Function and Regulation of Alx4 in Limb Development: Complex Genetic Interactions with Gli3 and Shh

View metadata, citation and similar papers at core.ac.uk brought to you by CORE

provided by Elsevier - Publisher Connector

Developmental Biology 285 (2005) 533 – 544 www.elsevier.com/locate/ydbio Genomes & Developmental Control Function and regulation of Alx4 in limb development: Complex genetic interactions with Gli3 and Shh

Sanne Kuijper, Harma Feitsma, Rushikesh Sheth, Jeroen Korving, Mark Reijnen, Frits Meijlink*

Hubrecht Laboratory, The Netherlands Institute for Developmental Biology, Uppsalalaan 8, 3584CT Utrecht, The Netherlands

Received for publication 8 March 2005, revised 6 June 2005, accepted 11 June 2005 Available online 21 July 2005

Abstract

The role of the aristaless-related homeobox gene Alx4 in antero-posterior (AP-) patterning of the developing vertebrate limb has remained somewhat elusive. Polydactyly of Alx4 mutant mice is known to be accompanied by ectopic anterior expression of genes like Shh, Fgf4 and 5VHoxd. We reported previously that polydactyly in Alx4 mutant mice requires SHH signaling, but we now show that in early Alx4À/À limb buds the anterior ectopic expression of Fgf4 and Hoxd13, and therefore disruption of AP-patterning, occurs independently of SHH signaling. To better understand how Alx4 functions in the pathways that regulate AP-patterning, we also studied genomic regulatory sequences that are capable of directing expression of a reporter gene in a pattern corresponding to endogenous Alx4 expression in anterior limb bud mesenchyme. We observed, as expected for authentic Alx4 expression, expansion of reporter construct expression in a ShhÀ/À background. Total lack of reporter expression in a Gli3À/À background confirms the existence of Gli3-dependent and -independent Alx4 expression in the limb bud. Apparently, these two modules of Alx4 expression are linked to dissimilar functions. D 2005 Elsevier Inc. All rights reserved.

Keywords: Limb development; Alx4; Gli3; Antero-posterior patterning; Gene regulation; Sonic hedgehog; FGF4; Polydactyly

Introduction onist GREMLIN has been found to transduce the SHH signal to the AER, which results in activation of FGF4. During development of the vertebrate limb, interacting FGF4, in turn, signals back to the ZPA to maintain SHH signals from the apical ectodermal ridge (AER) and zone of (Zuniga et al., 1999). polarizing activity (ZPA) direct its outgrowth and pattern- Prior to Shh expression, the early limb bud is prepat- ing. The AER is located at the distal edge of the limb bud terned by mutual genetic antagonism between the anteriorly and multiple fibroblast growth factors (FGFs), including expressed zinc-finger transcription factor GLI3 and the Fgf4, Fgf8, Fgf9 and Fgf17, are expressed in the AER posteriorly expressed bHLH protein dHAND. This mecha- (reviewed in Martin, 1998). The ZPA is located in posterior nism polarizes the nascent limb bud mesenchyme to mesenchyme of the limb bud and produces the signaling establish the proper posterior localization of Shh (Te factor Sonic hedgehog (SHH), which is essential for Welscher et al., 2002a; reviewed in Panman and Zeller, subsequent antero-posterior (AP) patterning (reviewed in 2003). Furthermore, the 5VHoxd genes are activated prior to Capdevila and Izpisua Belmonte, 2001; Niswander, 2003). Shh expression and were found to be essential for establish- To assure proper outgrowth and patterning of the limb bud, ment of AP-polarity of the developing limb bud as well a SHH/FGF4 feedback loop is established in the posterior (Zakany et al., 2004). limb bud. The Bone Morphogenetic Protein (BMP) antag- Loss of function of Shh leads to severe truncations of the limbs (Chiang et al., 1996, 2001; Kraus et al., 2001), and ectopic expression of Shh in the anterior part of the limb * Corresponding author. Fax: +31 30 251 6464. induces digit duplications (Yang et al., 1997; Johnson and E-mail address: [email protected] (F. Meijlink). Tabin, 1997), demonstrating that the correct localization of

Shh in the posterior part of the limb bud is essential for limb P.A. Beachy (Chiang et al., 1996). Genotyping was done by outgrowth and AP-patterning. PCR on ear cuts or yolk sac DNA. Alx4 embryos were Analysis of preaxial-polydactylous mouse mutants genotypes as described by Beverdam et al. (2001), Gli3 and showed that Shh transcription is negatively regulated by Shh mutants were genotyped as described by Te Welscher et several genes that are expressed in the anterior mesen- al. (2002b). chyme of the limb bud. These include Gli3, which is mutated in the Extra toes (Xt) mouse mutant and the Reporter construct preparation and generation of aristaless-related gene Alx4, which is linked to the mouse transgenic mice mutant Strong’s luxoid (Lst)(Masuya et al., 1995; Buscher et al., 1997; Mo et al., 1997; Qu et al., 1997; Takahashi et Genomic clones from the RPCI-21 PAC library al., 1998). Polydactyly in Alx4 mutant mice is associated (Osoegawa et al., 2000), containing at least 20 kb of with, and dependent on anterior ectopic expression of Shh genomic DNA sequences upstream of the first exon of (Chan et al., 1995; Qu et al., 1997; Takahashi et al., 1998; Alx4, were obtained from the Leiden Genome Technol- Te Welscher et al., 2002b). In contrast, Gli3À/À linked ogy Center (Leiden University Medical Center, Leiden, polydactyly occurs independently of SHH signaling (Te The Netherlands). Genomic fragments from PAC clone Welscher et al., 2002b; Litingtung et al., 2002). In RPCI21-447D21 were subcloned in pBluescript, sequen- addition, when SHH beads were inserted anteriorly in ced and then inserted into the HindIII site of the vector chick limb buds, Alx4 expression was rapidly down- pSDKLacZpA. This plasmid (a generous gift of J. regulated, revealing a reciprocal negative interaction Rossant, Toronto) contains the E. coli LacZ gene linked between Alx4 and Shh (Takahashi et al., 1998). Expression to a translation initiation site which is adapted for studies in mice have shown that a part of the Alx4 eukaryotic expression. These genomic fragments included expression domain is downregulated in Gli3 deficient limb a 17.4-kb XhoI–EagI fragment to generate construct 1; a buds, whereas expression of Gli3 is normal in Alx4À/À 6-kb KpnI–EagI fragment to generate construct 2 and a limb buds (Te Welscher et al., 2002a). To better under- 385-bp BglII–EagI fragment to generate construct 3. stand the roles of Alx4 in limb patterning, we studied both Linearized constructs were gel-purified using Geneclean its upstream and downstream genetic interactions. We Spin Kit (BIO101, Carlsbad, CA, USA) prior to micro- asked whether induction of ectopic SHH signaling is the injection into the male pronucleus of fertilized mouse only molecular mechanism by which Alx4 deficiency eggs following standard procedures (Hogan et al., 1994). disrupts AP-patterning in the limb bud, as early Alx4 Integration of the transgene was confirmed by PCR on expression in the presumptive limb region (Qu et al., ear cuts or yolk sac DNA using primers directed to LacZ 1997), as well as late emergence of ectopic Shh expression (forward primer 5V-GCATCGAGCTGGGTAATAAGC- in Alx4 mutant embryos (Qu et al., 1998) suggested GTTGGCAAT, reverse primer 5V-GACACCAGACCAAC- otherwise. We analyzed Alx4/Shh compound mutants and TGGTAATGGTAGCGAC). found that the initial disruption of antero-posterior All animal experiments were conducted under the patterning in Alx4 deficient limb buds occurs independ- approval of the animal care committee of the K.N.A.W. ently of Shh. (Netherlands Royal Academy of Arts and Sciences). In addition, a genomic fragment upstream of Alx4 directs expression of h-galactosidase in limb bud mesen- In situ hybridization and b-galactosidase staining chyme, mimicking the typical anterior pattern of Alx4. Authenticity of this pattern was corroborated by its Whole mount in situ hybridization using digoxygenin- expansion in a Shh mutant background. Expression of labeled RNA probes was performed as previously the reporter construct was completely turned off in a described (Leussink et al., 1995; ten Berge et al., 1998). Gli3À/À background, indicating that it corresponds Probes used included Alx4 (Beverdam and Meijlink, specifically to the GLI3-dependent part of endogenous 2001), Hoxd11 (Izpisua-Belmonte et al., 1991), Hoxd13 Alx4 expression. We suggest that the two components of (Dolle et al., 1991a), Fgf4 (Hebert et al., 1990), Gli1 (Hui the Alx4 expression pattern are linked to dissimilar et al., 1994), Ptch1 (Goodrich et al., 1996), Shh and functions. Gremlin (Zuniga et al., 1999). Whole-mount h-galactosidase staining was done essentially as described previously (Hogan et al., 1994). Materials and methods In silico sequence analysis Mutant mice lines Comparative genomic analysis was carried out using Alx4 (Lst J) and Gli3 (Xt J) mutant mice were obtained VISTA (http://www-gsd.lbl.gov/vista; Mayor et al., 2000) from The Jackson Laboratory (Bar Harbor, Maine, United and Multiz (http://genome.ucsc.edu; Blanchette et al., States) and Shh mutant mice were originally obtained from 2004). S. Kuijper et al. / Developmental Biology 285 (2005) 533–544 535

