DEVELOPMENT. Lia Panman 1,2, *,, Antonella Galli 1, *, Nadege Lagarde 1, Odysse Michos 1, Gwen Soete 2,, Aimee Zuniga 1, and Rolf Zeller 1,

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1 RESEARCH ARTICLE 3419 Development 133, (2006) doi: /dev Differential regulation of gene expression in the digit forming area of the mouse limb bud by SHH and gremlin 1/FGF-mediated epithelial-mesenchymal signalling Lia Panman 1,2, *,, Antonella Galli 1, *, Nadege Lagarde 1, Odysse Michos 1, Gwen Soete 2,, Aimee Zuniga 1, and Rolf Zeller 1, Spatially and temporally coordinated changes in gene expression are crucial to orderly progression of embryogenesis. We combine mouse genetics with experimental manipulation of signalling to analyze the kinetics by which the SHH morphogen and the BMP antagonist gremlin 1 (GREM1) control gene expression in the digit-forming mesenchyme of mouse limb buds. Although most mesenchymal cells respond rapidly to SHH signalling, the transcriptional upregulation of specific SHH target signals in the mesenchyme occurs with differential temporal kinetics and in a spatially restricted fashion. In particular, the expression of the BMP antagonist Grem1 is always upregulated in mesenchymal cells located distal to the SHH source and acts upstream of FGF signalling by the apical ectodermal ridge. GREM1/FGF-mediated feedback signalling is, in turn, required to propagate SHH and establish the presumptive digit expression domains of the Notch ligand jagged 1 (Jag1) and 5 Hoxd genes in the distal limb bud mesenchyme. Their establishment is significantly delayed in Grem1-deficient limb buds and cannot be rescued by specific restoration of SHH signalling in mutant limb buds. This shows that GREM1/FGF feedback signalling is required for regulation of the temporal kinetics of the mesenchymal response to SHH signalling. Finally, inhibition of SHH signal transduction at distinct time points reveals the differential temporal dependence of Grem1, Jag1 and 5 Hoxd gene expression on SHH signalling. In particular, the expression of Hoxd13 depends on SHH signal transduction significantly longer than does Hoxd11 expression, revealing that the reverse co-linear establishment, but not maintenance of their presumptive digit expression domains, depends on SHH signalling. KEY WORDS: BMP antagonist, Cyclopamine, Feedback signalling, FGF, Gremlin1, Hox gene expression, Limb development, Sonic hedgehog, SU5402 INTRODUCTION In vertebrate embryos, the expression of morphoregulatory genes is highly dynamic and their expression levels and spatial distributions change during progression of embryo- and organogenesis. In particular, the temporally and spatially coordinated expression of members of the four Hox gene clusters regulates various embryonic patterning processes, including limb bud morphogenesis. The socalled co-linear expression of 5 Hoxd and 5 Hoxa genes is crucial to correct limb skeletal patterning, as alterations of their expression kinetics causes dysmorphic phenotypes (reviewed by Kmita and Duboule, 2003; Zeller and Deschamps, 2002). In particular, the expression of 5 Hoxd genes can be divided in two phases (reviewed by Deschamps, 2004): their posteriorly nested early expression domains in the limb bud mesenchyme (phase I) are established prior to activation of morphogenetic signalling by sonic hedgehog (SHH) (Chiang et al., 2001; Kraus et al., 2001). In fact, Hoxd and Hoxa genes have been implicated in Shh activation in the posterior limb bud mesenchyme (Kmita et al., 2005; Zakany et al., 2004). However, 1 Developmental Genetics, DKBW Centre for Biomedicine, University of Basel Medical School, Mattenstrasse 28, CH-4058 Basel, Switzerland. 2 Department of Developmental Biology, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands. *These authors contributed equally to this work Present address: Ludwig Institute for Cancer Research, Department of Cell and Molecular Biology, Karolinska Institute, Box 240, S Stockholm, Sweden Present address: NIOB/KNAW Hubrecht Laboratorium, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands Joint senior authors for correspondence ( aimee.zuniga@unibas.ch; rolf.zeller@unibas.ch) Accepted 5 July 2006 subsequent upregulation and distoanterior expansion of 5 Hoxd gene expression depends on SHH signalling and results in establishment of their presumptive digit expression domains in the distal limb bud mesenchyme (late domains/phase II). Spitz et al. (Spitz et al., 2003) have identified the large cis-regulatory landscape that regulates their expression in the digit-forming area, while the relevant transacting signals and factors regulating their dynamic expression remained largely unknown. Furthermore, extensive lossand gain-of-function analysis in the mouse has established that their expression in the presumptive digit domains is indeed essential for patterning of the distal limb skeleton and specification of digit identities (Dollé et al., 1993; Zakany et al., 1997). Shh is expressed by the polarizing region (or ZPA) (Riddle et al., 1993) and mainly specifies identities along the anteroposterior limb bud axis. In limb buds lacking Shh [e.g. mouse (Chiang et al., 1996)] distal development is disrupted and posterior identities are lost such that only one zeugopodal element and one digit form. Furthermore, anterior grafts of SHH-producing cells induce mirror-image digit duplications, in agreement with the proposal that the distal limb buds is patterned by long-range morphogenetic signalling (Riddle et al., 1993; Yang et al., 1997). However, genetic marking of Shhexpressing cells and their descendants has revealed that the ulna and digits 3 to 5 derive largely from descendants of cells that previously expressed Shh. In addition, the study by Harfe et al. (Harfe et al., 2004) revealed that an expansion-based temporal gradient of exposure to SHH probably specifies digits 3 to 5. In particular, the posterior mesenchymal cells that express Shh for the longest time period were shown to give rise to digit 5. Interestingly, this and an earlier study (Lewis et al., 2001) led to the conclusion that only specification of digit 2 would require long-range SHH signalling.

