Function and regulation of Alx4 in limb development: Complex genetic interactions with Gli3 and Shh

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 Alx4 / 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 Shh / background. Total lack of reporter expression in a Gli3 / 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 During development of the vertebrate limb, interacting signals from the apical ectodermal ridge (AER) and zone of polarizing activity (ZPA) direct its outgrowth and patterning. The AER is located at the distal edge of the limb bud and multiple fibroblast growth factors (FGFs), including Fgf4, Fgf8, Fgf9 and Fgf17, are expressed in the AER (reviewed in Martin, 1998). The ZPA is located in posterior mesenchyme of the limb bud and produces the signaling factor Sonic hedgehog (SHH), which is essential for subsequent antero-posterior (AP) patterning (reviewed in Capdevila and Izpisua Belmonte, 2001; Niswander, 2003). To assure proper outgrowth and patterning of the limb bud, a SHH/FGF4 feedback loop is established in the posterior limb bud. The Bone Morphogenetic Protein (BMP) antag-

* Corresponding author. Fax: +31 30 251 6464. E-mail address: [email protected] (F. Meijlink). 0012-1606/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2005.06.017

onist GREMLIN has been found to transduce the SHH signal to the AER, which results in activation of FGF4. FGF4, in turn, signals back to the ZPA to maintain SHH (Zuniga et al., 1999). Prior to Shh expression, the early limb bud is prepatterned by mutual genetic antagonism between the anteriorly expressed zinc-finger transcription factor GLI3 and the posteriorly expressed bHLH protein dHAND. This mechanism polarizes the nascent limb bud mesenchyme to establish the proper posterior localization of Shh (Te Welscher et al., 2002a; reviewed in Panman and Zeller, 2003). Furthermore, the 5VHoxd genes are activated prior to Shh expression and were found to be essential for establishment of AP-polarity of the developing limb bud as well (Zakany et al., 2004). 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 induces digit duplications (Yang et al., 1997; Johnson and Tabin, 1997), demonstrating that the correct localization of

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Shh in the posterior part of the limb bud is essential for limb outgrowth and AP-patterning. Analysis of preaxial-polydactylous mouse mutants showed that Shh transcription is negatively regulated by several genes that are expressed in the anterior mesenchyme of the limb bud. These include Gli3, which is mutated in the Extra toes (Xt) mouse mutant and the aristaless-related gene Alx4, which is linked to the mouse mutant Strong’s luxoid (Lst) (Masuya et al., 1995; Buscher et al., 1997; Mo et al., 1997; Qu et al., 1997; Takahashi et al., 1998). Polydactyly in Alx4 mutant mice is associated with, and dependent on anterior ectopic expression of Shh (Chan et al., 1995; Qu et al., 1997; Takahashi et al., 1998; Te Welscher et al., 2002b). In contrast, Gli3 / linked polydactyly occurs independently of SHH signaling (Te Welscher et al., 2002b; Litingtung et al., 2002). In addition, when SHH beads were inserted anteriorly in chick limb buds, Alx4 expression was rapidly downregulated, revealing a reciprocal negative interaction between Alx4 and Shh (Takahashi et al., 1998). Expression studies in mice have shown that a part of the Alx4 expression domain is downregulated in Gli3 deficient limb buds, whereas expression of Gli3 is normal in Alx4 / limb buds (Te Welscher et al., 2002a). To better understand the roles of Alx4 in limb patterning, we studied both its upstream and downstream genetic interactions. We asked whether induction of ectopic SHH signaling is the only molecular mechanism by which Alx4 deficiency disrupts AP-patterning in the limb bud, as early Alx4 expression in the presumptive limb region (Qu et al., 1997), as well as late emergence of ectopic Shh expression in Alx4 mutant embryos (Qu et al., 1998) suggested otherwise. We analyzed Alx4/Shh compound mutants and found that the initial disruption of antero-posterior patterning in Alx4 deficient limb buds occurs independently of Shh. In addition, a genomic fragment upstream of Alx4 directs expression of h-galactosidase in limb bud mesenchyme, mimicking the typical anterior pattern of Alx4. Authenticity of this pattern was corroborated by its expansion in a Shh mutant background. Expression of the reporter construct was completely turned off in a Gli3 / background, indicating that it corresponds specifically to the GLI3-dependent part of endogenous Alx4 expression. We suggest that the two components of the Alx4 expression pattern are linked to dissimilar functions.