Results in the ZPA during limb development (Laufer et al., 1994; Niswander et al., 1994; Zuniga et al., 1999). In Alx4À/À Late ectopic SHH signaling in Alx4 deficient limb buds limb buds, an ectopic domain of Fgf4 expression is observed in the anterior part of the AER at E10.5, prior to Preaxial polydactyly in mutant chick or mice, including ectopic Shh expression (yellow arrowhead in Fig. 2B). Lst and Xt mice, which are caused by loss-of-function Moreover, Fgf4 expression was extended throughout the mutations in Alx4 and Gli3, respectively, is frequently AER of Alx4À/À hindlimb buds at this stage (Fig. 2F). associated with ectopic anterior expression of Shh (Chan et The BMP antagonist GREMLIN is required for main- al., 1995; Masuya et al., 1995, 1997; Buscher et al., 1997). tenance of SHH and FGF4 signaling during limb develop- Takahashi et al. (1998) reported that ectopic activation of Shh ment (Khokha et al., 2003; Michos et al., 2004). It is did not occur until E11.5. To verify this, we followed normally expressed, prior to Shh activation, in the posterior expression of Shh between E10.5 and E11.5 in Alx4 deficient part of the distal limb bud mesenchyme, close to Fgf4 limb buds and confirmed that ectopic Shh expression was not expression in the AER (Fig. 2A; Zuniga et al., 1999). In detected until E11.5 (Figs. 1A–D). Notably, at this stage, a Alx4 mutant limb buds, Gremlin is upregulated in the prospective extra digit is already emerging (Fig. 1D). anterior part of the limb bud, concomitant with ectopic Fgf4 To rule out the possibility that SHH signaling is present expression (black arrowhead in Figs. 2B and F). ectopically at earlier stages, but not detectable by our in situ The Hoxd genes are activated in a colinear fashion in hybridization techniques, we analyzed the expression of posterior mesenchyme of the normal limb bud. These Patched 1 (Ptch1)(Ingham and McMahon, 2001) and Gli1, overlapping expression patterns also emerge before Shh which are early transcriptional targets of hedgehog signaling expression. Hoxd11 –Hoxd13 have been shown to play a (Marigo et al., 1996; Buscher and Ruther, 1998) and are role in limb outgrowth and digit patterning independently of generally considered to be sensitive indicators of SHH Shh (Zakany et al., 2004). In Alx4À/À fore and hindlimb signaling. Both Ptch1 and Gli1 were not detected ectopi- buds, Hoxd11 and Hoxd13 are ectopically expressed at cally in Alx4 deficient limb buds until E11.5 (Figs. 1E–H E10.5, again prior to ectopic Shh expression (data not and I–L), consistent with absence of ectopic SHH signaling shown; Fig. 2J; Takahashi et al., 1998). at earlier stages. Therefore, even in the absence of detectable hedgehog Fgf4 is normally expressed in the posterior part of the signaling, three different markers of the posterior part of the AER, prior to Shh activation, and maintains Shh expression limb bud were detected anteriorly in Alx4À/À limb buds.

Fig. 1. Ectopic SHH signaling is not detected until E11.5 in Alx4 mutant limb buds. (A, B) Expression of Shh in wildtype (A) and Alx4À/À (B) forelimb buds at E10.5. (C, D) Expression of Shh in wildtype (C) and Alx4À/À (D) forelimb buds at E11.5. Note that the expression is normal at E10.5 in Alx4À/À limb buds, whereas at E11.5 an ectopic domain of Shh is present in Alx4 mutant limb buds (black arrowhead in D). At this stage, a presumptive anterior digit is apparent. (E–H) Ptch1 expression at E10.5 (E, F) and E11.5 (G, H) in wildtype (E, G) and Alx4À/À (F, H) limb buds shows that ectopic SHH signaling is not detected until E11.5 (black arrowhead in H). (I–L) Expression of Gli1 in wildtype (I, K) and Alx4À/À (J, L) limbs at E10.5 (I, J) and at E11.5 (K, L). Black arrowhead indicates ectopic Gli1 expression in Alx4À/À limb buds at E11.5. 536 S. Kuijper et al. / Developmental Biology 285 (2005) 533–544

