TBX3 is essential for establishment of the posterior boundary of anterior genes and upregulation of posterior genes together with HAND2 during the onset of limb bud development.

During limb bud formation, axis polarities are established as evidenced by the spatially restricted expression of key regulator genes. In particular, the mutually antagonistic interaction between the GLI3 repressor and HAND2 results in distinct and non-overlapping anterior-distal Gli3 and posterior Hand2 expression domains. This is a hallmark of the establishment of antero-posterior limb axis polarity, together with spatially restricted expression of homeodomain and other transcriptional regulators. Here, we show that TBX3 is required for establishment of the posterior expression boundary of anterior genes in mouse limb buds. ChIP-seq and differential gene expression analysis of wild-type and mutant limb buds identifies TBX3-specific and shared TBX3-HAND2 target genes. High sensitivity fluorescent whole-mount in situ hybridisation shows that the posterior expression boundaries of anterior genes are positioned by TBX3-mediated repression, which excludes anterior genes such as Gli3, Alx4, Hand1 and Irx3/5 from the posterior limb bud mesenchyme. This exclusion delineates the posterior mesenchymal territory competent to establish the Shh-expressing limb bud organiser. In turn, HAND2 is required for Shh activation and cooperates with TBX3 to upregulate shared posterior identity target genes in early limb buds.

SMAD4 target genes are part of a transcriptional network that integrates the response to BMP and SHH signaling during early limb bud patterning.

SMAD4 regulates gene expression in response to BMP and TGFß signal transduction, and is required for diverse morphogenetic processes, but its target genes have remained largely elusive. Here, we identify the SMAD4 target genes in mouse limb buds using an epitope-tagged Smad4 allele for ChIP-seq analysis in combination with transcription profiling. This analysis shows that SMAD4 predominantly mediates BMP signal transduction during early limb bud development. Unexpectedly, the expression of cholesterol biosynthesis enzymes is precociously downregulated and intracellular cholesterol levels are reduced in Smad4-deficient limb bud mesenchymal progenitors. Most importantly, our analysis reveals a predominant function of SMAD4 in upregulating target genes in the anterior limb bud mesenchyme. Analysis of differentially expressed genes shared between Smad4- and Shh-deficient limb buds corroborates this function of SMAD4 and also reveals the repressive effect of SMAD4 on posterior genes that are upregulated in response to SHH signaling. This analysis uncovers opposing trans-regulatory inputs from SHH- and SMAD4-mediated BMP signal transduction on anterior and posterior gene expression during the digit patterning and outgrowth in early limb buds.

Molecular signatures identify immature mesenchymal progenitors in early mouse limb buds that respond differentially to morphogen signaling.

The key molecular interactions governing vertebrate limb bud development are a paradigm for studying the mechanisms controlling progenitor cell proliferation and specification during vertebrate organogenesis. However, little is known about the cellular heterogeneity of the mesenchymal progenitors in early limb buds that ultimately contribute to the chondrogenic condensations prefiguring the skeleton. We combined flow cytometric and transcriptome analyses to identify the molecular signatures of several distinct mesenchymal progenitor cell populations present in early mouse forelimb buds. In particular, jagged 1 (JAG1)-positive cells located in the posterior-distal mesenchyme were identified as the most immature limb bud mesenchymal progenitors (LMPs), which crucially depend on SHH and FGF signaling in culture. The analysis of gremlin 1 (Grem1)-deficient forelimb buds showed that JAG1-expressing LMPs are protected from apoptosis by GREM1-mediated BMP antagonism. At the same stage, the osteo-chondrogenic progenitors (OCPs) located in the core mesenchyme are already actively responding to BMP signaling. This analysis sheds light on the cellular heterogeneity of the early mouse limb bud mesenchyme and on the distinct response of LMPs and OCPs to morphogen signaling.

Tissue remodelling through branching morphogenesis.