P.A. Beachy (Chiang et al., 1996). Genotyping was done by PCR on ear cuts or yolk sac DNA. Alx4 embryos were genotypes as described by Beverdam et al. (2001), Gli3 and Shh mutants were genotyped as described by Te Welscher et al. (2002b). Reporter construct preparation and generation of transgenic mice Genomic clones from the RPCI-21 PAC library (Osoegawa et al., 2000), containing at least 20 kb of genomic DNA sequences upstream of the first exon of Alx4, were obtained from the Leiden Genome Technology Center (Leiden University Medical Center, Leiden, The Netherlands). Genomic fragments from PAC clone RPCI21-447D21 were subcloned in pBluescript, sequenced and then inserted into the HindIII site of the vector pSDKLacZpA. This plasmid (a generous gift of J. Rossant, Toronto) contains the E. coli LacZ gene linked to a translation initiation site which is adapted for eukaryotic expression. These genomic fragments included a 17.4-kb XhoI –EagI fragment to generate construct 1; a 6-kb KpnI– EagI fragment to generate construct 2 and a 385-bp BglII –EagI fragment to generate construct 3. Linearized constructs were gel-purified using Geneclean Spin Kit (BIO101, Carlsbad, CA, USA) prior to microinjection into the male pronucleus of fertilized mouse eggs following standard procedures (Hogan et al., 1994). Integration of the transgene was confirmed by PCR on ear cuts or yolk sac DNA using primers directed to LacZ (forward primer 5V-GCATCGAGCTGGGTAATAAGCGTTGGCAAT, reverse primer 5V-GACACCAGACCAACTGGTAATGGTAGCGAC). All animal experiments were conducted under the approval of the animal care committee of the K.N.A.W. (Netherlands Royal Academy of Arts and Sciences). In situ hybridization and b-galactosidase staining Whole mount in situ hybridization using digoxygeninlabeled RNA probes was performed as previously described (Leussink et al., 1995; ten Berge et al., 1998). Probes used included Alx4 (Beverdam and Meijlink, 2001), Hoxd11 (Izpisua-Belmonte et al., 1991), Hoxd13 (Dolle et al., 1991a), Fgf4 (Hebert et al., 1990), Gli1 (Hui et al., 1994), Ptch1 (Goodrich et al., 1996), Shh and 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 Alx4 (Lst J ) and Gli3 (Xt J ) mutant mice were obtained from The Jackson Laboratory (Bar Harbor, Maine, United States) and Shh mutant mice were originally obtained from

Comparative genomic analysis was carried out using VISTA (http://www-gsd.lbl.gov/vista; Mayor et al., 2000) and Multiz (http://genome.ucsc.edu; Blanchette et al., 2004).

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Results Late ectopic SHH signaling in Alx4 deficient limb buds Preaxial polydactyly in mutant chick or mice, including Lst and Xt mice, which are caused by loss-of-function mutations in Alx4 and Gli3, respectively, is frequently associated with ectopic anterior expression of Shh (Chan et al., 1995; Masuya et al., 1995, 1997; Buscher et al., 1997). Takahashi et al. (1998) reported that ectopic activation of Shh did not occur until E11.5. To verify this, we followed expression of Shh between E10.5 and E11.5 in Alx4 deficient limb buds and confirmed that ectopic Shh expression was not detected until E11.5 (Figs. 1A – D). Notably, at this stage, a prospective extra digit is already emerging (Fig. 1D). To rule out the possibility that SHH signaling is present ectopically at earlier stages, but not detectable by our in situ hybridization techniques, we analyzed the expression of Patched 1 (Ptch1) (Ingham and McMahon, 2001) and Gli1, which are early transcriptional targets of hedgehog signaling (Marigo et al., 1996; Buscher and Ruther, 1998) and are generally considered to be sensitive indicators of SHH signaling. Both Ptch1 and Gli1 were not detected ectopically in Alx4 deficient limb buds until E11.5 (Figs. 1E – H and I– L), consistent with absence of ectopic SHH signaling at earlier stages. Fgf4 is normally expressed in the posterior part of the AER, prior to Shh activation, and maintains Shh expression