Fig. 2. Ectopic expression of Fgf4 and Hoxd13 in Alx4À/À mice occurs initially independent of SHH signaling. (A–D) Simultaneous expression of Fgf4 and Gremlin at E10.5 in wildtype (A), Alx4À/À (B), ShhÀ/À (C, CV) and Alx4À/ÀShhÀ/À (D, DV) forelimb buds. Yellow arrowhead (B) indicates ectopic expression of Fgf4 in Alx4À/À limb buds. Black arrowhead (B) indicates ectopic Gremlin expression in Alx4À/À limb buds. (C, D) Dorsal view of ShhÀ/À (C) and Alx4À/À ShhÀ/À (D) forelimb buds at E10.5. (CV,DV) Ventral view of ShhÀ/À (CV) and Alx4À/ÀShhÀ/À (DV) limb buds. Red arrowhead (CV,DV) indicates posterior Fgf4 expression. Yellow arrowhead (DV) indicates anterior ectopic spot of Fgf4 expression in Alx4À/ÀShhÀ/À limb buds. Note that Gremlin is not expressed ectopically in Alx4À/ÀShhÀ/À limb buds (D). (E–H) Expression of Fgf4 and Gremlin in wildtype (E), Alx4À/À (F), ShhÀ/À (G) and Alx4À/ÀShhÀ/À (H) hindlimb buds at E10.5. Note the ectopic expression of Fgf4 in Alx4À/À and Alx4À/ÀShhÀ/À hindlimb buds (yellow arrowhead). Black arrowhead in panel F indicates ectopic Gremlin expression in Alx4À/À hindlimb buds. (I–L) Expression of Hoxd13 in wildtype (I), Alx4À/À (J), ShhÀ/À (K) and Alx4À/ÀShhÀ/À (L) forelimb buds at E10.5 shows that Hoxd13 is ectopically expressed in Alx4À/À and Alx4À/ÀShhÀ/À limb buds (yellow arrowhead in panels J and L). (M–P) Expression of dHand in wildtype (M), Alx4À/À (N), ShhÀ/À (O), and Alx4À/ÀShhÀ/À (P) forelimb buds at E10.5.

In view of the known role of the dHand gene in early approach. We studied the expression of markers in limb buds polarization of the limb field (Te Welscher et al., 2002a), we lacking both Alx4 and Shh.InShh deficient limb buds, compared its expression in Alx4À/ÀShhÀ/À limb buds with expression of Fgf4 and Gremlin is normally activated around that in wildtype and either single mutant. As shown in Figs. E9, but is subsequently downregulated, and at E10.5 only low 2M–P, downregulation of dHand expression in Shh mutants levels of Gremlin and Fgf4 are visible in the posterior part of was indistinguishable in absence or presence of an intact Alx ShhÀ/À limb buds (Zuniga et al., 1999; Fig. 2C and ventral gene. view in Fig. 2CV). When expression of Fgf4 and Gremlin was simultaneously assayed in Alx4À/ÀShhÀ/À limb buds, using SHH-independent disruption of AP-patterning in Alx4À/À mixed probes, we found reduced expression of both genes limb buds posteriorly in the limb bud (Figs. 2D and DV). Strikingly, we did observe a small anterior domain of Fgf4 expression in To completely rule out methodological limitations as a Alx4/Shh mutant forelimb buds (yellow arrowhead in Fig. cause for the failure to detect ectopic hedgehog signaling in 2DV), and even more clearly in hindlimb buds (yellow Alx4 mutant limb buds prior to E11.5, we resorted to a genetic arrowhead in Fig. 2H), presumably linked to the higher S. Kuijper et al. / Developmental Biology 285 (2005) 533–544 537 penetrance of hindlimb polydactyly in Alx4 mutants (For- explained by some Fleakage_ during X-Gal staining, or sthoefel, 1963). differential sensitivity near the border of the expression In contrast to Fgf4 expression, Gremlin was not upregu- domain. The more proximal part of the Alx4 expression lated in the anterior part of Alx4À/ÀShhÀ/À fore and hindlimb domain, extending into the flank (compare Figs. 5A and I) buds at this stage (Figs. 2C and H). SHH-dependence of was, however, not part of the LacZ expression domain, Gremlin in this context would be surprising, as Gremlin is although the ventral body wall did express LacZ (Fig. 3L; normally activated prior to SHH signaling (Zuniga et al., compare Manley et al., 2001). 1999) and in particular because in Gli3 mutant – as well as in An unexpected and currently unexplained observation Gli3/Shh double mutant embryos – it is ectopically was that, in all five lines carrying construct 1, dorsal expressed (Te Welscher et al., 2002b). Although we cannot ectoderm also expressed h-galactosidase (black arrowhead exclude that ectopic Gremlin expression was merely too low in Figs. 3B and F). This expression was already visible at to be detected, it should be noted that ectopic expression of E9.5 and varied in strength, but was otherwise consistent. both Fgf4 and Hoxd13 in Alx4À/ÀShhÀ/À embryos was about Expression in ventral ectoderm was never seen. We have as clearly visible as the remainder of their posterior never observed Alx4 expression in dorsal limb bud expression (Figs. 2DVand L). ectoderm, and neither did several investigators who reported Normal posterior expression of the 5VHoxd genes is on embryonic Alx4 expression (Qu et al., 1997; Hudson et initiated but not maintained in Shh deficient limb buds al., 1998; Takahashi et al., 1998; Beverdam and Meijlink, (Chiang et al., 2001; Fig. 2K). At E10.5, a small domain of 2001). Hoxd13 expression is present in the anterior part of Alx4/ We unexpectedly observed expression in the nephric duct Shh deficient forelimb buds (arrowhead in Fig. 2L), (Figs. 3B, I, J and L) and ureteric bud in whole-mount h- showing that disturbed AP-patterning due to Alx4 defi- galactosidase stainings of E10.5 and E11.5 transgenic ciency leads to ectopic 5VHoxd expression irrespective of the embryos from all expressing lines. Although also not presence of SHH signaling. previously reported, in this case in situ hybridization on These results show that even though polydactyly in sections did confirm Alx4 expression in the nephric duct at Alx4À/À mice requires SHH signaling, initial disruption of E10.5. This is illustrated in Figs. 3M–P by direct AP-patterning in Alx4 deficient limb buds does not. comparison of sections of h-galactosidase expressing transgenic embryos with similar sections of wildtype embryos A 17-kb fragment upstream of Alx4 directs expression in hybridized with an Alx4 probe, showing expression in anterior limb bud mesenchyme nephric duct as well as mesonephric tubules. Furthermore, Fig. 3L shows that the genomic fragment The above results establish a role for Alx4 upstream of present in construct 1 does not direct h-galactosidase Fgf4, Gremlin, 5VHoxd and Shh in the anterior part of the expression into mesenchyme in the genital tubercle at early limb bud. To better understand this anterior restrictive E11.5, where endogenous Alx4 expression is normally cascade, and more in particular how Alx4 functions in it, we observed (Fig. 3K). Many genes that are expressed in limbs also wanted to investigate events upstream of Alx4 that are are also expressed in the genital tubercle (Dolle et al., responsible for its activation in anterior mesenchyme. 1991b; Bitgood and McMahon, 1995; Yamaguchi et al., To study transcriptional regulation of Alx4, we isolated 1999; Haraguchi et al., 2000), but apparently this expression from a mouse genomic PAC library a clone containing at is not necessarily mediated by the same regulatory elements. least 20 kb of upstream sequences flanking the Alx4 gene. A An important aspect of Alx4 is its expression and 17.4-kb DNA fragment that overlaps with the first 235 bp of function in the developing craniofacial primordia (Qu et exon 1 of Alx4 and contained about 17 kb of sequence al., 1997, 1999; Beverdam and Meijlink, 2001). In spite of upstream of the putative transcription start site of Alx4, was occasional patches of non-consistent expression in the head, then linked to the LacZ gene (construct 1; see Fig. 4A). Six there was no evidence that the 17-kb upstream region independent stable transgenic mouse lines bearing construct mediates craniofacial expression. 1 were generated and transgenic embryos from E9.5 to We generated several additional deletion constructs to E11.5 were examined for the expression of h-galactosidase. narrow down the region that could contain the regulatory Five lines showed consistent h-galactosidase expression, element(s) that mediate transcriptional control of Alx4. although some variation in staining was observed between Constructs 2 and 3 contain 6 and 0.385 kb, respectively, and different lines, presumably due to differences in integration have the same overlap with exon 1 (see Fig. 4A). Construct site and possibly the number of integrated copies. In all five 2 and even construct 3 contain sufficient information to lines, reporter gene expression was expressed in anterior direct Alx4 expression in the anterior part of the limb bud limb bud mesenchyme of the fore and hindlimb buds, (Figs. 3C, D, G and H). However, expression was much apparently reflecting the highly distinctive endogenous Alx4 weaker then obtained with construct 1, in particular activity expression domain (compare Figs. 3A and B with E and F). of construct 3 being barely detectable. In addition, the While h-galactosidase expression appeared to be somewhat frequency of expression among transgenic embryos of wider then endogenous Alx4 expression, this might be constructs 2 and 3 was less then 10%. 538 S. Kuijper et al. / Developmental Biology 285 (2005) 533–544