Branched structures are evident at all levels of organization in living organisms. Many organs, such as the vascular system, lung, kidney and mammary gland, are heavily branched. In each of these cases, equally fascinating questions have been put forward, including those that address the cellular and molecular mechanisms that regulate the branching process itself, such as where the branches are initiated and how they extend and grow in the right direction. Recent experiments suggest that cell competition and cell rearrangements might be conserved key features in branch formation and might be controlled by local cell signalling.

Attenuated sensing of SHH by Ptch1 underlies evolution of bovine limbs.

The large spectrum of limb morphologies reflects the wide evolutionary diversification of the basic pentadactyl pattern in tetrapods. In even-toed ungulates (artiodactyls, including cattle), limbs are adapted for running as a consequence of progressive reduction of their distal skeleton to symmetrical and elongated middle digits with hoofed phalanges. Here we analyse bovine embryos to establish that polarized gene expression is progressively lost during limb development in comparison to the mouse. Notably, the transcriptional upregulation of the Ptch1 gene, which encodes a Sonic hedgehog (SHH) receptor, is disrupted specifically in the bovine limb bud mesenchyme. This is due to evolutionary alteration of a Ptch1 cis-regulatory module, which no longer responds to graded SHH signalling during bovine handplate development. Our study provides a molecular explanation for the loss of digit asymmetry in bovine limb buds and suggests that modifications affecting the Ptch1 cis-regulatory landscape have contributed to evolutionary diversification of artiodactyl limbs.

To BMP or not to BMP during vertebrate limb bud development.

The analysis of vertebrate limb bud development provides insight of general relevance into the signaling networks that underlie the controlled proliferative expansion of large populations of mesenchymal progenitors, cell fate determination and initiation of differentiation. In particular, extensive genetic analysis of mouse and experimental manipulation of chicken limb bud development has revealed the self-regulatory feedback signaling systems that interlink the main morphoregulatory signaling pathways including BMPs and their antagonists. It this review, we showcase the key role of BMPs and their antagonists during limb bud development. This review provides an understanding of the key morphoregulatory interactions that underlie the highly dynamic changes in BMP activity and signal transduction as limb bud development progresses from initiation and setting-up the signaling centers to determination and formation of the chondrogenic primordia for the limb skeletal elements.

Smad4 is required to induce digit ray primordia and to initiate the aggregation and differentiation of chondrogenic progenitors in mouse limb buds.

SMAD4 is an essential mediator of canonical TGFß/BMP signal transduction and we inactivated Smad4 in mouse limb buds from early stages onward to study its functions in the mesenchyme. While this Smad4 inactivation did not alter the early Sox9 distribution, prefiguring the chondrogenic primordia of the stylopod and zeugopod, it disrupted formation of all Sox9-positive digit ray primordia. Specific inactivation of Smad4 during handplate development pointed to its differential requirement for posterior and anterior digit ray primordia. At the cellular level, Smad4 deficiency blocked the aggregation of Sox9-positive progenitors, thereby preventing chondrogenic differentiation as revealed by absence of collagen type II. The progressive loss of SOX9 due to disrupting digit ray primordia and chondrogenesis was paralleled by alterations in genes marking other lineages. This pointed to a general loss of tissue organization and diversion of mutant cells toward non-specific connective tissue. Conditional inactivation of Bmp2 and Bmp4 indicated that the loss of digit ray primordia and increase in connective tissue were predominantly a consequence of disrupting SMAD4-mediated BMP signal transduction. In summary, our analysis reveals that SMAD4 is required to initiate: (1) formation of the Sox9-positive digit ray primordia; and (2) aggregation and chondrogenic differentiation of all limb skeletal elements.

Islet1 regulates establishment of the posterior hindlimb field upstream of the Hand2-Shh morphoregulatory gene network in mouse embryos.