2 3420 RESEARCH ARTICLE Development 133 (17) Taken together, anteroposterior identities in the limb bud mesenchyme seem to be largely specified by a kinetic memory that integrates response to both autocrine and paracrine SHH signalling (Harfe et al., 2004). In addition, the cellular responsiveness to SHH signalling is modulated locally as the cells exposed to the highest SHH levels reduce their sensitivity to SHH over time (Ahn and Joyner, 2004). All these studies emphasize the importance of identifying the molecular circuits that regulate the temporal and spatial kinetics of gene expression during progression of vertebrate limb bud development (reviewed by Zeller, 2004). During limb bud morphogenesis, upregulation and maintenance of Shh expression depends on the secreted BMP antagonist gremlin 1 (GREM1), which promotes epithelial-mesenchymal (EM) feedback signalling by the apical ectodermal ridge [AER, expressing several FGF genes (Sun et al., 2002)]. GREM1 acts in the posteriordistal limb bud mesenchyme downstream of the initial, GLImediated cellular response to SHH signalling (Zuniga et al., 1999). Experimental and genetic evidence indicates that the SHH/GREM1/FGF feedback loop upregulates and maintains the expression of Shh, Grem1 and 5 Hoxd genes in the posterodistal mesenchyme and of FGF genes in the AER (Haramis et al., 1995; Laufer et al., 1994; Niswander et al., 1994). Analysis of mouse embryos lacking Grem1 showed that GREM1-mediated BMP antagonism in the mesenchyme is essential to induce and/or upregulate the expression of FGF and BMP genes in the overlying AER (Khokha et al., 2003; Michos et al., 2004). As a consequence, the upregulation and maintenance of SHH signalling are disrupted, which is indicative of the failure to establish EM feedback signalling (Haramis et al., 1995; Michos et al., 2004). These molecular alterations result in the characteristic ld phenotype, which includes loss of posterior digit identities and fusion (ulna with radius) or loss (fibula) of the posterior zeugopodal element. By contrast, single and compound mutant mouse embryos lacking FGF genes in the AER of their limb buds did not display phenotypes, as would have been expected from disrupting EM feedback signalling (see Lewandoski et al., 2000; Sun et al., 2000; Sun et al., 2002). These studies left some doubts with respect to the requirements of FGF signalling from the AER for regulation of gene expression in the distal limb bud mesenchyme and specification of digit identities. In the present study, we analyse the interactions of SHH, GREM1 and FGFs in the distal limb bud mesenchyme by combining analysis loss-of-function mutations in the mouse with manipulation of mouse limb buds in culture. First, we establish that SHH-dependent transcriptional upregulation of antagonists and signals such as Grem1, Bmp2 and Jag1 are controlled by localised and differential mesenchymal responsiveness to SHH signalling. The BMP antagonist Grem1 is an early transcriptional target of SHH signalling in the posterior limb bud mesenchyme, while the Notch ligand Jag1 is identified as a relatively late SHH target in the posterodistal mesenchyme. Grafts of SHH-producing cells into Shh / mouse limb buds reveals that the spatially restricted competence to express a particular target signal is an inherent, SHH-independent property of the mesenchyme. Second, grafts of FGF producing cells into Grem1 deficient limb buds restore the expression of Shh and Hoxd13. In addition, blocking FGF signal transduction with SU5402 in wildtype limb buds results alters gene expression in a similar manner as is observed in Grem1 / limb buds. These results establish that GREM1-mediated BMP antagonism acts via FGF signalling to propagate gene expression in the distal limb bud mesenchyme. Third, SHH grafts are unable to restore Jag1 and Hoxd13 expression in the distal limb bud mesenchyme of Grem1-deficient embryos. These results indicate that GREM1 is part of a timing mechanism that regulates expression kinetics in response to SHH signalling. Finally, to analyse the temporal requirement of SHH signalling, we block SHH signal transduction from specific time points onwards by treating mouse limb buds with cyclopamine. Rather unexpectedly, these studies reveal the differential and limited dependence of the expression of particular genes on SHH signalling. Upregulation of Grem1 expression requires SHH signalling, while its anterior expansion in the distal mesenchyme appears to be SHH independent. Jag1 expression depends on SHH only transiently during transiently and its expression in the presumptive digit area is largely SHH independent. Rather unexpectedly, only the establishment but not maintenance of the Hoxd11 expression domain in the distal limb bud mesenchyme requires SHH signalling. By contrast, establishment of the presumptive digit expression domain of Hoxd13 requires SHH signal transduction for much longer than Hoxd11. These studies show that the differential temporal dependence of 5 Hoxd genes on SHH signalling correlates well with the reverse co-linear establishment of their late expression domains (phase II) (reviewed by Deschamps, 2004). MATERIALS AND METHODS Mouse strains and embryos Heterozygous mice were intercrossed to obtain homozygous embryos for analysis. Noon of the day of vaginal plug detection was considered as day 0.5 (E0.5). Wild-type and mutant embryos were age matched according to their somite numbers (variation of ±1 somite). Shh / embryos were genotyped by PCR as described (St-Jacques et al., 1998). Mice homozygous for the Grem1-ld In2 mutation (Grem1 In2 ) were intercrossed to generate embryos lacking Grem1 expression specifically in limb buds (Zuniga et al., 2004). RNA in situ hybridization Whole-mount in situ hybridization using digoxigenin-labelled antisense riboprobes was performed as described by Haramis et al. (Haramis et al., 1995). Three or more independent embryos were analysed per stage and genotype, and yielded comparable results. In vitro grafting and culturing of mouse limb buds (trunk cultures) Mouse forelimb buds were grafted and cultured as described (Michos et al., 2004; Zuniga et al., 1999) with the following modifications. Trunks with forelimb buds attached were isolated from wild-type, Grem1 ln2/in2 and Shh / mutant embryos from embryonic day E10.25 after counting their somites (32-34 somites). After isolation, spherical aggregates of cell beads expressing the desired signalling molecule were grafted in forelimb buds. SHH signalling was blocked by supplementing the culture medium with 10 M cyclopamine (final, dissolved in ethanol) and FGF signal transduction was blocked with 10 M SU5402 (final, dissolved in DMSO). Controls were treated with 0.16% ethanol (final) or 0.03% DMSO (final), which is equal to the solvent content in the respective experimental samples. Trunks were cultured between 3 and 32 hours in serum-free, high-glucose DMEM medium (GIBCO-Invitrogen), supplemented with penicillin/streptomycin, L-glutamine, non-essential amino acids, sodium pyruvate, D-glucose, L- ascorbic acid, lactic acid, d-biotin, vitamin B12 and PABA in 6.5% CO 2 at 37 C. When culturing for 32 hours, the medium was exchanged after hours. Following culturing, samples were rinsed in PBS and fixed overnight with 4% PFA at 4 C. Each result shown is representative of minimally three independent embryos per genotype and type of manipulation, the analysis of which yielded identical results (in many studies significantly more embryos per result were analysed). QT6 fibroblast cells expressing Shh, Grem1, Fgf4 or Fgf9 (full-length coding sequences cloned into the prc/cmv vector, Invitrogen) under control of the CMV promoter were generated by standard calcium phosphate transfection. About 24 hours after transfection, cells were plated at high density on bacterial plates, which results in formation of spherical cell aggregates. The following day, cell aggregates were treated with mitomycin- C to completely block their proliferation (Zuniga et al., 1999) and washed extensively before grafting into forelimb buds.

