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.

The formation of the thumb requires direct modulation of Gli3 transcription by Hoxa13.

In the tetrapod limb, the digits (fingers or toes) are the elements most subject to morphological diversification in response to functional adaptations. However, despite their functional importance, the mechanisms controlling digit morphology remain poorly understood. Here we have focused on understanding the special morphology of the thumb (digit 1), the acquisition of which was an important adaptation of the human hand. To this end, we have studied the limbs of the Hoxa13 mouse mutant that specifically fail to form digit 1. We show that, consistent with the role of Hoxa13 in Hoxd transcriptional regulation, the expression of Hoxd13 in Hoxa13 mutant limbs does not extend into the presumptive digit 1 territory, which is therefore devoid of distal Hox transcripts, a circumstance that can explain its agenesis. The loss of Hoxd13 expression, exclusively in digit 1 territory, correlates with increased Gli3 repressor activity, a Hoxd negative regulator, resulting from increased Gli3 transcription that, in turn, is due to the release from the negative modulation exerted by Hox13 paralogs on Gli3 regulatory sequences. Our results indicate that Hoxa13 acts hierarchically to initiate the formation of digit 1 by reducing Gli3 transcription and by enabling expansion of the 5'Hoxd second expression phase, thereby establishing anterior-posterior asymmetry in the handplate. Our work uncovers a mutual antagonism between Gli3 and Hox13 paralogs that has important implications for Hox and Gli3 gene regulation in the context of development and evolution.

Evolution of Hoxa11 regulation in vertebrates is linked to the pentadactyl state.

The fin-to-limb transition represents one of the major vertebrate morphological innovations associated with the transition from aquatic to terrestrial life and is an attractive model for gaining insights into the mechanisms of morphological diversity between species. One of the characteristic features of limbs is the presence of digits at their extremities. Although most tetrapods have limbs with five digits (pentadactyl limbs), palaeontological data indicate that digits emerged in lobed fins of early tetrapods, which were polydactylous. How the transition to pentadactyl limbs occurred remains unclear. Here we show that the mutually exclusive expression of the mouse genes Hoxa11 and Hoxa13, which were previously proposed to be involved in the origin of the tetrapod limb, is required for the pentadactyl state. We further demonstrate that the exclusion of Hoxa11 from the Hoxa13 domain relies on an enhancer that drives antisense transcription at the Hoxa11 locus after activation by HOXA13 and HOXD13. Finally, we show that the enhancer that drives antisense transcription of the mouse Hoxa11 gene is absent in zebrafish, which, together with the largely overlapping expression of hoxa11 and hoxa13 genes reported in fish, suggests that this enhancer emerged in the course of the fin-to-limb transition. On the basis of the polydactyly that we observed after expression of Hoxa11 in distal limbs, we propose that the evolution of Hoxa11 regulation contributed to the transition from polydactyl limbs in stem-group tetrapods to pentadactyl limbs in extant tetrapods.

Decoupling the function of Hox and Shh in developing limb reveals multiple inputs of Hox genes on limb growth.

Limb development relies on an exquisite coordination between growth and patterning, but the underlying mechanisms remain elusive. Anterior-posterior and proximal-distal specification initiates in early limb bud concomitantly with the proliferative expansion of limb cells. Previous studies have shown that limb bud growth initially relies on fibroblast growth factors (FGFs) produced in the apical ectodermal ridge (AER-FGFs), the maintenance of which relies on a positive-feedback loop involving sonic hedgehog (Shh) and the BMP antagonist gremlin 1 (Grem1). The positive cross-regulation between Shh and the HoxA and HoxD clustered genes identified an indirect effect of Hox genes on the maintenance of AER-FGFs but the respective function of Shh and Hox genes in this process remains unknown. Here, by uncoupling Hox and Shh function, we show that HoxA and HoxD genes are required for proper AER-FGFs expression, independently of their function in controlling Shh expression. In addition, we provide evidence that the Hox-dependent control of AER-FGF expression is achieved through the regulation of key mesenchymal signals, namely Grem1 and Fgf10, ensuring proper epithelial-mesenchymal interactions. Notably, HoxA and HoxD genes contribute to both the initial activation of Grem1 and the subsequent anterior expansion of its expression domain. We propose that the intricate interactions between Hox genes and the FGF and Shh signaling pathways act as a molecular network that ensures proper limb bud growth and patterning, probably contributing to the coordination of these two processes.