Fig. 3. Analysis of genomic sequences flanking the Alx4 gene using reporter gene expression in vivo and comparison to endogenous Alx4 expression. (A, E) Whole mount in situ hybridization with an Alx4 probe on wildtype embryo (A) and higher magnification of forelimb of the same embryo (E). (B, F) h- galactosidase expression in whole embryo (B) and forelimb (F) containing construct 1 at E10.5. h-galactosidase expression is comparable to Alx4 expression in limb bud mesenchyme and nephric duct (yellow arrowhead in panels A and B). Note the LacZ positive cells in the dorsal ectoderm of the limb where Alx4 is not expressed (black arrowhead in panels B and F). (C, G) The 6-kb upstream fragment present in construct 2 contains sufficient information to direct expression in the anterior part of the limb bud. (C) Whole embryo at E10.5; (G) higher magnification of the forelimb of the same embryo. (D, H) h- galactosidase expression of the 385-bp fragment (construct 3) in E10.5 embryo shows very weak staining in the anterior part of the limb (black arrow). (H) LacZ expression in the forelimb of the same embryo depicted in (D). (I) LacZ expression directed by 17-kb regulatory region is visible in the nephric duct at E10.5 (yellow arrowhead). (J) Higher magnification of the ureteric bud (blue arrowhead) and nephric duct (yellow arrowhead) of the same embryo depicted in panel I. Note that for clarity the body wall is partly removed. (K) Alx4 expression in the hindlimb region of an E11.5 wildtype embryo. Green arrowhead indicates Alx4 expression in the body wall, whereas yellow arrow points to Alx4 expression in the genital tubercle. (L) h-galactosidase expression directed by construct 1 extends into the body wall (green arrowhead), but is not detected in the genital tubercle at E11.5 (yellow arrow). Red arrow indicates the proximal anterior domain of the limb bud in which LacZ expression is missing, and yellow arrowhead indicates the expression in the nephric duct. (M, O) In situ hybridization using digoxygenin-labeled Alx4 probe on transversal sections of E10.5 wildtype embryo. Blue arrow indicates the nephric duct and green arrow indicates mesonephric tubules. (N, P) Transversal section of E10.5 embryo stained with neutral red to indicate h-galactosidase expression directed by 17-kb regulatory region in the nephric duct (blue arrow in panels N, P) and mesonephric tubules (green arrow in panel P).

Apparently, elements mediating the quintessential Alx4 lowest. Similarity in the chicken genome was significant, expression in anterior limb bud mesenchyme are present in but in frog and two fish species essentially undetectable. the 385-bp fragment of construct 3, but more upstream In addition to high conservation near the transcription sequences are indispensable for robust expression. start site and including the first exon, two large blocks of As a first step to further analyze the 17.5-kb region remarkably strong sequence conservation in all mamma- upstream from and overlapping with mouse Alx4,we lian genomes tested were found in the region between compared this sequence to nine other sequenced verte- À10 kb and À12 kb. Only one of these blocks was also brate genomes present in the ENSEMBL database, using detected in the chicken genome. In the region between a Multiz alignment analysis (Blanchette et al., 2004). The 0.4 and 6 kb upstream, several dispersed short areas of results are shown Fig. 4B. As expected, the highest high conservation were found with strongly related degree of similarity was found in the four mammalian species, but at most 2 areas were conserved in all species, the more distant opossum, a marsupial, being vertebrate genomes. S. Kuijper et al. / Developmental Biology 285 (2005) 533–544 539

Fig. 4. Several regions of sequence conservation are located in the 17.4-kb sequence 5Vupstream of the Alx4 gene. (A) Schematic representation of the reporter constructs used in this study. Constructs 1, 2 and 3 are generated from PAC clone RPCI21-447D21 (see Materials and methods). They contain genomic fragments that are located upstream from Alx4, including an overlap of 235 bp with exon 1. The restriction enzymes used to isolate the fragments are depicted. X, XhoI; Ea, EagI; K, KpnI; Bg, BglII. The first two exons are indicated (grey boxes), as are the translational start site (ATG) and presumptive promoter (black arrow). (B) 10-way vertebrate Multiz alignment and conservation scheme showing peaks of sequence similarity between 10 different vertebrates, using the mouse as a reference. The y-axis represents the percent identity; the x-axis represents the base sequences corresponding to genomic sequences containing 17-kb of upstream sequence directly 5Vof the Alx4 gene and the first exon of Alx4.

