of a 14.5-day mouse embryo. mesenchyme (Figs. 6E and 6F). Black squares in left panels indicate the areas which have been shown at higher magnification in right panels. (A and B) Alx3 expression. (C, D) hXBP expression. Alx3 is absent from pancreatic Other Expression primordia, whereas hXBP is easily detectable. Apart from the expression in head and pharyngeal arches, most other expression of Alx3 was in lateral plate the presence of intact mRNA. Alx3 mRNA levels in the mesoderm and its derivatives. This was described above pancreas must be at least 20-fold lower than in total embryo for limbs and genital eminence. In addition, between 10 (days 10–14). and 14 days of gestation, we saw high expression in the body wall at all anteroposterior levels. Examples are Comparison to Cart1 and Other aristaless-Related shown in Figs. 6B and 6C in cross-sections at the level of Genes the liver near the diaphragm at 10.5 days and at 12.5 days As shown above, Alx3 and Cart1 are structurally highly of gestation at the level of the umbilical hernia, respec- related homeobox genes. Comparison of published expres- tively. At this latter site and stage, the body wall is still sion patterns of Cart1 (Zhao et al., 1994, 1996) with the open, which was reflected by an interruption in the Alx3 Alx3 pattern (this paper) shows that their expression pat- expression domain (Fig. 6C). terns overlap partially, but differ in certain aspects. To The flank mesoderm is continuous with a number of study the relation between Cart1 and Alx3 expression we serous membranes that form the sacs separating internal directly compared these patterns by whole-mount in situ organs. These also showed low to moderate, but significant hybridization, as well as by hybridization of nearby sections levels of Alx3 expression. We saw for instance at 10.5 and of embryos using a mouse Cart1 probe (see Materials and 11.5 days expression in the pericardial sac membrane, in the Methods). Figures 4G and 4H show a comparison of Alx3 pleuroperitoneal septum, and in the peritoneal membrane and Cart1 expression in 10.5-day embryos, demonstrating in which the liver is contained (not shown). Figure 6G similar expression in the frontonasal process, the mandib- shows expression in the progenitor of the pleuroperitoneal ular arch (see also Figs. 8A and 8B), and lateral plate membrane. Apart from weak expression in mesenchyme mesoderm, but lower Cart1 expression in the limbs. Expres- surrounding the mesonephric duct (Fig. 6G), there were no sion in the mesonephric and paramesonephric ducts, which other examples of expression in or near internal organs. We is characteristic of Cart1, is clearly visible in Fig. 4H. Figure paid special attention to the progenitors of the pancreas 8 shows sections of 10.5- and 13.5-day embryos demonstrat- because Alx3 is known to be expressed in pancreatic cell ing similar expression of Cart1 and Alx3 in pharyngeal lines as well as in the adult pancreas (Rudnick et al., 1994). arches, jaws, body wall, and tail. At several locations Cart1 We did not detect any Alx3 signal from the developing is expressed where Alx3 is barely detectable: the interme- pancreas at the stages investigated and shown for a 14.5-day diate mesoderm (Figs. 8D and 8E) and around the pharyn- embryo (Figs. 7A and 7B), under conditions allowing us to geal pouches (Figs. 8F and 8G). A distinct characteristic of detect high levels of expression of the murine hXBP-1 gene Cart1, to which the gene owes its name, is its persisting (Clauss et al., 1993) in near-adjacent sections of the same expression in cartilage. This is seen at many locations, for embryos (Figs. 7C and 7D). Apparently, Alx3 does not have instance the sclerotomes (see Zhao et al., 1994), Meckel’s a role in pancreatic development. Pancreas RNA from an cartilage, and the rib primordia (shown in Fig. 8H). Major adult mouse was also included in the Northern blot analy- quantitative differences are seen in the limbs and tail where sis shown in Fig. 3. Under the conditions used, Alx3 mRNA Cart1 expression is much lower and in the stomach mes- was undetectable, although control hybridizations showed enterium where it is higher (Figs. 8D and 8E).