"Self-regulation," a new facet of Hox genes' function.

BACKGROUND: Precise temporal and spatial expression of the clustered Hox genes is essential for patterning the developing embryo. Temporal activation of Hox genes was shown to be cluster-autonomous. However, gene clustering appears dispensable for spatial colinear expression. Generally, a set of Hox genes expressed in a group of cells instructs these cells about their fate such that the differential expression of Hox genes results in morphological diversity. The spatial colinearity is considered to rely both on local and long-range cis regulation. RESULTS: Here, we report on the global deregulation of HoxA and HoxD expression patterns upon inactivation of a subset of HOXA and HOXD proteins. CONCLUSIONS: Our data suggest the existence of a "self-regulation" mechanism, a process by which HOX proteins establish and/or maintain the spatial domains of the Hox gene family and we propose that the functionally dominant HOX proteins could contribute to generating the spatial parameters of Hox expression in a given tissue, i.e., HOX controlling the establishment of the ultimate HOX code.

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.

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 BMP-Shh negative-feedback loop restricts Shh expression during limb development.

Normal patterning of tissues and organs requires the tight restriction of signaling molecules to well-defined organizing centers. In the limb bud, one of the main signaling centers is the zone of polarizing activity (ZPA) that controls growth and patterning through the production of sonic hedgehog (SHH). The appropriate temporal and spatial expression of Shh is crucial for normal limb bud patterning, because modifications, even if subtle, have important phenotypic consequences. However, although there is a lot of information about the factors that activate and maintain Shh expression, much less is known about the mechanisms that restrict its expression to the ZPA. In this study, we show that BMP activity negatively regulates Shh transcription and that a BMP-Shh negative-feedback loop serves to confine Shh expression. BMP-dependent downregulation of Shh is achieved by interfering with the FGF and Wnt signaling activities that maintain Shh expression. We also show that FGF induction of Shh requires protein synthesis and is mediated by the ERK1/2 MAPK transduction pathway. BMP gene expression in the posterior limb bud mesoderm is positively regulated by FGF signaling and finely regulated by an auto-regulatory loop. Our study emphasizes the intricacy of the crosstalk between the major signaling pathways in the posterior limb bud.

Spatial regulation by multiple Gremlin1 enhancers provides digit development with cis-regulatory robustness and evolutionary plasticity.

Precise cis-regulatory control of gene expression is essential for normal embryogenesis and tissue development. The BMP antagonist Gremlin1 (Grem1) is a key node in the signalling system that coordinately controls limb bud development. Here, we use mouse reverse genetics to identify the enhancers in the Grem1 genomic landscape and the underlying cis-regulatory logics that orchestrate the spatio-temporal Grem1 expression dynamics during limb bud development. We establish that transcript levels are controlled in an additive manner while spatial regulation requires synergistic interactions among multiple enhancers. Disrupting these interactions shows that altered spatial regulation rather than reduced Grem1 transcript levels prefigures digit fusions and loss. Two of the enhancers are evolutionary ancient and highly conserved from basal fishes to mammals. Analysing these enhancers from different species reveal the substantial spatial plasticity in Grem1 regulation in tetrapods and basal fishes, which provides insights into the fin-to-limb transition and evolutionary diversification of pentadactyl limbs.

HOX13-dependent chromatin accessibility underlies the transition towards the digit development program.

Hox genes encode transcription factors (TFs) that establish morphological diversity in the developing embryo. The similar DNA-binding motifs of the various HOX TFs contrast with the wide-range of HOX-dependent genetic programs. The influence of the chromatin context on HOX binding specificity remains elusive. Here, we used the developing limb as a model system to compare the binding specificity of HOXA13 and HOXD13 (HOX13 hereafter), which are required for digit formation, and HOXA11, involved in forearm/leg development. We find that upon ectopic expression in distal limb buds, HOXA11 binds sites normally HOX13-specific. Importantly, these sites are loci whose chromatin accessibility relies on HOX13. Moreover, we show that chromatin accessibility specific to the distal limb requires HOX13 function. Based on these results, we propose that HOX13 TFs pioneer the distal limb-specific chromatin accessibility landscape for the proper implementation of the distal limb developmental program.