3 SHH/GREM1-mediated gene regulation in limbs RESEARCH ARTICLE 3421 RESULTS The kinetics of SHH-mediated transcriptional regulation in limb bud mesenchymal cells SHH signal transduction is required for positive transcriptional regulation of a variety of mesenchymal signals in the limb bud mesenchyme (data not shown). We had previously identified the BMP antagonist GREM1 as a signal activated prior to SHH, but its continued expression in the distal limb bud mesenchyme becomes rapidly dependent on SHH signalling (Zuniga et al., 1999). Like Grem1, the expression of signals such as Bmp2 and Jag1 is dependent on SHH signalling (Laufer et al., 1994; McGlinn et al., 2005). To gain insight into the temporal and spatial kinetics by which SHH regulates the expression of mesenchymal signals, SHHproducing cell aggregates were grafted into the forelimb bud mesenchyme of Shh-deficient and wild-type embryos (E10.25, somites, Fig. 1). In response to SHH, an anterior ectopic ring of Gli1 expression is induced within 6 hours in wild-type forelimb buds (blue arrowheads, Fig. 1A). These results establish that the initial transcriptional response to SHH signalling is rapid and comparable with endogenous Gli1 expression levels (open arrowheads, Fig. 1A). Localized and strong ectopic Grem1 expression is detected within 9 hours in the mesenchyme distal to the graft (blue arrowhead, Fig. 1B). Ectopic Jag1 expression is also detected within 9 hours distally to the graft (blue arrowhead, Fig. 1C), but levels are much lower than the endogenous transcripts (big open arrowheads, Fig. 1C). After 15 hours the levels of ectopic Jag1 expression in the distal-anterior limb bud mesenchyme have increased significantly (blue arrowhead, Fig. 1D) and continue to rise (Fig. 4A and data not shown). These results reveal that mesenchymal cells respond to SHH signalling by differential and localized transcriptional upregulation of secondary signals. Indeed, Grem1 is expressed locally by the dorsal and ventral mesenchyme, while Jag1 (like 5 Hoxd genes) is expressed throughout the distal limb bud mesenchyme (see Fig. S1 in the supplementary material). To analyse the responsiveness of nascent mesenchyme, SHHexpressing cells were grafted into the posterior limb bud mesenchyme of Shh-deficient limb buds. As a fraction of Shh / mouse embryos die prematurely, a series of pilot experiments established that culturing mutant limb buds for 15 hours allows reliable and reproducible assessment of gene expression levels (Fig. 1E-H; data not shown). As in wild-type limb buds (Fig. 1A), the general response to SHH signalling is revealed by upregulation of Gli1 expression around the graft (Fig. 1E). Despite this widespread initial response, the transcriptional upregulation of the BMP antagonist Grem1 and the Notch ligand Jag1 is always restricted to cells located distal to the SHH graft (blue arrowheads, Fig. 1F,G). By contrast, expression of Bmp2 is mostly upregulated in cells located proximally to the graft (blue arrowhead, Fig. 1H) and the AER (broken arrowhead, Fig. 1H). Restoration of SHH signalling in Shh / limb buds shows that the differential and spatial restricted competence to activate these SHH target signals is maintained in Shh / limb buds. Restoration of FGF signalling is sufficient to rescue the distal expression of Jag1 and 5 Hoxd genes in Grem1-deficient limb buds Our previous genetic analysis indicated that Grem1 is required to establish EM feedback signalling and to propagate Shh expression in the distal mesenchyme (Michos et al., 2004; Zuniga et al., 1999). Likewise, the expression of Jag1 in distal limb bud core mesenchyme depends on GREM1-mediated EM feedback Fig. 1. Analysis of the spatial competence to activate and the temporal kinetics to upregulate the expression of SHH target signals in the limb bud mesenchyme. Left panels: contralateral control limb buds (not grafted). Right panels: contralateral, wild-type (A-D) or Shh / mutant (E-H) forelimb buds received posterior grafts of SHH-producing cell aggregates at E10.25 (31-33 somites). The red circles indicate the positions and approximate sizes of the grafts after culturing. White arrowheads indicate the endogenous gene expression domains. Blue arrowheads indicate the induced gene expression in response to a SHH graft. Limb buds are oriented with anterior towards the top. (A-D) Analysis of the temporal kinetics of the differential transcriptional response to SHH signalling in wild-type forelimb buds. (A) A ring of mesenchymal cells responds to SHH signalling by activating expression of the downstream target Gli1 within 6 hours of grafting. (B) Within 9 hours, Grem1 expression is expanded distoanteriorly in response to SHH signalling. (C,D) Jag1 expression is activated in the distal anterior mesenchyme after 9 hours (C), but upregulation of its expression in the distoanterior mesenchyme requires at least 15 hours (D). The small white arrowheads indicate localized endogenous Jag1 expression in the anterior limb bud mesenchyme. (E-H) Shh-deficient limb buds were cultured for 15 hours after receiving a graft of SHH-producing cells to detect strong expression of the gene of interest in all cases. Asterisks indicate the posteroproximal margins of the limb buds after culture. (E) SHH signalling induces transcriptional response in a large area of mesenchymal cells surrounding the graft as assessed by Gli1 expression. (F) Grem1 expression is upregulated in mesenchymal cells that are always located distally to the SHH graft. (G) Jag1 expression is activated in mesenchymal cells located also always distally to the SHH graft. The weak and speckled AER staining corresponds to crossreactivity that appears in a fraction of cultured limb buds. (H) A posterior SHH graft upregulates Bmp2 expression in mesenchymal cells located proximal to the graft (blue arrowhead). Upregulation of Bmp2 expression can be seen in the AER (striped arrowhead).