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in the ZPA during limb development (Laufer et al., 1994; Niswander et al., 1994; Zuniga et al., 1999). In Alx4 / limb buds, an ectopic domain of Fgf4 expression is observed in the anterior part of the AER at E10.5, prior to ectopic Shh expression (yellow arrowhead in Fig. 2B). Moreover, Fgf4 expression was extended throughout the AER of Alx4 / hindlimb buds at this stage (Fig. 2F). The BMP antagonist GREMLIN is required for maintenance of SHH and FGF4 signaling during limb development (Khokha et al., 2003; Michos et al., 2004). It is normally expressed, prior to Shh activation, in the posterior part of the distal limb bud mesenchyme, close to Fgf4 expression in the AER (Fig. 2A; Zuniga et al., 1999). In Alx4 mutant limb buds, Gremlin is upregulated in the anterior part of the limb bud, concomitant with ectopic Fgf4 expression (black arrowhead in Figs. 2B and F). The Hoxd genes are activated in a colinear fashion in posterior mesenchyme of the normal limb bud. These overlapping expression patterns also emerge before Shh expression. Hoxd11 – Hoxd13 have been shown to play a role in limb outgrowth and digit patterning independently of Shh (Zakany et al., 2004). In Alx4 / fore and hindlimb buds, Hoxd11 and Hoxd13 are ectopically expressed at E10.5, again prior to ectopic Shh expression (data not shown; Fig. 2J; Takahashi et al., 1998). Therefore, even in the absence of detectable hedgehog signaling, three different markers of the posterior part of the limb bud were detected anteriorly in Alx4 / 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 Alx4 / (B) forelimb buds at E10.5. (C, D) Expression of Shh in wildtype (C) and Alx4 / (D) forelimb buds at E11.5. Note that the expression is normal at E10.5 in Alx4 / 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 Alx4 / (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 Alx4 / (J, L) limbs at E10.5 (I, J) and at E11.5 (K, L). Black arrowhead indicates ectopic Gli1 expression in Alx4 / limb buds at E11.5.

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Fig. 2. Ectopic expression of Fgf4 and Hoxd13 in Alx4 / mice occurs initially independent of SHH signaling. (A – D) Simultaneous expression of Fgf4 and Gremlin at E10.5 in wildtype (A), Alx4 / (B), Shh / (C, CV) and Alx4 / Shh / (D, DV) forelimb buds. Yellow arrowhead (B) indicates ectopic expression of Fgf4 in Alx4 / limb buds. Black arrowhead (B) indicates ectopic Gremlin expression in Alx4 / limb buds. (C, D) Dorsal view of Shh / (C) and Alx4 / Shh / (D) forelimb buds at E10.5. (CV, DV) Ventral view of Shh / (CV) and Alx4 / Shh / (DV) limb buds. Red arrowhead (CV, DV) indicates posterior Fgf4 expression. Yellow arrowhead (DV) indicates anterior ectopic spot of Fgf4 expression in Alx4 / Shh / limb buds. Note that Gremlin is not expressed ectopically in Alx4 / Shh / limb buds (D). (E – H) Expression of Fgf4 and Gremlin in wildtype (E), Alx4 / (F), Shh / (G) and Alx4 / Shh / (H) hindlimb buds at E10.5. Note the ectopic expression of Fgf4 in Alx4 / and Alx4 / Shh / hindlimb buds (yellow arrowhead). Black arrowhead in panel F indicates ectopic Gremlin expression in Alx4 / hindlimb buds. (I – L) Expression of Hoxd13 in wildtype (I), Alx4 / (J), Shh / (K) and Alx4 / Shh / (L) forelimb buds at E10.5 shows that Hoxd13 is ectopically expressed in Alx4 / and Alx4 / Shh / limb buds (yellow arrowhead in panels J and L). (M – P) Expression of dHand in wildtype (M), Alx4 / (N), Shh / (O), and Alx4 / Shh / (P) forelimb buds at E10.5.