Upstream interactions of Alx4 with SHH and GLI3 similar to that in Gli3 single mutant limbs (Fig. 5D). Apparently, the expansion of Alx4 expression caused by To validate the expression in vivo of the reporter gene, it deficient SHH signaling requires the presence of Gli3. More is essential to establish that it responds to mutations in precisely, it requires the GLI3 repressor form (GLI3R), to interacting genes in the same way as the endogenous Alx4 which the transcription factor encoded by Gli3 is processed in gene. We therefore compared the impact of Shh and Gli3 the absence of SHH signaling (Wang et al., 2000). loss of function on endogenous Alx4 expression with that on We then analyzed Gli3À/À embryos carrying construct 1 h-galactosidase expression from construct 1. for h-galactosidase expression. Strikingly, expression was In ShhÀ/À limb buds, Alx4 expression was upregulated completely downregulated in these embryos (Fig. 5L). This and expanded (Fig. 5B), which is consistent with data of strongly suggests that it is the Gli3-regulated part of the Alx4 Takahashi et al. (1998) who showed that overexpression of expression domain that is mediated by the genomic sequen- SHH downregulates Alx4 in the chick limb. To compare this ces present in construct 1. Apparently, Gli3R prevents the result with the behavior of construct 1, we generated downregulation of Alx4 in the medial anterior part of the limb embryos that were homozygous for a null mutation in Shh bud, and expression is mediated by a regulatory element in and that were also transgenic for the reporter construct. the 17-kb fragment present in construct 1. The more proximal Expression of h-galactosidase in these E10.5 embryos was part of the Alx4 expression domain must be regulated in a expanded to the posterior mesenchyme in ShhÀ/À limb buds different, Gli3-independent way through regulatory ele- (compare Figs. 5E and I to F and J), demonstrating that the ment(s) outside the 17 kb of genomic DNA. unusual negative response of Alx4 expression to SHH signaling is mirrored by the 17-kb reporter construct. It has been shown previously that Alx4 expression is Discussion downregulated in Gli3 deficient limb buds, although the proximal part of the Alx4 domain is not affected. This implies Alx4 is a component of a signaling cascade that is active that proximal-anterior and medial-anterior limb expressions in the anterior margin of the limb bud and counteracts the of Alx4 are under different transcriptional control (Te expression of posterior genes. Disruption of this cascade Welscher et al., 2002a; see also Fig. 5C). In addition, Alx4 usually leads to anterior ectopic expression of Shh, which is expression in ShhÀ/ÀGli3À/À mutant limb buds was very the essential posterior marker of the limb bud, and to the 540 S. Kuijper et al. / Developmental Biology 285 (2005) 533–544

Fig. 5. Expression of endogenous Alx4 and of construct 1 is responsive to Gli3 and Shh. (A–D) Whole mount in situ hybridization with Alx4 probe on forelimb buds at E10.5. Expression of Alx4 in ShhÀ/À limb buds is spread throughout the limb bud (B), when compared to wildtype (A). (C) Medial anterior expression of Alx4 is downregulated in Gli3À/À limb buds, whereas the proximal anterior expression is still present. (D) In ShhÀ/ÀGli3À/À forelimb buds, proximal anterior expression is slightly more downregulated when compared to Gli3 single mutant limb buds (compare panels C and D). (E–H) Whole mount h-galactosidase stainings of E10.5 embryos transgenic for construct 1. (I–L) Higher magnifications of embryos in panels E–H showing details of expression in forelimb. Embryos shown in panels E, F, I and J are from a different line then the ones in panels G, H, K and L, explaining variation in number of LacZ positive cells in dorsal ectoderm. (E, I, G, K) Embryos wildtype for Shh and Gli3 are compared to ShhÀ/À embryos (F, J) and to Gli3À/À embryos (H, L), respectively. (I–L) Ventral view of E10.5 forelimb buds that show h-galactosidase expression directed by 17.4-kb regulatory region. The limb buds are shown from the ventral side to have a better view of the mesenchymal expression of LacZ. Yellow arrow (I, K) indicates the proximal anterior domain of the limb bud in which h-galactosidase expression is missing. (I) In wildtype forelimb buds, expression of h-galactosidase is restricted anteriorly, but this restriction is lost in ShhÀ/À (J) forelimb buds. (K, L) h-galactosidase expression is absent in Gli3À/À (L) forelimb buds when compared to wildtype expression (K). Yellow arrowhead indicates LacZ positive cells in the AER, which are presumably caused by integration site of the reporter construct in this line, because only transgenic embryos obtained from this line show LacZ expression in the AER. formation of extra fingers. In this study, we address both activated anteriorly in the limb bud. This leads to activation upstream and downstream interactions of Alx4 with other of Shh, which is essential to ultimately produce super- players in the pathways that regulate AP-patterning. These numerary digits. Polydactyly is presaged by an emerging interactions are highly diverse and context-dependent. For outgrowth of the limb bud at E11.5, when ectopic Shh instance, at relatively late stages, SHH signaling is required expression is barely detectable (Fig. 1D). It seems likely that for polydactyly to develop in Alx4 mutants, whereas at FGF-signaling and Hoxd gene expression are at this stage slightly earlier stages, it restricts normal Alx4 expression. responsible for this outgrowth. Similarly, in Gli3 mutants However, we show that initial disruption of AP-polarity in anterior extra digits form even in the absence of Shh, which Alx4À/À limb buds occurs independently of Shh expression. is accompanied by ectopic Hoxd and Fgf4 expression. The genetic relationship with Gli3 demonstrates a different Unfortunately, expression analysis of Alx4À/ÀShhÀ/À limb type of complexity, since, as we will discuss below, Gli3- buds beyond E10.5 is hampered because of their deterio- dependent and -independent expression appears to be linked rated state at such stages. to different functions in the limb bud. In wildtype limb buds, GREMLIN-mediated BMP-antagonism is essential to relay the signal of SHH to the posterior Role of SHH signaling in initial disruption of AP-patterning AER, which results in activation of FGF4 and outgrowth of in Alx4À/À limb buds the limb (Zuniga et al., 1999; Khokha et al., 2003; Michos et al., 2004). It seems likely that this mechanism is also In the absence of Alx4, posterior genes, including Hoxd responsible for the ectopic induction of Fgf4 in the anterior genes and components of the FGF4/SHH feedback loop are AER of polydactylous mutants. The absence of ectopic S. Kuijper et al. / Developmental Biology 285 (2005) 533–544 541