4 3422 RESEARCH ARTICLE Development 133 (17) signalling (see Fig. S1 in the supplementary material). However, the analysis of Htu homozygous mouse embryos [carrying a point mutation in the Jag1 gene (Kiernan et al., 2001)] shows that JAG1 itself is does not regulate the expression of 5 Hoxd and other morphoregulatory genes in the developing limb bud. Rather, this Notch ligand seems to regulate aspects of cell proliferation in the limb bud mesenchyme (L.P. and R.Z., unpublished). For the purpose of the present study, Jag1 is used as an additional marker expressed within the digit-forming territory of the distal limb bud mesenchyme (see Fig. S1 in the supplementary material). To gain insight into the temporal and spatial kinetics by which GREM1-mediated EM feedback signalling regulates gene expression in the distal limb bud mesenchyme, GREM1 and FGF producing cell aggregates were grafted into the posterior limb bud mesenchyme of Gre1 In2/In2 embryos (E10.25, somites). These experiments establish that Shh expression in the posterior mesenchyme is restored (red arrowhead, Fig. 2A) and Fgf4 expression in the AER activated within 9 hours (red arrowhead, Fig. 2B). In limb buds lacking Grem1, grafts of FGF4 and FGF9 expressing cells restore Shh expression with identical kinetics (Fig. 2C; data not shown). This initial restoration of EM feedback signalling (Fig. 2A,B) is followed by upregulation of Jag1 expression in the distal mesenchyme within 15 hours (between graft and AER, Fig. 2D). The expression of 5 Hoxd genes in the distal mesenchyme is restored within 15 hours (Hoxd13, red arrowhead, Fig. 2E; Hoxd11 and Hoxd12: data not shown). Furthermore, grafts of FGF4 producing cells into Grem1 deficient limb buds restore Jag1 and Hoxd13 expression with identical kinetics (Fig. 2F,G). As FGFs are able to propagate mesenchymal gene expression in the absence of Grem1 (Fig. 2), these results provide good evidence that FGF signalling is required downstream of GREM1. This is an important finding as the requirement of FGF signalling for patterning of the distal limb bud mesenchyme has been a matter of some debate (see Introduction). To investigate the requirement of FGF signal transduction further, limb buds (E10.5, somites) were treated with the inhibitor SU5402 in culture (Montero et al., 2001). Culturing wild-type limb buds in the presence of 10 M SU5402 blocks FGF signal transduction (Fig. 3A-D). Such treatment interferes with transcriptional upregulation of Jag1 and partly inhibits anterior expansion of its expression (Fig. 3A,B). Similarly, upregulation and anterior expansion of Hoxd13 expression, i.e. establishment of its late, presumptive digit expression domain is disrupted by treatment with SU5402 (Fig. 3C,D). By contrast, Grem1 expression is not significantly altered by blocking FGF signal transduction (Zuniga et al., 2004). In summary, treatment of limb buds with SU5402 results in similar alterations of the Jag1 and Hoxd13 expression domains, as observed in Grem1- deficient limb buds at E11.0 (42 somites, Fig. 3E,F; compare 3E with 3A,B and 3F with 3C,D). These results further support the proposal that GREM1 acts up-stream of and via FGF signalling during limb bud morphogenesis. Fig. 2. Restoration of GREM1/FGF mediated EM feedback signalling in Grem1 In2/In2 limb buds rapidly rescues expression of Shh, Jag1 and Hoxd13 in the distal mesenchyme. Cell aggregates producing either gremlin 1 (GREM1, green circles) or FGF4 (FGF4, blue circles) were grafted into the posterior mesenchyme of Grem1 In2/In2 forelimb buds (E10.25, somites). In these studies, the grafts were placed more distally and further away from the posterior AER than in previous experiments in order to avoid disruption of the posterior mesenchyme competent to express Shh. Circles indicate the approximate size and positions of the grafts after culturing, coloured arrowheads indicate induced/ upregulated gene expression. White arrowheads indicate endogenous gene expression in contralateral control limb buds. (A) GREM1 rescues Shh expression (red arrowhead) within 9 hours, indicative of restored EM feedback signalling. (B) GREM1 induces Fgf4 expression (arrowhead) in the posterior AER within 9 hours. (C) Grafts of FGF4-expressing cells in turn upregulate the expression of Shh within 9 hours (red arrowhead). (D,E) GREM1 upregulates the expression of the SHH targets Jag1 (D; blue arrowhead) and Hoxd13 (E; pink arrowhead) in the distal mesenchyme within 15 hours. (F,G) FGF4 also induces the upregulation of Jag1 (F; blue arrowhead) and Hoxd13 expression (G; pink arrowhead) in the distal mesenchyme of Grem1-deficient limb buds within 15 hours. GREM1 is required to regulate the temporal kinetics of Jag1 and Hoxd13 expression in the distal limb bud mesenchyme To further dissect the functional relevance of GREM1-mediated EM feedback signalling with respect to SHH-dependent regulation of gene expression, wild-type and Grem1 In2/In2 limb buds (E10.25, somites) received anterior grafts of SHH-producing cells (Fig. 4) to clearly discriminate induced (blue arrowheads) from endogenous gene expression (open arrowheads). In wild-type limb buds, ectopic SHH signalling induces significant anterior expansion and upregulation of Jag1 and Hoxd13 gene expression in the distal mesenchyme within 15 to 22 hours (Fig. 4A,C; see also Fig. 1D and data not shown). By contrast, no or only little transcriptional upregulation of ectopic Jag1 and Hoxd13 transcripts is observed in the distal mesenchyme of Grem1-deficient limb buds after 22 hours (blue arrowheads, Fig. 4B,D; left panels). In fact, the ectopic Jag1 expression levels in Grem1-deficient limb buds after 22 hours are reproducibly lower than the ones in wild-type limb buds after 9 hours (compare Fig. 1C with Fig. 4B). Only after 32 hours, the levels of ectopic anterior Jag1 and Hoxd13 transcripts (blue arrowheads, Fig. 4B,D; right panels) become comparable with ones in wild-type limb buds (blue arrowheads, Fig. 4A,C; right panels). This indicates an at least 12 hours delay in efficient transcriptional response to SHH signalling in Grem1-deficient limb buds. This delay is probably due to the fact that grafts of SHH-producing cells into Grem1-deficient limb buds do not restore FGF gene expression by the mutant AER and thereby fail to restore EM feedback signalling

5 SHH/GREM1-mediated gene regulation in limbs RESEARCH ARTICLE 3423 (Michos et al., 2004; Zuniga et al., 1999). These results establish that GREM1-mediated EM feedback signalling regulates aspects of the temporal kinetics of the transcriptional response to SHH signalling. Fig. 4. GREM1 is required to time the mesenchymal response to SHH signalling. Forelimb buds of E10.25 (31-33 somites) embryos received anterior grafts of SHH cell aggregates (red circles indicate approximate positions and sizes of grafts after culture) and the temporal kinetics of inducing/expanding the distal expression domains of Jag1 and Hoxd13 were assessed. Anterior grafts were used to clearly distinguish endogenous (white arrowheads) from exogenous SHH induced gene expression (blue arrowheads). All whole-mount in situ results were developed for the same amount of time to clearly reveal the weak ectopic Jag1 and Hoxd13 expression in Grem1In2/In2 limb buds after 22 hours. (A) Wild-type limb buds. SHH induces ectopic Jag1 expression (blue arrowhead) within 22 hours. (B) Grem1-deficient limb buds. Left panel: only low levels of SHH induced ectopic Jag1 expression (blue arrowhead) are detected after 22 hours. Right panel: significant ectopic Jag1 expression (blue arrowhead) is detected only after 32 hours. (C) Wild-type limb buds. SHH induces significant anterior expansion of the Hoxd13 expression domain (blue arrowhead) within 22 hours. (D) Grem1-deficient limb buds. Left panel: only low levels of ectopic Hoxd13 expression (blue arrowhead) are detected after 22 hours. Right panel: significant levels of SHH induced anterior ectopic Hoxd13 expression (blue arrowhead) are detected after 32 hours. Differential dependence of Grem1, Jag1 and 5 Hoxd gene expression on SHH signalling in the distal limb