In view of the known role of the dHand gene in early polarization of the limb field (Te Welscher et al., 2002a), we compared its expression in Alx4 / Shh / limb buds with that in wildtype and either single mutant. As shown in Figs. 2M –P, downregulation of dHand expression in Shh mutants was indistinguishable in absence or presence of an intact Alx gene. SHH-independent disruption of AP-patterning in Alx4 limb buds

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To completely rule out methodological limitations as a cause for the failure to detect ectopic hedgehog signaling in Alx4 mutant limb buds prior to E11.5, we resorted to a genetic

approach. We studied the expression of markers in limb buds lacking both Alx4 and Shh. In Shh deficient limb buds, expression of Fgf4 and Gremlin is normally activated around E9, but is subsequently downregulated, and at E10.5 only low levels of Gremlin and Fgf4 are visible in the posterior part of Shh / limb buds (Zuniga et al., 1999; Fig. 2C and ventral view in Fig. 2CV). When expression of Fgf4 and Gremlin was simultaneously assayed in Alx4 / Shh / limb buds, using mixed probes, we found reduced expression of both genes posteriorly in the limb bud (Figs. 2D and DV). Strikingly, we did observe a small anterior domain of Fgf4 expression in Alx4/Shh mutant forelimb buds (yellow arrowhead in Fig. 2DV), and even more clearly in hindlimb buds (yellow arrowhead in Fig. 2H), presumably linked to the higher

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penetrance of hindlimb polydactyly in Alx4 mutants (Forsthoefel, 1963). In contrast to Fgf4 expression, Gremlin was not upregulated in the anterior part of Alx4 / Shh / fore and hindlimb buds at this stage (Figs. 2C and H). SHH-dependence of Gremlin in this context would be surprising, as Gremlin is normally activated prior to SHH signaling (Zuniga et al., 1999) and in particular because in Gli3 mutant – as well as in Gli3/Shh double mutant embryos – it is ectopically expressed (Te Welscher et al., 2002b). Although we cannot exclude that ectopic Gremlin expression was merely too low to be detected, it should be noted that ectopic expression of both Fgf4 and Hoxd13 in Alx4 / Shh / embryos was about as clearly visible as the remainder of their posterior expression (Figs. 2DVand L). Normal posterior expression of the 5VHoxd genes is initiated but not maintained in Shh deficient limb buds (Chiang et al., 2001; Fig. 2K). At E10.5, a small domain of Hoxd13 expression is present in the anterior part of Alx4/ Shh deficient forelimb buds (arrowhead in Fig. 2L), showing that disturbed AP-patterning due to Alx4 deficiency leads to ectopic 5VHoxd expression irrespective of the presence of SHH signaling. These results show that even though polydactyly in Alx4 / mice requires SHH signaling, initial disruption of AP-patterning in Alx4 deficient limb buds does not. A 17-kb fragment upstream of Alx4 directs expression in anterior limb bud mesenchyme The above results establish a role for Alx4 upstream of Fgf4, Gremlin, 5VHoxd and Shh in the anterior part of the early limb bud. To better understand this anterior restrictive cascade, and more in particular how Alx4 functions in it, we also wanted to investigate events upstream of Alx4 that are responsible for its activation in anterior mesenchyme. To study transcriptional regulation of Alx4, we isolated from a mouse genomic PAC library a clone containing at least 20 kb of upstream sequences flanking the Alx4 gene. A 17.4-kb DNA fragment that overlaps with the first 235 bp of exon 1 of Alx4 and contained about 17 kb of sequence upstream of the putative transcription start site of Alx4, was then linked to the LacZ gene (construct 1; see Fig. 4A). Six independent stable transgenic mouse lines bearing construct 1 were generated and transgenic embryos from E9.5 to E11.5 were examined for the expression of h-galactosidase. Five lines showed consistent h-galactosidase expression, although some variation in staining was observed between different lines, presumably due to differences in integration site and possibly the number of integrated copies. In all five lines, reporter gene expression was expressed in anterior limb bud mesenchyme of the fore and hindlimb buds, apparently reflecting the highly distinctive endogenous Alx4 expression domain (compare Figs. 3A and B with E and F). While h-galactosidase expression appeared to be somewhat wider then endogenous Alx4 expression, this might be

