The limb bud is of paradigmatic value to understanding vertebrate organogenesis. Recent genetic analysis in mice has revealed the existence of a largely self-regulatory limb bud signalling system that involves many of the pathways that are known to regulate morphogenesis. These findings contrast with the prevailing view that the main limb bud axes develop largely independently of one another. In this Review, we discuss models of limb development and attempt to integrate the current knowledge of the signalling interactions that govern limb skeletal development into a systems model. The resulting integrative model provides insights into how the specification and proliferative expansion of the anteroposterior and proximodistal limb bud axes are coordinately controlled in time and space.

Distal limb development and specification of digit identities in tetrapods are under the control of a mesenchymal organizer called the polarizing region. Sonic Hedgehog (SHH) is the morphogenetic signal produced by the polarizing region in the posterior limb bud. Ectopic anterior SHH signaling induces digit duplications and has been suspected as a major cause underlying congenital malformations that result in digit polydactyly. Here, we report that the polydactyly of Gli3-deficient mice arises independently of SHH signaling. Disruption of one or both Gli3 alleles in mouse embryos lacking Shh progressively restores limb distal development and digit formation. Our genetic analysis indicates that SHH signaling counteracts GLI3-mediated repression of key regulator genes, cell survival, and distal progression of limb bud development.

The spine apparatus is a cellular organelle that is present in many dendritic spines of excitatory neurons in the mammalian forebrain. Despite its discovery >40 years ago, the function of the spine apparatus is still unknown although calcium buffering functions as well as roles in synaptic plasticity have been proposed. We have recently shown that the 100-kDa protein synaptopodin is associated with the spine apparatus. Here, we now report that mice homozygous for a targeted deletion of the synaptopodin gene completely lack spine apparatuses. Interestingly, this absence of the spine apparatus is accompanied by a reduction in hippocampal long-term potentiation (LTP) in the CA1 region of the hippocampus and by an impairment of spatial learning in the radial arm maze test. This genetic analysis points to a role of the spine apparatus in synaptic plasticity.

Epithelial-mesenchymal feedback signaling is the key to diverse organogenetic processes such as limb bud development and branching morphogenesis in kidney and lung rudiments. This study establishes that the BMP antagonist gremlin (Grem1) is essential to initiate these epithelial-mesenchymal signaling interactions during limb and metanephric kidney organogenesis. A Grem1 null mutation in the mouse generated by gene targeting causes neonatal lethality because of the lack of kidneys and lung septation defects. In early limb buds, mesenchymal Grem1 is required to establish a functional apical ectodermal ridge and the epithelial-mesenchymal feedback signaling that propagates the sonic hedgehog morphogen. Furthermore, Grem1-mediated BMP antagonism is essential to induce metanephric kidney development as initiation of ureter growth,branching and establishment of RET/GDNF feedback signaling are disrupted in Grem1-deficient embryos. As a consequence, the metanephric mesenchyme is eliminated by apoptosis, in the same way as the core mesenchymal cells of the limb bud.

Turner syndrome is characterized by short stature and is frequently associated with a variable spectrum of somatic features including ovarian failure, heart and renal abnormalities, micrognathia, cubitus valgus, high-arched palate, short metacarpals and Madelung deformity. Madelung deformity is also a key feature of Leri-Weill syndrome. Defects of the pseudoautosomal homeobox gene SHOX were previously shown to lead to short stature and Leri-Weill syndrome, and haploinsufficiency of SHOX was implicated to cause the short stature phenotype in Turner syndrome. Despite exhaustive searches, no direct murine orthologue of SHOX is evident. SHOX is, however, closely related to the SHOX2 homeobox gene on 3q, which has a murine counterpart, Og12x. We analysed SHOX and SHOX2 expression during human embryonic development, and referenced the expression patterns against those of Og12x. The SHOX expression pattern in the limb and first and second pharyngeal arches not only explains SHOX -related short stature phenotypes, but also for the first time provides evidence for the involvement of this gene in the development of additional Turner stigmata. This is strongly supported by the presence of Turner-characteristic dysmorphic skeletal features in patients with SHOX nonsense mutations.