bud mesenchyme To analyse the requirement of SHH signalling during EM feedback signalling and establishment of the presumptive digit expression domains of 5 Hoxd genes, SHH signal transduction was blocked at specific time points by culturing limb buds in the presence of the inhibitor cyclopamine (Chen et al., 2002). This approach was chosen as the genes of interest are either not expressed or downregulated in limb buds of Shh-deficient embryos much prior to establishing GREM1-mediated feedback signalling (Jag1, 5 Hoxd genes) (Chiang et al., 2001; Kraus et al., 2001). The presence of 10 M cyclopamine in the culture medium significantly reduces Gli1 expression within 9 hours (Fig. 5A), indicative of blocking SHH signal transduction (E10.5, somites). After 15 hours of cyclopamine treatment, Gli1 is no longer detectable by whole-mount in situ hybridization, while cellular apoptosis is not yet significantly increased (Fig. 5A; data not shown). Therefore, embryonic trunks were cultured for 15 hours in the presence of cyclopamine prior to analysis (Fig. 5B,C and Figs 6, 7). Cyclopamine treatment induces loss of Fgf4 expression from the AER, indicating that Fgf4 requires sustained SHH signalling (Fig. 5B). By contrast, the expression of Fgf8 is only slightly reduced in comparison with wild-type controls (Fig. 5C). During limb bud morphogenesis, Grem1 expression is activated independently of SHH signalling in the posterior limb bud and its expression is upregulated and expands progressively from posterior to anterior within the distal limb bud mesenchyme under control of SHH signalling (Zuniga et al., 1999). Cyclopamine treatment from E10.25 (31-33 somites) onwards does not alter the anterior limit of the Grem1 expression domain (Figs 6A,B; broken lines indicate the approximate anterior limits), while expression is lost from the distal mesenchyme (brackets in Cyc panel in Fig. 6A,B). This failure to upregulate Grem1 expression in the distal-most mesenchyme is the likely cause of the loss of Fgf4 expression from the overlying AER (Fig. 5B). Furthermore, concurrent inhibition of both SHH and FGF signal transduction neither alters the anterior boundary of the Grem1 expression domain nor decreases expression levels further (data not shown). In light of this rather unexpected maintenance of the anterior expression boundary, Grem1 expression was re-assessed in Fig. 3. Blocking FGF signal transduction with SU5402 (10 M final) in wild-type limb buds (E10.5, somites) phenocopies aspects of the disruption of Jag1 and Hoxd13 expression in Grem1-deficient limb buds. (A-D) Asterisks indicate the anterior margins when necessary. (A) Jag1 expression in a wild-type forelimb bud cultured for 16 hours. Left panel: dorsal view with posterior towards the bottom and distal towards the left. Right panel: view onto the distal part of the same limb bud to reveal the anterior expansion of Jag1 expression at this stage. (B) Jag1 distribution in a wild-type forelimb bud cultured with SU5402 for 16 hours. Left panel: top view onto the distal limb to allow direct comparison with the control. Right panel: dorsal view. (C) Hoxd13 expression in a wild-type forelimb bud cultured for 16 hours. Left panel: dorsal view. Right panel: top view. (D) Hoxd13 distribution in a wild-type forelimb bud cultured with SU5402 for 16 hours. Left panel: top view of the distal limb to reveal the downregulation of Hoxd13 expression and lack of anterior expansion in the distal mesenchyme. Right panel: dorsal view. (E,F) Jag1 (E) and Hoxd13 expression (F) in wild-type and Grem1 deficient (Grem1In2/In2) forelimb buds at E11.0 (42 somites, not cultured), which corresponds to a stage similar to the limb buds shown in A-D.

6 3424 RESEARCH ARTICLE Development 133 (17) Shh deficient limb buds (Fig. 6C). As previously reported, Grem1 expression is rapidly downregulated in Shh-deficient limb buds (Fig. 6C, compare panel Shh / with wild type) (Zuniga et al., 1999). Although overall expression levels are low, Grem1 transcripts are reproducibly detected in the distoanterior mesenchyme of Shhdeficient limb buds at E9.75 (Fig. 6C, anterior limit indicated by broken line). By E10.5 (34-36 somites), Grem1 transcript levels are further reduced, but expression remains throughout in the distalmost mesenchyme in Shh-deficient limb buds (Fig. 6C). These results show that anterior expansion of the Grem1 expression domain occurs in the absence of SHH, while SHH signalling is required to upregulate Grem1 expression in the distal limb bud mesenchyme. In contrast to Grem1, transcriptional activation of Jag1 requires SHH, as it is not expressed in Shh / limb buds (Fig. 1G). Indeed, the initial upregulation of Jag1 expression is affected by blocking SHH signal transduction from E10.25 onwards (Fig. 6D). However, subsequent propagation of Jag1 expression does not depend on SHH signal transduction, as cyclopamine treatment from E10.5 onwards neither interferes with transcriptional upregulation nor with anterior expansion of the Jag1 expression domain in the distal limb bud mesenchyme (Fig. 6E,F). Finally, the effects of cyclopamine treatment on the establishment of the presumptive digit expression domains of 5 Hoxd genes were assessed (Fig. 7). In contrast to Grem1 and Jag1, SHH signalling is continuously required for establishment of the late, presumptive digit expression domains of 5 Hoxd genes. Cyclopamine-treatment from E10.25 onwards interferes with transcriptional upregulation and Fig. 6. Anterior expansion of Grem1 expression in the distal mesenchyme and propagation of Jag1 expression do not require continuous SHH signal transduction. T0: non-cultured control forelimb bud at the stage indicated. Wt: wild-type control forelimb bud cultured for 15 hours. Cyc: wild-type forelimb bud cultured for 15 hours in the presence of 10 M cyclopamine. E10.25, somites; E10.5, somites; E10.75, somites. All limb buds are oriented with anterior towards the top and posterior towards the bottom. Blue broken lines indicate the approximate anterior limits of Grem1 expression when necessary in A-C. (A) Inhibition of SHH signal transduction at E10.25 does not significantly alter Grem1 expression. (B) Inhibition of SHH signal transduction at E10.5 reduces Grem1 levels in the distal mesenchyme (indicated by bracket), but does not significantly alter its anterior expression limit. (C) Grem1 expression in Shh / forelimb buds is rapidly downregulated, but is clearly detectable in the distoanterior mesenchyme at E9.75 (28 somites) and at E10.25 (32 somites). (D-F) Effects of cyclopamine treatment on Jag1 expression. (D) Inhibition of SHH signal transduction at E10.25 interferes to some extent with upregulation of Jag1 expression in the posterior limb bud mesenchyme. (E) Inhibition of SHH signal transduction at E10.5 does not significantly alter the onset of anterior expansion of Jag1 expression in the distal mesenchyme, albeit expression levels remain slightly lower. (F) Similarly, cyclopamine treatment at E10.75 does not interfere with continued anterior expansion of Jag1 expression in the distal limb bud mesenchyme. Fig. 5. The kinetics of blocking SHH signal transduction in cultured wild-type forelimb buds (E10.5, somites) using 10 M cyclopamine. (A) Loss of Gli1 transcription within 9 to 15 hours. T0: Gli1 expression in a non-cultured control limb bud. Wt: Gli1 expression in a wild-type limb bud cultured for 15 hours as a control. Cyc (9 hours): Gli1 expression in a wild-type limb bud cultured for 9 hours in the presence of cyclopamine. Cyc (15 hours): Gli1 expression in a wild-type limb bud cultured for 15 hours in the presence of cyclopamine. Complete loss of Gli1 expression occurs. Therefore, all subsequent experiments were carried out by culturing limb bud as in the presence of 10 M cyclopamine for 15 hours. (B) Fgf4 expression in the AER is lost by culturing limb buds in the presence of cyclopamine for 15 hours (Cyc panel). (C) By contrast, Fgf8 remains expressed in the AER of cyclopamine treated limb buds (Cyc panel). All limb buds are oriented with anterior towards the top and posterior towards the bottom.