How divergent genetic systems regulate a common pathway during the development of two serial structures, forelimbs and hindlimbs, is not well understood. Specifically, HAND2 has been shown to regulate Shh directly to initiate its expression in the posterior margin of the limb mesenchyme. Although the Hand2-Shh morphoregulatory system operates in both the forelimb and hindlimb bud, a recent analysis suggested that its upstream regulation is different in the forelimb and hindlimb bud. A combination of all four Hox9 genes is required for Hand2 expression in the forelimb-forming region; however, it remains elusive what genetic system regulates the Hand2-Shh pathway in the hindlimb-forming region. By conditional inactivation of Islet1 in the hindlimb-forming region using the Hoxb6Cre transgene, we show that Islet1 is required for establishing the posterior hindlimb field, but not the forelimb field, upstream of the Hand2-Shh pathway. Inactivation of Islet1 caused the loss of posterior structures in the distal and proximal regions, specifically in the hindlimb. We found that Hand2 expression was downregulated in the hindlimb field and that Shh expression was severely impaired in the hindlimb bud. In the Hoxb6Cre; Islet1 mutant pelvis, the proximal element that is formed in a Shh-independent manner, displayed complementary defects in comparison with Pitx1(-/-) hindlimbs. This suggests that Islet1 and Pitx1 function in parallel during girdle development in hindlimbs, which is in contrast with the known requirement for Tbx5 in girdle development in forelimbs. Our studies have identified a role for Islet1 in hindlimb-specific development and have revealed Islet1 functions in two distinct processes: regulation upstream of the Hand2-Shh pathway and contributions to girdle development.

Vertebrate limb bud development: moving towards integrative analysis of organogenesis.

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.

SHH propagates distal limb bud development by enhancing CYP26B1-mediated retinoic acid clearance via AER-FGF signalling.

The essential roles of SHH in anteroposterior (AP) and AER-FGF signalling in proximodistal (PD) limb bud development are well understood. In addition, these morphoregulatory signals are key components of the self-regulatory SHH/GREM1/AER-FGF feedback signalling system that regulates distal progression of limb bud development. This study uncovers an additional signalling module required for coordinated progression of limb bud axis development. Transcriptome analysis using Shh-deficient mouse limb buds revealed that the expression of proximal genes was distally extended from early stages onwards, which pointed to a more prominent involvement of SHH in PD limb axis development. In particular, retinoic acid (RA) target genes were upregulated proximally, while the expression of the RA-inactivating Cyp26b1 enzyme was downregulated distally, pointing to increased RA activity in Shh-deficient mouse limb buds. Further genetic and molecular analysis established that Cyp26b1 expression is regulated by AER-FGF signalling. During initiation of limb bud outgrowth, the activation of Cyp26b1 expression creates a distal 'RA-free' domain, as indicated by complementary downregulation of a transcriptional sensor of RA activity. Subsequently, Cyp26b1 expression increases as a consequence of SHH-dependent upregulation of AER-FGF signalling. To better understand the underlying signalling interactions, computational simulations of the spatiotemporal expression patterns and interactions were generated. These simulations predicted the existence of an antagonistic AER-FGF/CYP26B1/RA signalling module, which was verified experimentally. In summary, SHH promotes distal progression of limb development by enhancing CYP26B1-mediated RA clearance as part of a signalling network linking the SHH/GREM1/AER-FGF feedback loop to the newly identified AER-FGF/CYP26B1/RA module.

The hedgehog target Vlk genetically interacts with Gli3 to regulate chondrocyte differentiation during mouse long bone development.

