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 0012-1606/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2005.06.017 534 S. Kuijper et al. / Developmental Biology 285 (2005) 533–544 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-galactosi- dase 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).