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explained by some Fleakage_ during X-Gal staining, or differential sensitivity near the border of the expression domain. The more proximal part of the Alx4 expression domain, extending into the flank (compare Figs. 5A and I) was, however, not part of the LacZ expression domain, although the ventral body wall did express LacZ (Fig. 3L; compare Manley et al., 2001). An unexpected and currently unexplained observation was that, in all five lines carrying construct 1, dorsal ectoderm also expressed h-galactosidase (black arrowhead in Figs. 3B and F). This expression was already visible at E9.5 and varied in strength, but was otherwise consistent. Expression in ventral ectoderm was never seen. We have never observed Alx4 expression in dorsal limb bud ectoderm, and neither did several investigators who reported on embryonic Alx4 expression (Qu et al., 1997; Hudson et al., 1998; Takahashi et al., 1998; Beverdam and Meijlink, 2001). We unexpectedly observed expression in the nephric duct (Figs. 3B, I, J and L) and ureteric bud in whole-mount hgalactosidase stainings of E10.5 and E11.5 transgenic embryos from all expressing lines. Although also not previously reported, in this case in situ hybridization on sections did confirm Alx4 expression in the nephric duct at E10.5. This is illustrated in Figs. 3M – P by direct comparison of sections of h-galactosidase expressing transgenic embryos with similar sections of wildtype embryos hybridized with an Alx4 probe, showing expression in nephric duct as well as mesonephric tubules. Furthermore, Fig. 3L shows that the genomic fragment present in construct 1 does not direct h-galactosidase expression into mesenchyme in the genital tubercle at E11.5, where endogenous Alx4 expression is normally observed (Fig. 3K). Many genes that are expressed in limbs are also expressed in the genital tubercle (Dolle et al., 1991b; Bitgood and McMahon, 1995; Yamaguchi et al., 1999; Haraguchi et al., 2000), but apparently this expression is not necessarily mediated by the same regulatory elements. An important aspect of Alx4 is its expression and function in the developing craniofacial primordia (Qu et al., 1997, 1999; Beverdam and Meijlink, 2001). In spite of occasional patches of non-consistent expression in the head, there was no evidence that the 17-kb upstream region mediates craniofacial expression. We generated several additional deletion constructs to narrow down the region that could contain the regulatory element(s) that mediate transcriptional control of Alx4. Constructs 2 and 3 contain 6 and 0.385 kb, respectively, and have the same overlap with exon 1 (see Fig. 4A). Construct 2 and even construct 3 contain sufficient information to direct Alx4 expression in the anterior part of the limb bud (Figs. 3C, D, G and H). However, expression was much weaker then obtained with construct 1, in particular activity of construct 3 being barely detectable. In addition, the frequency of expression among transgenic embryos of constructs 2 and 3 was less then 10%.

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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) hgalactosidase 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) hgalactosidase 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 expression in anterior limb bud mesenchyme are present in the 385-bp fragment of construct 3, but more upstream sequences are indispensable for robust expression. As a first step to further analyze the 17.5-kb region upstream from and overlapping with mouse Alx4, we compared this sequence to nine other sequenced vertebrate genomes present in the ENSEMBL database, using a Multiz alignment analysis (Blanchette et al., 2004). The results are shown Fig. 4B. As expected, the highest degree of similarity was found in the four mammalian species, the more distant opossum, a marsupial, being

lowest. Similarity in the chicken genome was significant, but in frog and two fish species essentially undetectable. In addition to high conservation near the transcription start site and including the first exon, two large blocks of remarkably strong sequence conservation in all mammalian genomes tested were found in the region between 10 kb and 12 kb. Only one of these blocks was also detected in the chicken genome. In the region between 0.4 and 6 kb upstream, several dispersed short areas of high conservation were found with strongly related species, but at most 2 areas were conserved in all vertebrate genomes.