Distal Limb Patterning Requires Modulation of cis-Regulatory Activities by HOX13.

The combinatorial expression of Hox genes along the body axes is a major determinant of cell fate and plays a pivotal role in generating the animal body plan. Loss of HOXA13 and HOXD13 transcription factors (HOX13) leads to digit agenesis in mice, but how HOX13 proteins regulate transcriptional outcomes and confer identity to the distal-most limb cells has remained elusive. Here, we report on the genome-wide profiling of HOXA13 and HOXD13 in vivo binding and changes of the transcriptome and chromatin state in the transition from the early to the late-distal limb developmental program, as well as in Hoxa13-/-; Hoxd13-/- limbs. Our results show that proper termination of the early limb transcriptional program and activation of the late-distal limb program are coordinated by the dual action of HOX13 on cis-regulatory modules.

Hox genes regulate digit patterning by controlling the wavelength of a Turing-type mechanism.

The formation of repetitive structures (such as stripes) in nature is often consistent with a reaction-diffusion mechanism, or Turing model, of self-organizing systems. We used mouse genetics to analyze how digit patterning (an iterative digit/nondigit pattern) is generated. We showed that the progressive reduction in Hoxa13 and Hoxd11-Hoxd13 genes (hereafter referred to as distal Hox genes) from the Gli3-null background results in progressively more severe polydactyly, displaying thinner and densely packed digits. Combined with computer modeling, our results argue for a Turing-type mechanism underlying digit patterning, in which the dose of distal Hox genes modulates the digit period or wavelength. The phenotypic similarity with fish-fin endoskeleton patterns suggests that the pentadactyl state has been achieved through modification of an ancestral Turing-type mechanism.

Hoxd and Gli3 interactions modulate digit number in the amniote limb.

During limb development, Sonic hedgehog (SHH) and HOX proteins are considered among the most important factors regulating digit number and identity. SHH signaling prevents the processing of GLI3 into a short form that functions as a strong transcriptional repressor. Gli3 mutant limbs are characterized by a severe polydactyly and associated ectopic anterior expression of 5'Hoxd genes. To genetically determine the involvement of 5'Hoxd genes in the polydactyly of Gli3 mutants, we have generated a compound mutant that simultaneously removes the three most 5'-located Hoxd genes and Gli3. Remarkably, the limbs that form in the absence of all four of these genes show the most severe polydactyly so far reported in the mouse. The analysis of gene expression performed in compound mutants allows us to propose that the increase in the number of digits is mediated by the gain in function of Hoxd10 and Hoxd9. Our results also support the notion that an adequate balance between positive and negative effects of different Hoxd genes is required for pentadactyly.

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

The role of the aristaless-related homeobox gene Alx4 in antero-posterior (AP-) patterning of the developing vertebrate limb has remained somewhat elusive. Polydactyly of Alx4 mutant mice is known to be accompanied by ectopic anterior expression of genes like Shh, Fgf4 and 5'Hoxd. We reported previously that polydactyly in Alx4 mutant mice requires SHH signaling, but we now show that in early Alx4-/- limb buds the anterior ectopic expression of Fgf4 and Hoxd13, and therefore disruption of AP-patterning, occurs independently of SHH signaling. To better understand how Alx4 functions in the pathways that regulate AP-patterning, we also studied genomic regulatory sequences that are capable of directing expression of a reporter gene in a pattern corresponding to endogenous Alx4 expression in anterior limb bud mesenchyme. We observed, as expected for authentic Alx4 expression, expansion of reporter construct expression in a Shh-/- background. Total lack of reporter expression in a Gli3-/- background confirms the existence of Gli3-dependent and -independent Alx4 expression in the limb bud. Apparently, these two modules of Alx4 expression are linked to dissimilar functions.