Gremlin expression in the same Alx4À/ÀShhÀ/À limb buds of Alx4 in its distal domain coincides consistently with the that did show an anterior domain of Fgf4 expression, may be presence of GLI3R. Conceivably, a direct repressor of Alx4 caused by failure to detect low levels but seems, as a transcription is negatively regulated by GLI3R, but the minimum to indicate highly different kinetics of induction of identity of this repressor remains presently elusive. Loebel Gremlin on one hand and Fgf4 and Hoxd13 on the other. et al. (2002) have proposed the functional importance of a number of conserved E-box sequences (CANNTG) scat- Regulation of Alx4 expression in the limb bud tered throughout the 17.4-kb upstream genomic region. E- boxes are recognized by bHLH transcription factors Full understanding of the position of Alx4 in the anterior (Massari and Murre, 2000), such as dHand and Twist. cascade in the limb bud necessitates insight in the Expression of Alx4 is shifted posteriorly in dHand mutants mechanisms underlying its anterior transcriptional activa- (Te Welscher et al., 2002a), and downregulated in Twist tion. Furthermore, an interesting aspect of Alx4 regulation is mutant forelimbs (Loebel et al., 2002). We were, however, its negative response to SHH signaling. Takahashi et al. unsuccessful in attempts to demonstrate interactions of (1998) have suggested that Alx4 expression is restricted by dHAND, TWIST or MyoD with Alx4 upstream sequences in SHH signaling in limb bud mesenchyme, but this was based cell transfection experiments (SK and L. Luong, unpub- on gain-of-function experiments. Likewise, Sun et al. (2002) lished data). attributed Alx4 expansion in limb buds of embryos lacking Our analysis of the transgenic mice led to the discovery both FGF4 and FGF8 to loss of SHH signaling. Here, we that Alx4 is expressed in the nephric duct and ureteric bud. show directly, using Shh mutants that SHH is necessary to A point of potential interest is that the highly similar restrict Alx4 expression anteriorly (Fig. 5B). aristaless-related gene Cart1 is also expressed in the The transgenic approach used in this study revealed that mesonephric duct (ten Berge et al., 1998). This opens the 17.4 kb of sequences flanking the Alx4 gene drives possibility that these highly redundant genes (Qu et al., expression of a reporter gene in a pattern in anterior limb 1999) function in kidney formation, which remains at this bud mesenchyme. Most likely, this pattern corresponds point to be investigated. exactly to the part of the Alx4 expression that is downregulated in Gli3 mutants. This was confirmed by the Dual function of Alx4 during limb development is reflected observation that in Gli3 deficient limb buds expression of by two separate enhancer regions the reporter gene was almost completely switched off. The analysis of smaller genomic fragments demonstrated that the We show that two subregions of the expression domain essential part of this 17-kb fragment was also present in the of Alx4 in the limb bud are differently regulated. In the 385-bp overlapping the promoter region, but expression of more distal part of its expression domain, in the medial this construct was very weak. Thus, sequence elements anterior part of the limb bud, control of Alx4 expression further upstream are necessary for the robust expression of is mediated by a regulatory region located in 17.4-kb Alx4, but do not necessarily contribute to the specificity of genomic sequence flanking the Alx4 gene, whereas the upstream region. Unfortunately, the weak expression regulatory elements located outside this sequence must and low frequency of expression obtained with constructs 2 regulate the proximal domain. Furthermore, we and others and 3 did not allow us to further test individual transcription (Te Welscher et al., 2002a; Fig. 5C) show that only distal factor binding sites. Alx4 expression, in the medial anterior part of the limb, is Our multialignment analysis of the region upstream under transcriptional regulation of Gli3, whereas the from Alx4, involving 10 vertebrate genomes, revealed proximal anterior domain is still present in Gli3À/À limb several stretches of strongly conserved sequences (Fig. buds. Additionally, expression analyses in Shh/Gli3 4B), presumably reflecting functional significance in double mutants show that only the distal Alx4 expression regulation of Alx4. The strikingly extensive blocks of domain is subject to negative regulation by SHH signaling high similarity in the À10 kb to À12 kb region are likely (Fig. 5D). to be responsible for the difference in robustness and These results confirm that Alx4, at least partially, reproducibility of transgene expression between con- functions genetically downstream of Gli3. Strikingly, recent structs. The difference between constructs 2 and 3 is results by Panman et al. (2005) reveal that polydactyly in characterized by a series of dispersed short stretches of Gli3 mutant mice depends on the function of Alx4: Gli3À/À high similarity, only one of which is recognized in the embryos have limbs with up to eight digits, whereas Gli3/ chicken promoter. Obviously, these data set the stage for Alx4 compound mutant limb buds have five to six digits future functional research that should eventually lead to (Panman et al., 2005). Since the distal expression domain of identification of transcription factors directly interacting Alx4 is lost in Gli3À/À limb buds, the only difference with Alx4. between Gli3À/À and Alx4/Gli3 double mutant limbs, as far We do not consider Gli3 to be a candidate for such direct as Alx4 expression is concerned, is the loss of the proximal regulation. Downregulation of Alx4 in Gli3 loss-of-function domain of Alx4 expression in the latter. This leads to the mutants must be indirect, because transcriptional activation paradoxical conclusion that proximal expression of Alx4 is 542 S. Kuijper et al. / Developmental Biology 285 (2005) 533–544