7 SHH/GREM1-mediated gene regulation in limbs RESEARCH ARTICLE 3425 Fig. 7. Differential temporal requirement of SHH signal transduction for the establishment of the presumptive digit expression domains of 5 Hoxd genes. T 0 : non-cultured control forelimb bud at the stage indicated. Wt: wild-type control forelimb bud cultured for 15 hours. Cyc: wild-type forelimb bud cultured for 15 hours in the presence of 10 M cyclopamine. E10.25, somites; E10.5, somites; E10.75, somites. All limb buds are oriented with anterior towards the top and posterior towards the bottom. (A-C) Effects on Hoxd11 expression. (A) Cyclopamine treatment starting at E10.25 interferes with upregulation and distoanterior expansion of Hoxd11 expression. (B) By E10.5, the late Hoxd11 expression domain has been established and inhibition of SHH signal transduction no longer alters the spatial distribution, while Hoxd11 expression levels are still affected. (C) Inhibition of SHH signal transduction from E10.75 onwards no longer alters Hoxd11 expression. (D-F) Effects on Hoxd12 expression. (D) Cyclopamine treatment starting at E10.25 significantly interferes with upregulation and expansion of Hoxd12 expression. (E) Similarly, inhibition of SHH signal transduction at E10.5 blocks the ongoing distoanterior expansion of Hoxd12 expression (compare Cyc with Wt). (F) From E10.75 onwards, cyclopamine treatment no longer affects Hoxd12 expression significantly. (G,H) Effects on Hoxd13 expression. (G) At E10.5, the establishment of the distal Hoxd13 expression domain has been initiated (compare T 0 with Wt). Cyclopamine treatment blocks the onset of anterior expansion of the Hoxd13 expression domain in the distal mesenchyme and upregulation of its expression (Cyc panel). (H) Cyclopamine treatment at E10.75 still efficiently blocks the ongoing anterior expansion of the Hoxd13 expression domain and upregulation of its expression levels. anterior expansion of the late expression domains of 5 most Hoxd genes (Fig. 7A,D; data not shown). By E10.5, the late Hoxd11 domain has been established and maintenance of its spatial distribution no longer requires SHH, while expression levels are lower in cyclopamine-treated animals than in wild-type controls (Fig. 7B). Blocking SHH signal transduction from E10.75 onwards (37-39 somites) no longer affects Hoxd11 expression, despite the fact that its presumptive digit expression domain continues to enlarge (Fig. 7C, compare T 0 panel with wild type and Cyc panels). These results reveal that Hoxd11 expression depends on SHH until about E10.5, while subsequent maintenance and propagation of its presumptive digit expression domain occurs independently of SHH signal transduction. The expression of Hoxd12 depends slightly longer on SHH as treatment at both E10.25 (Fig. 7D) and E10.5 efficiently blocks its transcriptional upregulation and establishment of the late expression domain (compare Fig. 7E with 7B). Only around E10.75, is the establishment of the late Hoxd12 expression domain largely independent of SHH signalling. By contrast, Hoxd13 expression, the presumptive digit domain of which is established last and extends most anterior (Dollé et al., 1993), requires SHH signal transduction for longer (i.e. beyond E10.75, Fig. 7G,H). The results shown in Fig. 7 reveal the graded dependence of 5 Hoxd genes on SHH signal transduction. This differential temporal dependence correlates well with the kinetics by which their presumptive digit expression domains are established. In particular, the expression of the 5 most Hoxd13 gene (Fig. 7G,H) depends significantly longer on SHH signal transduction than do the ones of the more 3 located Hoxd12 and Hoxd11 genes (Fig. 7A-F), in agreement with reverse co-linear establishment of their presumptive digit expression domains. DISCUSSION Activation and dynamic regulation of gene expression in the posterior-distal limb bud mesenchyme We have analysed the temporal and spatial requirements of the SHH, GREM1 and FGF feedback signalling interactions for upregulation and propagation of their own expression, and for establishment of the presumptive digit expression domains of 5 Hoxd genes and Jag1. In contrast to the primary mesenchymal response to SHH signalling, secondary signals are activated and/or upregulated in a spatially restricted fashion. Our study shows that this spatially restricted competence is an inherent property of the limb bud mesenchyme,

8 3426 RESEARCH ARTICLE Development 133 (17) which is retained in Shh-deficient limb buds. This spatially restricted competence may be established by the mutual antagonistic interaction of GLI3 with HAND2, which pre-patterns the mouse limb bud mesenchyme prior to SHH signalling (te Welscher et al., 2002a). GLI3-mediated restriction of Hand2 expression to the posterior limb bud mesenchyme seems to regulate the activation of Shh, Grem1 and, possibly, 5 Hoxd genes (te Welscher et al., 2002a; Charite et al., 2000; Yelon et al., 2000; Zuniga et al., 1999; Zuniga and Zeller, 1999). In addition, the transcriptional regulators Hoxd13 (Chen et al., 2004), Tbx3 (Rallis et al., 2005) and Twist1 (Firulli et al., 2005) interact with GLI and Hand2, respectively. In addition, genetic studies have implicated retinoic acid, 5 Hoxa and 5 Hoxd genes in activation of Shh expression in the posterior limb bud mesenchyme (Kmita et al., 2005; Mic et al., 2004; Niederreither et al., 2002). Subsequently, SHH