Dickkopf-1 (dkk-1) is member of a novel family of secreted proteins and functions in head induction during Xenopus embryogenesis, acting as a potent inhibitor of Wnt signalling. Here we report: (1) the isolation of two additional murine members of the dkk family, dkk-2 and dkk-3; and (2) analysis of adult and embryonic gene expression of mouse dkk-1,-2, and -3, Xenopus dkk-1 as well as chicken dkk-3. Comparative developmental analyses of the dkk-1, dkk-2 and dkk-3 in mice indicate that these genes are both temporally and spatially regulated. They define overlapping deep domains in mesenchymal lineages suggesting a co-ordinated mode of action. All dkks show distinct and elevated expression patterns in tissues that mediate epithelial- mesenchyme transformations suggesting that they may participate in heart, tooth, hair and whisker follicle, limb and bone induction. In the limb buds expression of these genes are found in regions of programmed cell death. In a given organ, dkk-1 tends to be the earliest member expressed. Comparison with Xenopus dkk-1 and chicken dkk-3 shows evolutionarily conserved expression patterns. Our observations indicate that dkk genes constitute a new family of secreted proteins that may mediate inductive interactions between epithelial and mesenchymal cells.

Embryogenesis depends on self-regulatory interactions between spatially separated signaling centers, but few of these are well understood. Limb development is regulated by epithelial-mesenchymal (e-m) feedback loops between sonic hedgehog (SHH) and fibroblast growth factor (FGF) signaling involving the bone morphogenetic protein (BMP) antagonist Gremlin1 (GREM1). By combining mouse molecular genetics with mathematical modeling, we showed that BMP4 first initiates and SHH then propagates e-m feedback signaling through differential transcriptional regulation of Grem1 to control digit specification. This switch occurs by linking a fast BMP4/GREM1 module to the slower SHH/GREM1/FGF e-m feedback loop. This self-regulatory signaling network results in robust regulation of distal limb development that is able to compensate for variations by interconnectivity among the three signaling pathways.

The mouse limb deformity (ld) mutations cause limb malformations by disrupting epithelial-mesenchymal signaling between the polarizing region and the apical ectodermal ridge. Formin was proposed as the relevant gene because three of the five ld alleles disrupt its C-terminal domain. In contrast, our studies establish that the two other ld alleles directly disrupt the neighboring Gremlin gene, corroborating the requirement of this BMP antagonist for limb morphogenesis. Further doubts concerning an involvement of Formin in the ld limb phenotype are cast, as a targeted mutation removing the C-terminal Formin domain by frame shift does not affect embryogenesis. In contrast, the deletion of the corresponding genomic region reproduces the ld limb phenotype and is allelic to mutations in Gremlin. We resolve these conflicting results by identifying a cis-regulatory region within the deletion that is required for Gremlin activation in the limb bud mesenchyme. This distant cis-regulatory region within Formin is also altered by three of the ld mutations. Therefore, the ld limb bud patterning defects are not caused by disruption of Formin, but by alteration of a global control region (GCR) required for Gremlin transcription. Our studies reveal the large genomic landscape harboring this GCR, which is required for tissue-specific coexpression of two structurally and functionally unrelated genes.[Keywords: cis regulation; Formin; global control region; Gremlin; limb development; regulatory landscape] Supplemental material is available at http://www.genesdev.org. Received February 8, 2004; revised version accepted May 5, 2004. The mouse is the genetic model of choice to study mammalian development and disease. In addition to alteration of specific genes by gene targeting and transgenesis, mutant mouse strains identified by phenotypic screens are commonly used to analyze developmental and disease processes (for reviews, see Justice 2000; Perkins 2002). A significant fraction of spontaneous mutations in mice (and humans) cause congenital limb malformations and have proven crucial to unravel the molecular mechanisms regulating vertebrate limb bud morphogenesis (for review, see Gurrieri et al. 2002). In particular, several alleles of the recessive mouse limb deformity (ld) mutation disrupt patterning of the distal limb skeleton. Over the years, a total of five ld alleles have been identified by phenotypic and genetic complementation analysis. All ld homozygous newborn mice display limb patterning defects characterized by synostosis of the zeugopod in combination with oligo-and syndactyly of metacarpal bones and digits (for review, see Zeller et al. 1999). In addition, ld homozygous newborn mice display varying degrees of uni-and bilateral renal aplasias depending on allele "strength" (Maas et al. 1994). Molecular analysis showed that the two ld alleles (ld