Endochondral bone development is orchestrated by the spatially and temporally coordinated differentiation of chondrocytes along the longitudinal axis of the cartilage anlage. Initially, the slowly proliferating, periarticular chondrocytes give rise to the pool of rapidly dividing columnar chondrocytes, whose expansion determines the length of the long bones. The Indian hedgehog (IHH) ligand regulates both the proliferation of columnar chondrocytes and their differentiation into post-mitotic hypertrophic chondrocytes in concert with GLI3, one of the main transcriptional effectors of HH signal transduction. In the absence of Hh signalling, the expression of Vlk (vertebrate lonesome kinase, also called Pkdcc) is increased. We now show that the shortening of limb long bones in Vlk-deficient mouse embryos is aggravated by additional inactivation of Gli3. Our analysis establishes that Vlk and Gli3 synergize to control the temporal kinetics of chondrocyte differentiation during long bone development. Whereas differentiation of limb mesenchymal progenitors into chondrocytes and the initial formation of the cartilage anlagen of the limb skeleton are not altered, Vlk and Gli3 are required for the temporally coordinated differentiation of periarticular into columnar and ultimately hypertrophic chondrocytes in long bones. In limbs lacking both Vlk and Gli3, the appearance of columnar and hypertrophic chondrocytes is severely delayed and zones of morphologically distinct chondrocytes are not established until E16.5. At the molecular level, these morphological alterations are reflected by delayed activation and lowered expression of Ihh, Pth1r and Col10a1 in long bone rudiments of double mutant limbs. In summary, our genetic analysis establishes that VLK plays a role in the IHH/GLI3 interactions and that Vlk and Gli3 cooperate to regulate long bone development by modulating the temporal kinetics of establishing columnar and hypertrophic chondrocyte domains.

Dual requirement of ectodermal Smad4 during AER formation and termination of feedback signaling in mouse limb buds.

BMP signaling is pivotal for normal limb bud development in vertebrate embryos and genetic analysis of receptors and ligands in the mouse revealed their requirement in both mesenchymal and ectodermal limb bud compartments. In this study, we genetically assessed the potential essential functions of SMAD4, a mediator of canonical BMP/TGFß signal transduction, in the mouse limb bud ectoderm. Msx2-Cre was used to conditionally inactivate Smad4 in the ectoderm of fore- and hindlimb buds. In hindlimb buds, the Smad4 inactivation disrupts the establishment and signaling by the apical ectodermal ridge (AER) from early limb bud stages onwards, which results in severe hypoplasia and/or aplasia of zeugo- and autopodal skeletal elements. In contrast, the developmentally later inactivation of Smad4 in forelimb buds does not alter AER formation and signaling, but prolongs epithelial-mesenchymal feedback signaling in advanced limb buds. The late termination of SHH and AER-FGF signaling delays distal progression of digit ray formation and inhibits interdigit apoptosis. In summary, our genetic analysis reveals the temporally and functionally distinct dual requirement of ectodermal Smad4 during initiation and termination of AER signaling.

Dual RMCE for efficient re-engineering of mouse mutant alleles.

We have developed dual recombinase-mediated cassette exchange (dRMCE) to efficiently re-engineer the thousands of available conditional alleles in mouse embryonic stem cells. dRMCE takes advantage of the wild-type loxP and FRT sites present in these conditional alleles and in many gene-trap lines. dRMCE is a scalable, flexible tool to introduce tags, reporters and mutant coding regions into an endogenous locus of interest in an easy and highly efficient manner.

Recapitulation of endochondral bone formation using human adult mesenchymal stem cells as a paradigm for developmental engineering.

Mesenchymal stem/stromal cells (MSC) are typically used to generate bone tissue by a process resembling intramembranous ossification, i.e., by direct osteoblastic differentiation. However, most bones develop by endochondral ossification, i.e., via remodeling of hypertrophic cartilaginous templates. To date, endochondral bone formation has not been reproduced using human, clinically compliant cell sources. Here, we aimed at engineering tissues from bone marrow-derived, adult human MSC with an intrinsic capacity to undergo endochondral ossification. By analogy to embryonic limb development, we hypothesized that successful execution of the endochondral program depends on the initial formation of hypertrophic cartilaginous templates. Human MSC, subcutaneously implanted into nude mice at various stages of chondrogenic differentiation, formed bone trabeculae only when they had developed in vitro hypertrophic tissue structures. Advanced maturation in vitro resulted in accelerated formation of larger bony tissues. The underlying morphogenetic process was structurally and molecularly similar to the temporal and spatial progression of limb bone development in embryos. In particular, Indian hedgehog signaling was activated at early stages and required for the in vitro formation of hypertrophic cartilage. Subsequent development of a bony collar in vivo was followed by vascularization, osteoclastic resorption of the cartilage template, and appearance of hematopoietic foci. This study reveals the capacity of human MSC to generate bone tissue via an endochondral program and provides a valid model to study mechanisms governing bone development. Most importantly, this process could generate advanced grafts for bone regeneration by invoking a "developmental engineering" paradigm.