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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 To validate the expression in vivo of the reporter gene, it is essential to establish that it responds to mutations in interacting genes in the same way as the endogenous Alx4 gene. We therefore compared the impact of Shh and Gli3 loss of function on endogenous Alx4 expression with that on h-galactosidase expression from construct 1. In Shh / limb buds, Alx4 expression was upregulated and expanded (Fig. 5B), which is consistent with data of Takahashi et al. (1998) who showed that overexpression of SHH downregulates Alx4 in the chick limb. To compare this result with the behavior of construct 1, we generated embryos that were homozygous for a null mutation in Shh and that were also transgenic for the reporter construct. Expression of h-galactosidase in these E10.5 embryos was expanded to the posterior mesenchyme in Shh / limb buds (compare Figs. 5E and I to F and J), demonstrating that the 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 downregulated in Gli3 deficient limb buds, although the proximal part of the Alx4 domain is not affected. This implies that proximal-anterior and medial-anterior limb expressions of Alx4 are under different transcriptional control (Te Welscher et al., 2002a; see also Fig. 5C). In addition, Alx4 expression in Shh / Gli3 / mutant limb buds was very

similar to that in Gli3 single mutant limbs (Fig. 5D). Apparently, the expansion of Alx4 expression caused by deficient SHH signaling requires the presence of Gli3. More precisely, it requires the GLI3 repressor form (GLI3R), to which the transcription factor encoded by Gli3 is processed in the absence of SHH signaling (Wang et al., 2000). We then analyzed Gli3 / embryos carrying construct 1 for h-galactosidase expression. Strikingly, expression was completely downregulated in these embryos (Fig. 5L). This strongly suggests that it is the Gli3-regulated part of the Alx4 expression domain that is mediated by the genomic sequences present in construct 1. Apparently, Gli3R prevents the downregulation of Alx4 in the medial anterior part of the limb bud, and expression is mediated by a regulatory element in the 17-kb fragment present in construct 1. The more proximal part of the Alx4 expression domain must be regulated in a different, Gli3-independent way through regulatory element(s) outside the 17 kb of genomic DNA.

Discussion Alx4 is a component of a signaling cascade that is active in the anterior margin of the limb bud and counteracts the expression of posterior genes. Disruption of this cascade usually leads to anterior ectopic expression of Shh, which is the essential posterior marker of the limb bud, and to the

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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 Shh / limb buds is spread throughout the limb bud (B), when compared to wildtype (A). (C) Medial anterior expression of Alx4 is downregulated in Gli3 / limb buds, whereas the proximal anterior expression is still present. (D) In Shh / Gli3 / 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 Shh / embryos (F, J) and to Gli3 / 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 Shh / (J) forelimb buds. (K, L) h-galactosidase expression is absent in Gli3 / (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 upstream and downstream interactions of Alx4 with other players in the pathways that regulate AP-patterning. These interactions are highly diverse and context-dependent. For instance, at relatively late stages, SHH signaling is required for polydactyly to develop in Alx4 mutants, whereas at slightly earlier stages, it restricts normal Alx4 expression. However, we show that initial disruption of AP-polarity in Alx4 / limb buds occurs independently of Shh expression. The genetic relationship with Gli3 demonstrates a different type of complexity, since, as we will discuss below, Gli3dependent and -independent expression appears to be linked to different functions in the limb bud. Role of SHH signaling in initial disruption of AP-patterning in Alx4 / limb buds In the absence of Alx4, posterior genes, including Hoxd genes and components of the FGF4/SHH feedback loop are