signalling is required to upregulate and propagate mesenchymal gene expression in the distal mesenchyme as the expression of many genes is rapidly downregulated and/or lost in Shh-deficient limb buds (Chiang et al., 2001; Kraus et al., 2001; Litingtung et al., 2002; te Welscher et al., 2002b; Zuniga et al., 1999). Our study establishes that SHH is required for transcriptional upregulation but not distoanterior expansion of Grem1 expression during limb bud patterning. Therefore, the SHH-independent anterior expansion of Grem1 expression in early limb buds could be regulated by the pre-patterning mechanism acting upstream of SHH signalling (see before). patterning of the anteroposterior limb axis (Ahn and Joyner, 2004; Harfe et al., 2004). In the present study, we uncover the temporal requirement of SHH signal transduction for establishment of the late, presumptive digit expression domains of 5 Hoxd genes. Their co-linear expression is reversed in the distal limb bud mesenchyme, such that expression of the 5 most gene, Hoxd13 extends most anterior and is essential for digit patterning (Dollé et al., 1993). Genetic manipulation of the Hoxd complex in mice has revealed the existence of a large global control region (GCR) that regulates the establishment of their late expression domains in the limb bud mesenchyme (Spitz et al., 2003). A distant digit I Setup phase: Pre-patterning Gli3R dhand II SHH/GREM1/FGF feedback signalling GREM1 acts via FGF-mediated EM signalling to regulate the temporal kinetics of gene expression in response to SHH signalling In Grem1-deficient limb buds, the propagation of Shh expression is disrupted and there is a significant temporal delay in establishing the 5 Hoxd digit expression domains (Haramis et al., 1995; Michos et al., 2004). This delay cannot be rescued by grafts of SHH-expressing cells into the posterior limb bud mesenchyme (this study) and probably results in mesenchymal cells not receiving their positional identities at the correct time. This achronism provides a plausible explanation for the observed loss of posterior identities in Grem1-deficient limbs (Michos et al., 2004). In contrast to SHH, grafts of FGF-producing cells upregulate mesenchymal gene expression in Grem1-deficient limb buds with temporal kinetics comparable with GREM1-producing cells. Furthermore, inhibition of FGF signal transduction in wild-type limb buds phenocopies aspects of the molecular alterations observed in Grem1-deficient limb buds. These results provide good evidence that GREM1-mediated BMP antagonism regulates the mesenchymal response to SHH signalling indirectly via FGF signalling from the AER. Hence, our studies support an essential role of GREM1/FGFmediated EM feedback signalling in regulation of the temporal kinetics of gene expression and patterning. As GREM1 is required to induce Fgf4, Fgf9 and Fgf17 expression in the posterior AER and to upregulate Fgf8 expression (Michos et al., 2004) (L.P. and R.Z., unpublished), the overall strength of FGF signalling by the AER may be most relevant to FGF-mediated EM feedback signalling. Indeed, transgene-mediated overexpression of Fgf4 in the AER of mouse limb buds lacking Fgf8 completely restores their development, which indicates that FGF4 functionally replace FGF8 in the mutant AER (Lu et al., 2006). SHH differentially regulates the expression of the 5 Hoxd genes in the digit forming area of the limb bud The importance of the temporal control of gene expression is emphasized by the fact that an expansion-based temporal gradient of SHH and modulation of SHH responsiveness over time control III Independence of the feedback loop SHH Shh Descendents GREM1 FGFs 5'HoxD Fig. 8. The signalling interactions that control dynamic regulation of gene expression in the distal, digit-forming area of the limb bud. The scheme depicts three distinct phases of limb bud morphogenesis in a simplified manner (for details see text). Limb buds are drawn schematically with anterior towards the top and posterior towards the bottom. Only genes relevant to the present study are indicated. Phase I: setting up the signalling centres and differential mesenchymal responsiveness. The expression of various key regulator genes including Shh (dark blue), Grem1 (orange) and 5 Hoxd genes (green) in the mesenchyme and FGF genes in the AER (light blue) is activated locally and independent of SHH signalling. The interaction of GLI3R with HAND2 and other transcription factors pre-pattern the limb bud mesenchyme and regulate activation of their expression. Phase II: SHH signalling (dark blue) and GREM1/FGF-mediated feedback signalling (orange/light blue) are required to establish and propagate gene expression in the distal limb bud mesenchyme. Epithelialmesenchymal (EM) feedback signalling regulates the temporally coordinated anterior expansion (yellow arrow) of gene expression in the distal mesenchyme (Jag1 and 5 Hoxd genes) and FGF gene expression in the AER (light blue). Jag1 becomes independent of SHH signalling early, while 5 Hoxd genes are progressively rendered SHH independent (with 3 to 5 polarity) as their presumptive digit expression domains are being established. Phase III: the expanding population of Shh descendants increasingly separates SHH signalling from GREM1 producing mesenchymal cells, which probably causes breakdown of SHH/GREM1/FGF feedback signalling and terminates limb bud patterning.