Fig. 6. Schematic representation of molecular changes and autopod phenotypes in Alx4, Gli3 single and Alx4/Gli3 double mutants. In wildtype limb buds, Gli3R is present in the anterior part of the limb (gray). Alx4 is normally expressed in two contiguous domains in the anterior part of the limb bud. The proximal expression domain (Alx4-P; dark green) occurs independently of Gli3, whereas the distal expression domain (Alx4-D; light green) requires Gli3 functioning. The distal domain, possibly in cooperation with the proximal domain of Alx4 corresponds to a function that restricts 5VHoxd (yellow), Fgf4 (red) and Shh (blue) posteriorly. In Alx4À/À limb buds, Fgf4 and 5VHoxd are ectopically expressed at E10.5. At later stages, an ectopic digit will form (n m 1: extra digit has not the morphology of digit 1, but has otherwise unknown identity). In Gli3 deficient limb buds, the distal, but not the proximal, expression domain of Alx4 is lost. Alx4-P, proximal domain of Alx4. 5VHoxd genes are expressed symmetrically posterior and anterior, while Fgf4 is expressed throughout the AER (Zuniga and Zeller, 1999; Te Welscher et al., 2002a,b). At later stages, Gli3À/À limbs contain several extra digits of undetermined identity (n). The only difference between Gli3 single and Alx4/Gli3 double mutant limb buds, as far as Alx4 expression is concerned, is the loss of proximal expression of Alx4 in Alx4/Gli3 mutants. Only one or two extra digits form in these mutants at later stages, suggesting that the proximal domain of Alx4 corresponds to a function that in the context of Gli3À/À background supports the formation of extra digits (Panman et al., 2005). Note that ectopic, anterior, expression of HoxD and FGF4 accompanies the generation of extra anterior digits. In contrast, loss of function of Alx4 or Gli3 (either genetically or by downregulation of their expression) leads to extra digits in their region of expression anteriorly in the limb bud. However, specific removal of the proximal part of the Alx4 domain (i.e., the difference between the Gli3 and Alx4/Gli3 double mutants) leads to a decrease in the number of anterior digits. essential for the polydactylous phenotype seen in Gli3À/À polydactyly have ectopic expression of Fgf4 and Hoxd13 in limb buds. common, although these genes are expressed more sym- Alx4 seems therefore to function in two disparate ways metrically in Gli3 deficient limb buds (Te Welscher et al., during autopod development: the proximal expression 2002b and Fig. 2). In Fig. 6, a schematic overview is domain corresponds to a function that, in the context of depicted of the molecular changes in the various mutant Gli3 deficiency, supports formation of extra digits. The limb buds and their autopod phenotypes that are described distal domain, or possibly the combination of both distal and in this report and in Panman et al. (2005). proximal domains, corresponds to a function to repress It is conceivable that the mechanism that links Gli3 Fgf4, Hoxd13, Gremlin and Shh in the posterior part of the deficiency to polydactyly includes downregulation of limb. In this case, it is loss of function that leads to extra Fdistal_ Alx4. This hypothesis predicts that Gli3-polydactyly digits, provided that an intact Shh allele is present. These will be suppressed by distal over-expression of Alx4, for dissimilar functions of Alx4 emphasize that it functions in a instance driven by the promoter fragment described in this highly context-dependent way. Along the same lines, we paper. recently showed that Alx4 and Gli3 exert opposing functions Finally, specific knockouts of the regulatory regions during shoulder girdle development (Kuijper et al., 2005). mediating proximal and distal Alx4 expression are required It should be noted that both types of polydactyly, in to fully understand their significance. which Alx4 has such contradictory roles are different in nature. Typical Gli3-polydactyly is characterized by sym- metry due to loss of identity, and is Shh-independent. Acknowledgments Typical Alx4 polydactyly is marked by mirror-image duplications and a posterior morphology of anterior super- We thank Rolf Zeller and Aime´e Zuniga for support, numerary digits (see Litingtung et al., 2002). Both types of discussions and comments on the manuscript. We are S. Kuijper et al. / Developmental Biology 285 (2005) 533–544 543 grateful to Lambert Luong for performing part of the Hogan, B.L.M., Beddington, R., Costantini, F., Lacy, E., 1994. Manipulat- experimental work and to Antje Brouwer and Carla Kroon ing the Mouse Embryo: A Laboratory Manual, Second edition. Cold Spring Harbor press, Cold Spring Harbor, NY. for technical assistance. We thank Edwin Cuppen for Hudson, R., Taniguchi-Sidle, A., Boras, K., Wiggan, O., Hamel, P.A., 1998. making the Multiz alignment plot. We also thank Jacqueline Alx-4, a transcriptional activator whose expression is restricted to sites Deschamps for reading the manuscript. SK and FM are of epithelial-mesenchymal interactions. Dev. Dyn. 213, 159–169. supported by the Netherlands Research Organization NWO Hui, C.C., Slusarski, D., Platt, K.A., Holmgren, R., Joyner, A.L., 1994. (grant ALW 810.68.013). Expression of three mouse homologs of the Drosophila segment polarity gene cubitus interruptus, Gli, Gli-2, and Gli-3, in ectoderm- and mesoderm-derived tissues suggests multiple roles during post- implantation development. Dev. Biol. 162, 402–413. References Ingham, P.W., McMahon, A.P., 2001. Hedgehog signaling in animal development: paradigms and principles. Genes Dev. 15, 3059–3087. Beverdam, A., Meijlink, F., 2001. Expression patterns of group-I aristaless- Izpisua-Belmonte, J.C., Tickle, C., Dolle, P., Wolpert, L., Duboule, D., related genes during craniofacial and limb development. Mech. Dev. 1991. Expression of the homeobox Hox-4 genes and the specification of 107, 163–167. position in chick wing development. Nature 350, 585–589. Beverdam, A., Brouwer, A., Reijnen, M., Korving, J., Meijlink, F., 2001. Johnson, R.L., Tabin, C.J., 1997. Molecular models for vertebrate limb Severe nasal clefting and abnormal embryonic apoptosis in Alx3/Alx4 development. Cell 90, 979–990. double mutant mice. Development 128, 3975–3986. Khokha, M.K., Hsu, D., Brunet, L.J., Dionne, M.S., Harland, R.M., 2003. Bitgood, M.J., McMahon, A.P., 1995. Hedgehog and Bmp genes are Gremlin is the BMP antagonist required for maintenance of Shh and Fgf coexpressed at many diverse sites of cell–cell interaction in the mouse signals during limb patterning. Nat. Genet. 34, 303–307. embryo. Dev. Biol. 172, 126–138. Kraus, P., Fraidenraich, D., Loomis, C.A., 2001. Some distal limb structures Blanchette, M., Kent, W.J., Riemer, C., Elnitski, L., Smit, A.F.A., Roskin, develop in mice lacking Sonic hedgehog signaling. Mech. Dev. 100, K.M., Baertsch, R., Rosenbloom, K., Clawson, H., Green, E.D., 45–58. Haussler, D., Miller, M., 2004. Aligning multiple genomic sequences Kuijper, S., Beverdam, A., Kroon, C., Brouwer, A., Candille, S., Barsh, G., with the threaded blockset aligner. Genome Res. 14, 708–715. Meijlink, F., 2005. Genetics of shoulder girdle formation: roles of Buscher, D., Ruther, U., 1998. Expression profile of Gli family members Tbx15 and aristaless-like genes. Development 132, 1601–1610. and Shh in normal and mutant mouse limb development. Dev. Dyn. Laufer, E., Nelson, C.E., Johnson, R.L., Morgan, B.A., Tabin, C., 1994. 211, 88–96. Sonic hedgehog and Fgf-4 act through a signaling cascade and feedback Buscher, D., Bosse, B., Heymer, J., Ruther, U., 1997. Evidence for genetic loop to integrate growth and patterning of the developing limb bud. Cell control of Sonic hedgehog by Gli3 in mouse limb development. Mech. 79, 993–1003. Dev. 62, 175–182. Leussink, B., Brouwer, A., el Khattabi, M., Poelmann, R.E., Gitten- Capdevila, J., Izpisua Belmonte, J.C., 2001. Patterning mechanisms berger-de Groot, A.C., Meijlink, F., 1995. Expression patterns of the controlling vertebrate limb development. Annu. Rev. Cell Dev. Biol. paired-related homeobox genes MHox/Prx1 and S8/Prx2 suggest 17, 87–132. roles in development of the heart and the forebrain. Mech. Dev. 52, Chan, D.C., Laufer, E., Tabin, C., Leder, P., 1995. Polydactylous limbs in 51–64. Strong’s luxoid mice result from ectopic polarizing activity. Develop- Litingtung, Y., Dahn, R.D., Li, Y., Fallon, J.F., Chiang, C., 2002. Shh and ment 121, 1971–1978. Gli3 are dispensable for limb skeleton formation but regulate digit Chiang, C., Litingtung, Y., Lee, E., Young, K.E., Corden, J.L., Westphal, number and identity. Nature 418, 979–983. H., Beachy, P.A., 1996. Cyclopia and defective axial patterning in mice Loebel, D.A., O’Rourke, M.P., Steiner, K.A., Banyer, J., Tam, P.P., 2002. lacking Sonic hedgehog gene function. Nature 383, 407–413. Isolation of differentially expressed genes from wild-type and Twist Chiang, C., Litingtung, Y., Harris, M.P., Simandl, B.K., Li, Y., Beachy, mutant mouse limb buds. Genesis 33, 103–113. P.A., Fallon, J.F., 2001. Manifestation of the limb prepattern: limb Manley, N.R., Barrow, J.R., Zhang, T., Capecchi, M.R., 2001. Hoxb2 and development in the absence of sonic hedgehog function. Dev. Biol. 236, hoxb4 act together to specify ventral body wall formation. Dev. Biol. 421–435. 237, 130–144. Dolle, P., Izpisua-Belmonte, J.C., Boncinelli, E., Duboule, D., 1991a. The Marigo, V., Johnson, R.L., Vortkamp, A., Tabin, C.J., 1996. Sonic Hox-4.8 gene is localized at the 5Vextremity of the Hox-4 complex and hedgehog differentially regulates expression of GLI and GLI3 during is expressed in the most posterior parts of the body during development. limb development. Dev. Biol. 180, 273–283. Mech. Dev. 36, 3–13. Martin, G.R., 1998. The roles of FGFs in the early development of Dolle, P., Izpisua-Belmonte, J.C., Brown, J.M., Tickle, C., Duboule, D., vertebrate limbs. Genes Dev. 12, 1571–1586. 1991b. HOX-4 genes and the morphogenesis of mammalian genitalia. Massari, M.E., Murre, C., 2000. Helix–loop–helix proteins: regulators of Genes Dev. 5, 1767. transcription in eucaryotic organisms. Mol. Cell. Biol. 20, 429–440. Forsthoefel, P.F., 1963. The embryological development of the effects of Masuya, H., Sagai, T., Wakana, S., Moriwaki, K., Shiroishi, T., 1995. A Strong’s luxoid in the mouse. J. Morphol. 113, 427–451. duplicated zone of polarizing activity in polydactylous mouse mutants. Goodrich, L.V., Johnson, R.L., Milenkovic, L., McMahon, J.A., Scott, Genes Dev. 9, 1645–1653. M.P., 1996. Conservation of the hedgehog/patched signaling pathway Masuya, H., Sagai, T., Moriwaki, K., Shiroishi, T., 1997. Multigenic control from flies to mice: induction of a mouse patched gene by Hedgehog. of the localization of the zone of polarizing activity in limb morpho- Genes Dev. 10, 301–312. genesis in the mouse. Dev. Biol. 182, 42–51. Haraguchi, R., Suzuki, K., Murakami, R., Sakai, M., Kamikawa, M., Mayor, C., Brudno, M., Schwartz, J.R., Poliakov, A., Rubin, E.M., Frazer, Kengaku, M., Sekine, K., Kawano, H., Kato, S., Ueno, N., Yamada, G., K.A., Pachter, L.S., Dubchak, I., 2000. VISTA: visualizing global 2000. Molecular analysis of external genitalia formation: the role of DNA sequence alignments of arbitrary length. Bioinformatics 16, fibroblast growth factor (Fgf) genes during genital tubercle formation. 1046–1047. Development 127, 2471–2479. Michos, O., Panman, L., Vintersten, K., Beier, K., Zeller, R., Zuniga, A., Hebert, J.M., Basilico, C., Goldfarb, M., Haub, O., Martin, G.R., 1990. 2004. Gremlin-mediated BMP antagonism induces the epithelial– Isolation of cDNAs encoding four mouse FGF family members and mesenchymal feedback signaling controlling metanephric kidney and characterization of their expression patterns during embryogenesis. Dev. limb organogenesis. Development 131, 3401–3410. Biol. 138, 454–463. Mo, R., Freer, A.M., Zinyk, D.L., Crackower, M.A., Michaud, J., Heng, 544 S. Kuijper et al. / Developmental Biology 285 (2005) 533–544