activated anteriorly in the limb bud. This leads to activation of Shh, which is essential to ultimately produce supernumerary digits. Polydactyly is presaged by an emerging outgrowth of the limb bud at E11.5, when ectopic Shh expression is barely detectable (Fig. 1D). It seems likely that FGF-signaling and Hoxd gene expression are at this stage responsible for this outgrowth. Similarly, in Gli3 mutants anterior extra digits form even in the absence of Shh, which is accompanied by ectopic Hoxd and Fgf4 expression. Unfortunately, expression analysis of Alx4 / Shh / limb buds beyond E10.5 is hampered because of their deteriorated state at such stages. In wildtype limb buds, GREMLIN-mediated BMP-antagonism is essential to relay the signal of SHH to the posterior AER, which results in activation of FGF4 and outgrowth of the limb (Zuniga et al., 1999; Khokha et al., 2003; Michos et al., 2004). It seems likely that this mechanism is also responsible for the ectopic induction of Fgf4 in the anterior AER of polydactylous mutants. The absence of ectopic

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Gremlin expression in the same Alx4 / Shh / limb buds that did show an anterior domain of Fgf4 expression, may be caused by failure to detect low levels but seems, as a minimum to indicate highly different kinetics of induction of Gremlin on one hand and Fgf4 and Hoxd13 on the other. Regulation of Alx4 expression in the limb bud Full understanding of the position of Alx4 in the anterior cascade in the limb bud necessitates insight in the mechanisms underlying its anterior transcriptional activation. Furthermore, an interesting aspect of Alx4 regulation is its negative response to SHH signaling. Takahashi et al. (1998) have suggested that Alx4 expression is restricted by SHH signaling in limb bud mesenchyme, but this was based on gain-of-function experiments. Likewise, Sun et al. (2002) attributed Alx4 expansion in limb buds of embryos lacking both FGF4 and FGF8 to loss of SHH signaling. Here, we show directly, using Shh mutants that SHH is necessary to restrict Alx4 expression anteriorly (Fig. 5B). The transgenic approach used in this study revealed that 17.4 kb of sequences flanking the Alx4 gene drives expression of a reporter gene in a pattern in anterior limb bud mesenchyme. Most likely, this pattern corresponds exactly to the part of the Alx4 expression that is downregulated in Gli3 mutants. This was confirmed by the observation that in Gli3 deficient limb buds expression of the reporter gene was almost completely switched off. The analysis of smaller genomic fragments demonstrated that the essential part of this 17-kb fragment was also present in the 385-bp overlapping the promoter region, but expression of this construct was very weak. Thus, sequence elements further upstream are necessary for the robust expression of Alx4, but do not necessarily contribute to the specificity of the upstream region. Unfortunately, the weak expression and low frequency of expression obtained with constructs 2 and 3 did not allow us to further test individual transcription factor binding sites. Our multialignment analysis of the region upstream from Alx4, involving 10 vertebrate genomes, revealed several stretches of strongly conserved sequences (Fig. 4B), presumably reflecting functional significance in regulation of Alx4. The strikingly extensive blocks of high similarity in the 10 kb to 12 kb region are likely to be responsible for the difference in robustness and reproducibility of transgene expression between constructs. The difference between constructs 2 and 3 is characterized by a series of dispersed short stretches of high similarity, only one of which is recognized in the chicken promoter. Obviously, these data set the stage for future functional research that should eventually lead to identification of transcription factors directly interacting with Alx4. We do not consider Gli3 to be a candidate for such direct regulation. Downregulation of Alx4 in Gli3 loss-of-function mutants must be indirect, because transcriptional activation