FIG. 8. Direct comparisons of Alx3 and Cart1 hybridization. In situ hybridization analysis of whole-mount embryos (A–C) and sections (D–I). Probes used are indicated in the lower right corner. Stage is indicated in the upper right corner of panels in top row; bottom panels compare same stage in whole mounts and nearby sections of the same embryo in the radioactive in situs of sections. (A–C) Dissected mandibular arches of 10.5-day embryos. Alx3 and Cart1 expressions are visualized by whole-mount in situ hybridization; Prx2 expression by X-gal staining of an embryo heterozygous for a Prx2 allele in which a LacZ open reading frame has been targeted. It is worthwhile to compare these three panels to Fig. 6 of Qiu et al. (1997), who show expression patterns of a series of Dlx genes at 9.5 days of gestation. (D, E) Cross-sections demonstrating shared hybridization in body wall, hybridization to intermediate mesoderm of Cart1 probe (arrow), whereas Alx3 expression is minimal in this area. (F, G) Sagittal sections of embryo of same stage showing expression of Cart1 around pharyngeal pouches (yellow arrow) and in the mesonephric duct. (H, I) Sagittal sections of 13.5-day embryo, showing Cart1 expression in rib primordia (yellow arrows). 21 22 ten Berge et al.

Characteristic distal expression patterns of Cart1 and currently unknown, although Simeone et al. (1994) present Alx3 in dissected mandibular arches at 10.5 days are shown some evidence for a function as activation domain. in Figs. 8A–8C. Expression of a third aristaless-related gene Results of studies in vitro are consistent with prd/K50 that is expressed distally in the mandibular arch, Prx2, was and prd/Q50 homeodomains having an overlap in DNA- also included in this figure. In this case, expression was binding specificity (Wilson et al., 1996), leaving open the visualized by X-Gal staining of a mouse embryo heterozy- possibility that they share common targets. In these studies gous for a Prx2 allele in which a LacZ cassette was targeted binding specificity of cooperatively binding dimers of prd- in-frame (Ten Berge et al., manuscript in preparation). It class homeodomains to palindromic DNA sites was ana- appears that Prx2 is expressed over a larger proximodistal lyzed. Heterodimerization of prd-class homeodomains in range but the techniques used, in situ hybridization and vivo (Wilson et al., 1995) would be of capital significance for LacZ staining, do not allow a quantitative interpretation. the interpretation of their expression patterns: a specific The two remaining known members of this subgroup of biological function might then be linked to the overlap of aristaless-related genes, Prx1 and Alx4, are also expressed the expression domains of interacting homeodomain pro- similarly in the mandibular arch (Leussink et al., 1995; Qu teins. In this context a report of Mead et al. (1996) is of et al., 1997a,b). The overlapping proximodistal patterns of interest. Ectopic expression in Xenopus embryos of the these five vertebrate aristaless-related homeobox genes paired-class homeodomain protein, Siamois, induces a sec- suggest that they have a coordinated function in patterning ond axis, and this effect could be blocked by coexpression of of the pharyngeal arch mesenchyme. another paired-class homeodomain protein, Mix1; it was shown that this effect is likely to be a consequence of heterodimerization between these proteins. DISCUSSION Genetic Mapping of Alx3 Possible Functions of Alx3 We have mapped the Alx3 gene between 46.8 and 47.3 cM The expression domain of Alx3 that we describe here from the centromere of mouse Chr3 on the EUCIB genetic suggests functions in the patterning in the neural crest- map, using a panel of offsprings from this back-cross. This derived mesenchyme that shapes craniofacial structures locates the gene in the close vicinity of the droopy-ear and in formation of lateral plate mesoderm-derived struc- mutation (de). The order and position of the markers used tures and the tail. Alx3 expression is largely restricted to to localize Alx3 differ slightly on the consensus map for loose mesenchymal cells suggesting that differentiation of mouse chromosome 3 (Yui and Wakeland, 1997); in this mesenchymal cells leads to downregulation of Alx3. In the map, Cd2 is located at 46.2 cM from the centromere