Distinct roles of Hand2 in initiating polarity and posterior Shh expression during the onset of mouse limb bud development.

The polarization of nascent embryonic fields and the endowment of cells with organizer properties are key to initiation of vertebrate organogenesis. One such event is antero-posterior (AP) polarization of early limb buds and activation of morphogenetic Sonic Hedgehog (SHH) signaling in the posterior mesenchyme, which in turn promotes outgrowth and specifies the pentadactylous autopod. Inactivation of the Hand2 transcriptional regulator from the onset of mouse forelimb bud development disrupts establishment of posterior identity and Shh expression, which results in a skeletal phenotype identical to Shh deficient limb buds. In wild-type limb buds, Hand2 is part of the protein complexes containing Hoxd13, another essential regulator of Shh activation in limb buds. Chromatin immunoprecipitation shows that Hand2-containing chromatin complexes are bound to the far upstream cis-regulatory region (ZRS), which is specifically required for Shh expression in the limb bud. Cell-biochemical studies indicate that Hand2 and Hoxd13 can efficiently transactivate gene expression via the ZRS, while the Gli3 repressor isoform interferes with this positive transcriptional regulation. Indeed, analysis of mouse forelimb buds lacking both Hand2 and Gli3 reveals the complete absence of antero-posterior (AP) polarity along the entire proximo-distal axis and extreme digit polydactyly without AP identities. Our study uncovers essential components of the transcriptional machinery and key interactions that set-up limb bud asymmetry upstream of establishing the SHH signaling limb bud organizer.

Optimized protocol for whole-mount RNA fluorescent in situ hybridization using oxidation-mediated autofluorescence reduction on mouse embryos.

Tissue autofluorescence poses significant challenges for RNA and protein analysis using fluorescence-based techniques. Here, we present a protocol that combines oxidation-mediated autofluorescence reduction with detergent-based tissue permeabilization for whole-mount RNA-fluorescence in situ hybridization (FISH) on mouse embryonic limb buds. We describe the steps for embryo collection, fixation, photochemical bleaching, permeabilization, and RNA-FISH, followed by optical clearing of RNA-FISH and immunofluorescence samples for imaging. The protocol alleviates the need for digital image post-processing to remove autofluorescence and is applicable to other tissues, organs, and vertebrate embryos.

A spatio-temporally constrained gene regulatory network directed by PBX1/2 acquires limb patterning specificity via HAND2.

A lingering question in developmental biology has centered on how transcription factors with widespread distribution in vertebrate embryos can perform tissue-specific functions. Here, using the murine hindlimb as a model, we investigate the elusive mechanisms whereby PBX TALE homeoproteins, viewed primarily as HOX cofactors, attain context-specific developmental roles despite ubiquitous presence in the embryo. We first demonstrate that mesenchymal-specific loss of PBX1/2 or the transcriptional regulator HAND2 generates similar limb phenotypes. By combining tissue-specific and temporally controlled mutagenesis with multi-omics approaches, we reconstruct a gene regulatory network (GRN) at organismal-level resolution that is collaboratively directed by PBX1/2 and HAND2 interactions in subsets of posterior hindlimb mesenchymal cells. Genome-wide profiling of PBX1 binding across multiple embryonic tissues further reveals that HAND2 interacts with subsets of PBX-bound regions to regulate limb-specific GRNs. Our research elucidates fundamental principles by which promiscuous transcription factors cooperate with cofactors that display domain-restricted localization to instruct tissue-specific developmental programs.

Conserved cis-regulatory regions in a large genomic landscape control SHH and BMP-regulated Gremlin1 expression in mouse limb buds.