9 SHH/GREM1-mediated gene regulation in limbs RESEARCH ARTICLE 3427 enhancer is located far 5 to the Hoxd gene complex and strongest enhances the expression of Hoxd13, while the expression of the more 3 located Hoxd12 and Hoxd11 genes is enhanced progressively less (Kmita et al., 2002). Our study reveals that establishment but not maintenance of the late 5 Hoxd expression domains requires SHH signalling. In good correlation with their differential regulation by the digit enhancer, Hoxd13 requires SHH signal transduction for significantly longer than the more 3 located Hoxd12 and Hoxd11 genes. Therefore, their differential dependence on SHH signalling could be mediated by interaction of SHH targets such as the GLI transcriptional regulators with the digit enhancer (Kmita et al., 2002; Spitz et al., 2003). Our study shows that 5 Hoxd genes are rendered SHH-independent in a reverse co-linear fashion as their presumptive digit expression domains are being established. This progressive stabilization and SHH independent expression of 5 Hoxd genes may constitute a part of, or at least mark the kinetic memory that mesenchymal cells may acquire as a consequence of their overall exposure to SHH signalling (Harfe et al., 2004). SHH dependent and independent phases of vertebrate limb bud development Our analysis, together with previous studies allows division of limb bud patterning in three distinct phases. During the initial setup phase, the AER-FGF and ZPA-SHH signalling centres and differential mesenchymal responsiveness are established under the influence of the GLI3R-HAND2 pre-patterning mechanism (Fig. 8, phase I) (e.g. te Welscher et al., 2002a). During this initial phase, the differential responsiveness to SHH signalling is probably setup in the nascent mesenchyme (this study) and Grem1 expression is activated in the posterior mesenchyme (Zuniga et al., 1999). In addition, the early posteriorly nested expression domains of 5 Hoxd genes are established (e.g. Zuniga and Zeller, 1999) and participate in activation of Shh expression (Kmita et al., 2005). The second, dynamic phase is initiated by concurrent establishment of SHH and GREM1/FGF-mediated EM feedback signalling, which coordinates temporal progression with anterior expansion of gene expression in the distal mesenchyme (Fig. 8, phase II; yellow arrow). The present study leads us to conclude that SHH constitutes the main engine for this dynamic phase, while the temporal kinetics are regulated in concert with GREM1/FGF EM feedback signalling. During this dynamic phase, the presumptive digit expression domains of Jag1 and the 5 Hoxd genes are progressively established and rendered independent of SHH signalling. Mesenchymal cells probably acquire their kinetic memory of exposure to SHH signal transduction (Harfe et al., 2004) and the expanding population of Shh descendents displaces the posterior limit of the Grem1 expression domain towards anterior (Scherz et al., 2004). This widening gap between SHHproducing and Grem1-expressing cells eventually terminates SHH/GREM1/FGF-mediated feedback signalling and limb bud patterning (Fig. 8, phase III). The present study reveals the temporal and spatial kinetics by which mesenchymal SHH signalling and GREM1-mediated BMP antagonism function together with FGF signalling from the AER to pattern the distal limb bud mesenchyme. It will be of particular interest to identify the BMP ligands that are antagonized by GREM1 in the limb bud and participate in regulation of the gene expression kinetics. Finally, it will be important to gain more insight into how these highly dynamic morphoregulatory interactions are established (Zuniga et al., 1999; te Welscher, 2002a) and terminated at the appropriate developmental time points (Scherz et al., 2004). The authors are grateful to C. Lehmann and C. Torres de los Reyes for technical assistance, to A. Roulier for artwork, and to C. Müller-Thompson for help in preparing the manuscript. We thank A. Gossler and F. Guillemot for providing probes for in situ hybridization. We are grateful to N. Matt for experimental suggestions and to anonymous reviewers and our group members for critical input into the manuscript. This study was supported by the Swiss National Science Foundation (R.Z.), both cantons Basel (A.Z., R.Z.), the Dutch NWO (R.Z.), KNAW (A.Z.) and the Stichting Catharine van Tussenbroek (L.P.). Supplementary material Supplementary material for this article is available at References Ahn, S. and Joyner, A. L. (2004). Dynamic changes in the response of cells to positive hedgehog signaling during mouse limb patterning. Cell 118, Charite, J., McFadden, D. G. and Olson, E. N. (2000). The bhlh transcription factor dhand controls Sonic hedgehog expression and establishment of the zone of polarizing activity during limb development. Development 127, Chen, J. K., Taipale, J., Cooper, M. K. and Beachy, P. A. (2002). Inhibition of Hedgehog signaling by direct binding of cyclopamine to Smoothened. Genes Dev. 16, Chen, Y., Knezevic, V., Ervin, V., Hutson, R., Ward, Y. and Mackem, S. (2004). Direct interaction with Hoxd proteins reverses Gli3-repressor function to promote digit formation downstream of Shh. Development 131, Chiang, C., Litingtung, Y., Lee, E., Young, K. E., Corden, J. L., Westphal, H. and Beachy, P. A. (1996). Cyclopia and defective axial patterning in mice lacking Sonic hedgehog gene function. Nature 383, Chiang, C., Litingtung, Y., Harris, M. P., Simandl, B. K., Li, Y., Beachy, P. A. and Fallon, J. F. (2001). Manifestation of the limb prepattern: limb development in the absence of Sonic hedgehog function. Dev. Biol. 236, Deschamps, J. (2004). Developmental Biology. Hox genes in the limb: a play in two acts. Science 304, Dollé, P., Dierich, A., LeMeur, M., Schimmang, T., Schuhbaur, B., Chambon, P. and Duboule, D. (1993). Disruption of the Hoxd-13 gene induces localized heterochrony leading to mice with neotenic limbs. Cell 75, Firulli, B. A., Krawchuk, D., Centonze, V. E., Vargesson, N., Virshup, D. M., Conway, S. J., Cserjesi, P., Laufer, E. and Firulli, A. B. (2005). Altered Twist1 and Hand2 dimerization is associated with Saethre-Chotzen syndrome and limb abnormalities. Nat. Genet. 37, Haramis, A. G., Brown, J. M. and Zeller, R. (1995). The limb deformity mutation disrupts the SHH/FGF-4 feedback loop and regulation of 5 HoxD genes during limb pattern formation. Development 121, Harfe, B. D., Scherz, P. J., Nissim, S., Tian, H., McMahon, A. P. and Tabin, C. J. (2004). Evidence for an expansion-based temporal Shh gradient in specifying vertebrate digit identities. Cell 118, Khokha, M. K., Hsu, D., Brunet, L. J., Dionne, M. S. and Harland, R. M. (2003). Gremlin is the BMP antagonist required for maintenance of Shh and Fgf signals during limb patterning. Nat. Genet. 34, Kiernan, A. E., Ahituv, N., Fuchs, H., Balling, R., Avraham, K. B., Steel, K. P. and Hrabe de Angelis, M. (2001). The Notch ligand Jagged1 is required for inner ear sensory development. Proc. Natl. Acad. Sci. USA 98, Kmita, M. and Duboule, D. (2003). Organizing axes in time and space; 25 years of colinear tinkering. Science 301, Kmita, M., Fraudeau, N., Herault, Y. and Duboule, D. (2002). Serial deletions and duplications suggest a mechanism for the collinearity of Hoxd genes in limbs. Nature 420, Kmita, M., Tarchini, B., Zakany, J., Logan, M., Tabin, C. J. and Duboule, D. (2005). Early developmental arrest of mammalian limbs lacking HoxA/HoxD gene function. Nature 435, Kraus, P., Fraidenraich, D. and Loomis, C. A. (2001). Some distal limb structures develop in mice lacking Sonic hedgehog signaling. Mech. Dev. 100, Laufer, E., Nelson, C. E., Johnson, R. L., Morgan, B. A. and Tabin, C. (1994). Sonic hedgehog and Fgf-4 act through a signaling cascade and feedback loop to integrate growth and patterning of the developing limb bud. Cell 79, Lewandoski, M., Sun, X. and Martin, G. R. (2000). Fgf8 signalling from the AER is essential for normal limb development. Nat. Genet. 26, Lewis, P. M., Dunn, M. P., McMahon, J. A., Logan, M., Martin, J. F., St- Jacques, B. and McMahon, A. P. (2001). Cholesterol modification of sonic hedgehog is required for long-range signaling activity and effective modulation of signaling by Ptc1. Cell 105, Litingtung, Y., Dahn, R. D., Li, Y., Fallon, J. F. and Chiang, C. (2002). Shh and Gli3 are dispensable for limb skeleton formation but regulate digit number and identity. Nature 418, Lu, P., Minowada, G. and Martin, G. R. (2006). Increasing Fgf4 expression in the