and limbs, for instance, the expression pattern evolves from D3Mit102 at 49.7 cM, while Tshb is located between them broad diffuse areas to more sharply delineated regions in at 48.5 cM. Nonetheless, both Alx3 and de map within the remaining undifferentiated mesenchyme underneath the 3.5-cM interval defined by Cd2 and D3Mit102. Mice ho- surface ectoderm, not obviously related to cartilage primor- mozygous for de are characterized by a low setting of the dia (compare Figs. 4G and 4I; Fig. 6A). Similarly, no expres- ears on the sides of the head and lateral projection of the sion is seen in Meckel’s cartilage in the lower jaw nor in the pinnae, as a consequence of abnormalities of the cranial ossifying mandible (Fig. 5F). It seems likely that genes like skeleton. The skeleton of the occiput and shoulder girdle Alx3 exert their function in undifferentiated mesenchyme shows immaturity of form with disproportionate shorten- by influencing differentiation choices. ing of the limb bones, resulting from disturbed mesenchy- Early Alx3 expression in lateral mesoderm presaged out- mal condensation. This may derive from defective growth growth of limbs and genital tubercle. Alx3 expression of the cartilage itself (Curry, 1959). This phenotype is at extends into the flank anteriorly from the limbs and is at least in part in keeping with the expression of Alx3. early stages absent from the posterior part of the limb. This Therefore, Alx3 is a candidate gene for the de mutation. indicates that it does not merely reflect a difference in rate of cell proliferation between these sites and the rest of the flank mesoderm, but instead is more likely to depend on, Relation with Other al-Related Genes and possibly mediate, signals that specify positional cues. Prx1, Prx2, Cart1, Alx3, and Alx4, based on their pre- dominant expression in mesenchyme and lateral plate mesoderm, appear to constitute a subfamily of related Structural Aspects of aristaless-Related Genes aristaless-related homeobox genes, possibly also including aristaless-related proteins are defined by the presence of OG12 (also known as Prx3; Van Schaijk et al., 1997). the aristaless-domain and a homeodomain of either a prd/ Structurally, however, the Prx genes do not appear to be Q50- or a prd/K50-type. The combined presence of these more strongly related to the Alx genes and Cart1 than to conserved domains appears to embody a molecular link the genes predominantly expressed in neural tissues like between a set of homeodomain proteins that have different CHX10 and Rax (see Fig. 1B). biological roles but share potential for similar biochemical Based on sequence comparison, Alx3, Cart1, and Alx4 are interactions. The role of the aristaless-domain remains close relatives that share sequence similarity even outside

Copyright © 1998 by Academic Press. All rights of reproduction in any form reserved. Alx3 Expression in Mouse Embryos 23 the conserved homeo- and aristaless-domains, although arches (Robinson and Mahon, 1994; Qiu et al., 1997). conservation between the amino-terminal parts of the pro- Comparison of our data (Figs. 8A–8C) with those of Qiu teins encoded is minimal. In the embryo, the expression et al., however, shows a distinct difference between these patterns of Alx3 and Cart1 overlap to a considerable extent, two sets of patterns: Dlx expression is more lateral than for instance in the head and body wall, but a number of the expression patterns of the aristaless-related genes. differences are seen: Cart1 is expressed in the somites and aristaless-related and Distal-less-related genes appear to be sclerotomes (Zhao et al., 1994), whereas Alx3 is not ex- expressed, therefore, in parallel proximodistal patterns. pressed in the paraxial skeleton; expression in the meso- Alternatively, considering the tips of the arches that grow nephric duct is high for Cart1, but only very minor in the toward each other as the distal part of the arches would case of Alx3. Cart1 expression in the limb was much lower imply that the al-related genes are expressed generally more than that of Alx3, but the expression spot in the lateral distally than the Dlx genes. Mice carrying mutations in mesoderm at the level of the heart was equally high. The Dlx1 and -2 have defects in derivatives of the proximal part expression domains of both