BACKGROUND: Mouse limb bud is a prime model to study the regulatory interactions that control vertebrate organogenesis. Major aspects of limb bud development are controlled by feedback loops that define a self-regulatory signalling system. The SHH/GREM1/AER-FGF feedback loop forms the core of this signalling system that operates between the posterior mesenchymal organiser and the ectodermal signalling centre. The BMP antagonist Gremlin1 (GREM1) is a critical node in this system, whose dynamic expression is controlled by BMP, SHH, and FGF signalling and key to normal progression of limb bud development. Previous analysis identified a distant cis-regulatory landscape within the neighbouring Formin1 (Fmn1) locus that is required for Grem1 expression, reminiscent of the genomic landscapes controlling HoxD and Shh expression in limb buds. RESULTS: Three highly conserved regions (HMCO1-3) were identified within the previously defined critical genomic region and tested for their ability to regulate Grem1 expression in mouse limb buds. Using a combination of BAC and conventional transgenic approaches, a 9 kb region located ~70 kb downstream of the Grem1 transcription unit was identified. This region, termed Grem1 Regulatory Sequence 1 (GRS1), is able to recapitulate major aspects of Grem1 expression, as it drives expression of a LacZ reporter into the posterior and, to a lesser extent, in the distal-anterior mesenchyme. Crossing the GRS1 transgene into embryos with alterations in the SHH and BMP pathways established that GRS1 depends on SHH and is modulated by BMP signalling, i.e. integrates inputs from these pathways. Chromatin immunoprecipitation revealed interaction of endogenous GLI3 proteins with the core cis-regulatory elements in the GRS1 region. As GLI3 is a mediator of SHH signal transduction, these results indicated that SHH directly controls Grem1 expression through the GRS1 region. Finally, all cis-regulatory regions within the Grem1 genomic landscape locate to the DNAse I hypersensitive sites identified in this genomic region by the ENCODE consortium. CONCLUSIONS: This study establishes that distant cis-regulatory regions scattered through a larger genomic landscape control the highly dynamic expression of Grem1, which is key to normal progression of mouse limb bud development.

Wnt/ß-catenin signaling in the dental mesenchyme regulates incisor development by regulating Bmp4.

Loss- and gain-of function approaches modulating canonical Wnt/ß-catenin activity have established a role for the Wnt/ß-catenin pathway during tooth development. Here we show that Wnt/ß-catenin signaling is required in the dental mesenchyme for normal incisor development, as locally restricted genetic inactivation of ß-catenin results in a splitting of the incisor placode, giving rise to two incisors. Molecularly this is first associated with down-regulation of Bmp4 and subsequent splitting of the Shh domain at a subsequent stage. The latter phenotype can be mimicked by ectopic application of the BMP antagonist Noggin. Conditional genetic inactivation of Bmp4 in the mesenchyme reveals that mesenchymal BMP4 activity is required for maintenance of Shh expression in the dental ectoderm. Taken together our results indicate that ß-catenin together with Lef1 and Tcf1 are required to activate Bmp4 expression in order to maintain Shh expression in the dental ectoderm. This provides a mechanism whereby the number of incisors arising from one placode can be varied through local alterations of a mesenchymal signaling circuit involving ß-catenin, Lef1, Tcf1 and Bmp4.

Shh and Gremlin1 chromosomal landscapes in development and disease.

Two regulatory signals play major roles in digit patterning during vertebrate limb development, the SHH morphogen and the BMP antagonist Gremlin1. Their dynamic expression in limb buds is controlled by distant cis-regulatory elements embedded in unrelated neighboring genes, which has confused identification of the primary cause of different types of congenital limb malformations affecting mice and humans. Comparative and functional genomics have uncovered the large and complex chromosomal landscapes that control Shh and Gremlin1 expression, identified the molecular cause of the congenital malformations and provided insights into limb evolution. While most of the transacting factors remain unknown, Hoxd proteins have been shown to bind to the far upstream Shh cis-regulatory elements and activate their expression in limb buds.