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of Alx4 in its distal domain coincides consistently with the presence of GLI3R. Conceivably, a direct repressor of Alx4 transcription is negatively regulated by GLI3R, but the identity of this repressor remains presently elusive. Loebel et al. (2002) have proposed the functional importance of a number of conserved E-box sequences (CANNTG) scattered throughout the 17.4-kb upstream genomic region. Eboxes are recognized by bHLH transcription factors (Massari and Murre, 2000), such as dHand and Twist. Expression of Alx4 is shifted posteriorly in dHand mutants (Te Welscher et al., 2002a), and downregulated in Twist mutant forelimbs (Loebel et al., 2002). We were, however, unsuccessful in attempts to demonstrate interactions of dHAND, TWIST or MyoD with Alx4 upstream sequences in cell transfection experiments (SK and L. Luong, unpublished data). Our analysis of the transgenic mice led to the discovery that Alx4 is expressed in the nephric duct and ureteric bud. A point of potential interest is that the highly similar aristaless-related gene Cart1 is also expressed in the mesonephric duct (ten Berge et al., 1998). This opens the possibility that these highly redundant genes (Qu et al., 1999) function in kidney formation, which remains at this point to be investigated. Dual function of Alx4 during limb development is reflected by two separate enhancer regions We show that two subregions of the expression domain of Alx4 in the limb bud are differently regulated. In the more distal part of its expression domain, in the medial anterior part of the limb bud, control of Alx4 expression is mediated by a regulatory region located in 17.4-kb genomic sequence flanking the Alx4 gene, whereas regulatory elements located outside this sequence must regulate the proximal domain. Furthermore, we and others (Te Welscher et al., 2002a; Fig. 5C) show that only distal Alx4 expression, in the medial anterior part of the limb, is under transcriptional regulation of Gli3, whereas the proximal anterior domain is still present in Gli3 / limb buds. Additionally, expression analyses in Shh/Gli3 double mutants show that only the distal Alx4 expression domain is subject to negative regulation by SHH signaling (Fig. 5D). These results confirm that Alx4, at least partially, functions genetically downstream of Gli3. Strikingly, recent results by Panman et al. (2005) reveal that polydactyly in Gli3 mutant mice depends on the function of Alx4: Gli3 / embryos have limbs with up to eight digits, whereas Gli3/ Alx4 compound mutant limb buds have five to six digits (Panman et al., 2005). Since the distal expression domain of Alx4 is lost in Gli3 / limb buds, the only difference between Gli3 / and Alx4/Gli3 double mutant limbs, as far as Alx4 expression is concerned, is the loss of the proximal domain of Alx4 expression in the latter. This leads to the paradoxical conclusion that proximal expression of Alx4 is

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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 Alx4 / 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, Gli3 / 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 Gli3 / 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 Gli3 / limb buds. Alx4 seems therefore to function in two disparate ways during autopod development: the proximal expression domain corresponds to a function that, in the context of Gli3 deficiency, supports formation of extra digits. The distal domain, or possibly the combination of both distal and proximal domains, corresponds to a function to repress Fgf4, Hoxd13, Gremlin and Shh in the posterior part of the limb. In this case, it is loss of function that leads to extra digits, provided that an intact Shh allele is present. These dissimilar functions of Alx4 emphasize that it functions in a highly context-dependent way. Along the same lines, we recently showed that Alx4 and Gli3 exert opposing functions during shoulder girdle development (Kuijper et al., 2005). It should be noted that both types of polydactyly, in which Alx4 has such contradictory roles are different in nature. Typical Gli3-polydactyly is characterized by symmetry due to loss of identity, and is Shh-independent. Typical Alx4 polydactyly is marked by mirror-image duplications and a posterior morphology of anterior supernumerary digits (see Litingtung et al., 2002). Both types of

polydactyly have ectopic expression of Fgf4 and Hoxd13 in common, although these genes are expressed more symmetrically in Gli3 deficient limb buds (Te Welscher et al., 2002b and Fig. 2). In Fig. 6, a schematic overview is depicted of the molecular changes in the various mutant limb buds and their autopod phenotypes that are described in this report and in Panman et al. (2005). It is conceivable that the mechanism that links Gli3 deficiency to polydactyly includes downregulation of Fdistal_ Alx4. This hypothesis predicts that Gli3-polydactyly will be suppressed by distal over-expression of Alx4, for instance driven by the promoter fragment described in this paper. Finally, specific knockouts of the regulatory regions mediating proximal and distal Alx4 expression are required to fully understand their significance.

Acknowledgments We thank Rolf Zeller and Aime´e Zuniga for support, discussions and comments on the manuscript. We are

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grateful to Lambert Luong for performing part of the experimental work and to Antje Brouwer and Carla Kroon for technical assistance. We thank Edwin Cuppen for making the Multiz alignment plot. We also thank Jacqueline Deschamps for reading the manuscript. SK and FM are supported by the Netherlands Research Organization NWO (grant ALW 810.68.013).

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