genes in the head appear to be of the mandibular arch (Qiu et al., 1995, 1997), which was similar. Alx3 is probably expressed in the part of the head explained by compensation by the more distally expressed mesenchyme that is believed to be critical for the defect Dlx genes. Anyhow, these patterns are suggestive of com- caused by the Cart1 null mutation (Figs. 4F, 4G, and 4H; see plementary and/or overlapping roles of Dll- and al-related Zhao et al., 1996). Because of the probable biochemical genes in patterning of the pharyngeal arches. Intriguingly, similarities between the Cart1 and Alx3 transcription fac- Dll and aristaless have been reported to be expressed in the tors, and their overlapping expression patterns, a degree of fruit fly in patterns that have complementary as well as redundancy in their functions seems plausible. The obvious overlapping aspects. Dll is involved in initiation of append- way to investigate this possibility is the comparison of the age development (Cohen et al., 1989) and in proximodistal phenotype of an Alx3 null mutant (work currently in patterning of the limb (Lecuit and Cohen, 1997). The progress) with the phenotypes of the Cart1 null mutant and expression of al at embryonic and larval stages in areas a Cart1/Alx3 double knock-out. abutting or overlapping areas of Dll expression would be The expression patterns of Alx3 and Alx4 (Qu et al., consistent with a related function (Schneitz et al., 1993), as 1997a) are strikingly similar. The genes share characteristic is the phenotype of al loss-of-function and gain-of-function expression patterns in the limbs and in craniofacial struc- mutants (Campbell et al., 1993). Both genes mark in the tures and both are expressed in the body wall. Precisely in imaginal disks of the appendages the future tip, and both these three structures defects are found in the Alx4 mutant have been shown to be expressed at the intersection of mice that were reported to have polydactyly, (transiently) wingless and decapentaplegic expression areas (Cohen, decreased size of the parietal plate of the skull, and ventral 1990; Campbell et al., 1993). Although it is clear that al and body wall defects (gastroschesis) (Qu et al., 1997b). Alx4 Dll have a role in proximodistal patterning of the append- mutants have polydactyly caused by an ectopic zone of ages, their interdependent relation is unclear. It is possibly polarizing activity (ZPA), indicating that the presence of more than a coincidence that both genes are implicated in Alx4 somehow precludes the formation of an anterior ZPA. proximodistal patterning of appendages in the fly and that Qu et al. (1997b) suggested that the limited size of the some of their vertebrate counterparts may play a part in ectopic ZPA (based upon expression patterns of Shh, FGF4, proximodistal patterning of the pharyngeal arches. It is and Hoxd13 in the mutants) could be caused by compensa- tempting to speculate that Dll and al are part of a regulatory tion by genes like Cart1, Prx1 and -2, and Alx3. In view of genetic circuit which has been conserved throughout evo- its expression pattern, Alx3 now seems to be the prime lution. Earlier, Miura et al. (1997) speculated on this on the candidate to be responsible for partially compensating the basis of a comparison of expression patterns in the brain of Alx4 mutant phenotype. This would imply that mice defi- another aristaless-related gene, Arx, and Dlx1. cient in both Alx3 and Alx4 will have a much stronger limb phenotype; the same obvious prediction could be made concerning the body wall and cranial phenotypes. ACKNOWLEDGMENTS We thank B. Galliot for a mouse cDNA library, Truus Hoeijmakers Comparison with Dll-Related Genes and Rivka Slager for DNA sequencing, and Peter Lanser for histolog- ical assistance. We thank A. Reimold and L. Glimcher for the murine Alx3 expression in the mandibular and hyoid arches is HXBT probe. We also thank Kirstie A. Lawson for advice concerning early on, at 10.5 days, located in the distal tips (see for early embryos and Annemiek Beverdam and Jacqueline Deschamps instance Figs. 4E and 4G, Fig. 5B, and Fig. 8B). Later, upon for discussions and comments on the manuscript. fusion of the distal portions of the arches, this results in a median location. 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