H.H., Chik, K.W., Shi, X.M., Tsui, L.C., Cheng, S.H., Joyner, A.L., ment of anteroposterior polarity during vertebrate limb development. Hui, C., 1997. Specific and redundant functions of Gli2 and Gli3 zinc Development 125, 4417–4425. finger genes in skeletal patterning and development. Development 124, Te Welscher, P., Fernandez-Teran, M., Ros, M.A., Zeller, R., 2002a. Mutual 113–123. genetic antagonism involving GLI3 and dHAND prepatterns the Niswander, L., 2003. Pattern formation: old models out on a limb. Nat. vertebrate limb bud mesenchyme prior to SHH signaling. Genes Dev. Rev., Genet. 4, 133–143. 16, 421–426. Niswander, L., Jeffrey, S., Martin, G.R., Tickle, C., 1994. A positive Te Welscher, P., Zuniga, A., Kuijper, S., Drenth, T., Goedemans, H.J., feedback loop coordinates growth and patterning in the vertebrate limb. Meijlink, F., Zeller, R., 2002b. Progression of vertebrate limb Nature 371, 609–612. development through SHH-mediated counteraction of GLI3. Science Osoegawa, K., Tateno, M., Woon, P.Y., Frengen, E., Mammoser, A.G., 298, 827–830. Catanese, J.J., Hayashizaki, Y., de Jong, P.J., 2000. Bacterial artificial ten Berge, D., Brouwer, A., el Bahi, S., Guenet, J.L., Robert, B., Meijlink, chromosome libraries for mouse sequencing and functional analysis. F., 1998. Mouse Alx3: an aristaless-like homeobox gene expressed Genome Res. 10, 116–128. during embryogenesis in ectomesenchyme and lateral plate mesoderm. Panman, L., Zeller, R., 2003. Patterning the limb before and after SHH Dev. Biol. 199, 11–25. signalling. J. Anat. 202, 3–12. Wang, B., Fallon, J.F., Beachy, P.A., 2000. Hedgehog-regulated processing Panman, L., Drenth, T., Te Welscher, P., Zuniga, A., Zeller, R., 2005. of Gli3 produces an anterior/posterior repressor gradient in the Genetic interaction of Gli3 and Alx4 during limb development. Int. J. developing vertebrate limb. Cell 100, 423–434. Dev. Biol. 49, 443–448. Yamaguchi, T.P., Bradley, A., McMahon, A.P., Jones, S., 1999. A Wnt5a Qu, S., Niswender, K.D., Ji, Q., van der, M.R., Keeney, D., Magnuson, pathway underlies outgrowth of multiple structures in the vertebrate M.A., Wisdom, R., 1997. Polydactyly and ectopic ZPA formation in embryo. Development 126, 1211–1223. Alx-4 mutant mice. Development 124, 3999–4008. Yang, Y., Drossopoulou, G., Chuang, P.T., Duprez, D., Marti, E., Bumcrot, Qu, S., Tucker, S.C., Ehrlich, J.S., Levorse, J.M., Flaherty, L.A., Wisdom, D., Vargesson, N., Clarke, J., Niswander, L., McMahon, A., Tickle, C., R., Vogt, T.F., 1998. Mutations in mouse aristaless-like4 cause Strong’s 1997. Relationship between dose, distance and time in Sonic Hedge- luxoid polydactyly. Development 125, 2711–2721. hog-mediated regulation of anteroposterior polarity in the chick limb. Qu, S., Tucker, S.C., Zhao, Q., deCrombrugghe, B., Wisdom, R., 1999. Development 124, 4393–4404. Physical and genetic interactions between Alx4 and Cart1. Develop- Zakany, J., Kmita, M., Duboule, D., 2004. A dual role for Hox genes in ment 126, 359–369. limb anterior–posterior asymmetry. Science 304, 1669–1672. Sun, X., Mariani, F.V., Martin, G.R., 2002. Functions of FGF signalling Zuniga, A., Zeller, R., 1999. Gli3 (Xt) and formin (ld) participate in the from the apical ectodermal ridge in limb development. Nature 418, positioning of the polarising region and control of posterior limb-bud 501–508. identity. Development 126, 13–21. Takahashi, M., Tamura, K., Buscher, D., Masuya, H., Yonei-Tamura, S., Zuniga, A., Haramis, A.P., McMahon, A.P., Zeller, R., 1999. Signal relay Matsumoto, K., Naitoh-Matsuo, M., Takeuchi, J., Ogura, K., Shiroishi, by BMP antagonism controls the SHH/FGF4 feedback loop in T., Ogura, T., Belmonte, J.C., 1998. The role of Alx-4 in the establish- vertebrate limb buds. Nature 401, 598–602.