Marie Kmita

Serial deletions and duplications suggest a mechanism for the collinearity of Hoxd genes in limbs

Hox genes, located at one end of the HoxD cluster, are essential for the development of the extremities of our limbs; that is, the digits. This ‘collinear’ correspondence is accompanied by a gradual decrease in the transcriptional efficiency of the genes. To decipher the underlying regulatory mechanisms, and thus to understand better how digits develop, we engineered a series of deletions and duplications in vivo. We find that HoxD genes compete for a remote enhancer that recognizes the locus in a polar fashion, with a preference for the 5′ extremity. Modifications in either the number or topography of Hoxd loci induced regulatory reallocations affecting both the number and morphology of digits. These results demonstrate why genes located at the extremity of the cluster are expressed at the distal end of the limbs, following a gradual reduction in transcriptional efficiency, and thus highlight the mechanistic nature of collinearity in limbs.

Early developmental arrest of mammalian limbs lacking HoxA/HoxD gene function

Vertebrate HoxA and HoxD cluster genes are required for proper limb development. However, early lethality, compensation and redundancy have made a full assessment of their function difficult. Here we describe mice that are lacking all Hoxa and Hoxd functions in their forelimbs. We show that such limbs are arrested early in their developmental patterning and display severe truncations of distal elements, partly owing to the absence of Sonic hedgehog expression. These results indicate that the evolutionary recruitment of Hox gene function into growing appendages might have been crucial in implementing hedgehog signalling, subsequently leading to the distal extension of tetrapod appendages. Accordingly, these mutant limbs may be reminiscent of an ancestral trunk extension, related to that proposed for arthropods.

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

Digit Determination Pentadactyly has been an early and rapid innovation of tetrapods. Sheth et al. (p. 1476) report that a dramatic reduction in distally expressed Hox genes, in the absence of a functional morphogen signaling pathway, results in extreme polydactyly in mice. Mutant digits exhibited patterns reminiscent of the endoskeleton of fins, suggesting that an ancestral patterning mechanism has been deeply conserved in evolution. Mouse genetics and computer modeling suggest that a reaction-diffusion mechanism defines digit pattern. 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.

Localized and transient transcription of Hox genes suggests a link between patterning and the segmentation clock

During development, Hox gene transcription is activated in presomitic mesoderm with a time sequence that follows the order of the genes along the chromosome. Here, we show that Hoxd1 and other Hox genes display dynamic stripes of expression within presomitic mesoderm. The underlying transcriptional bursts may reflect the mechanism that coordinates Hox gene activation with somitogenesis. This mechanism appears to depend upon Notch signaling, as mice deficient for RBPJk, the effector of the Notch pathway, showed severely reduced Hoxd gene expression in presomitic mesoderm. These results suggest a molecular link between Hox gene activation and the segmentation clock. Such a linkage would efficiently keep in phase the production of novel segments with their morphological specification.

Clustering of tissue-specific sub-TADs accompanies the regulation of HoxA genes in developing limbs.

HoxA genes exhibit central roles during development and causal mutations have been found in several human syndromes including limb malformation. Despite their importance, information on how these genes are regulated is lacking. Here, we report on the first identification of bona fide transcriptional enhancers controlling HoxA genes in developing limbs and show that these enhancers are grouped into distinct topological domains at the sub-megabase scale (sub-TADs). We provide evidence that target genes and regulatory elements physically interact with each other through contacts between sub-TADs rather than by the formation of discreet “DNA loops”. Interestingly, there is no obvious relationship between the functional domains of the enhancers within the limb and how they are partitioned among the topological domains, suggesting that sub-TAD formation does not rely on enhancer activity. Moreover, we show that suppressing the transcriptional activity of enhancers does not abrogate their contacts with HoxA genes. Based on these data, we propose a model whereby chromatin architecture defines the functional landscapes of enhancers. From an evolutionary standpoint, our data points to the convergent evolution of HoxA and HoxD regulation in the fin-to-limb transition, one of the major morphological innovations in vertebrates.

Regulatory constraints in the evolution of The tetrapod limb anterior-posterior polarity

The anterior to posterior (A–P) polarity of the tetrapod limb is determined by the confined expression of Sonic hedgehog (Shh) at the posterior margin of developing early limb buds, under the control of HOX proteins encoded by gene members of both the HoxA and HoxD clusters. Here, we use a set of partial deletions to show that only the last four Hox paralogy groups can elicit this response: that is, precisely those genes whose expression is excluded from most anterior limb bud cells owing to their collinear transcriptional activation. We propose that the limb A–P polarity is produced as a collateral effect of Hox gene collinearity, a process highly constrained by its crucial importance during trunk development. In this view, the co-option of the trunk collinear mechanism, along with the emergence of limbs, imposed an A–P polarity to these structures as the most parsimonious solution. This in turn further contributed to stabilize the architecture and operational mode of this genetic system.

Targeted inversion of a polar silencer within the HoxD complex re-allocates domains of enhancer sharing.

Mammalian Hox genes are clustered at four genomic loci. During development, neighbouring genes are coordinately regulated by global enhancer sequences, which control multiple genes at once, as exemplified by the expression of series of contiguous Hoxd genes in either limbs or gut. The link between vertebrate Hox gene transcription and their clustered distribution is poorly understood. Experimental and comparative approaches have revealed that various mechanisms, such as gene clustering or global enhancer sequences, might have constrained this genomic organization and stabilized it throughout evolution. To understand what restricts the effect of a particular enhancer to a precise set of genes, we generated a loxP/Cre-mediated targeted inversion within the HoxD cluster. Mice carrying the inversion showed a reciprocal re-assignment of the limb versus gut regulatory specificities, suggesting the presence of a silencer element with a unidirectional property. This polar silencer appears to limit the number of genes that respond to one type of regulation and thus indicates how separate regulatory domains may be implemented within intricate gene clusters.

GLI3 constrains digit number by controlling both progenitor proliferation and BMP-dependent exit to chondrogenesis.

Inactivation of Gli3, a key component of Hedgehog signaling in vertebrates, results in formation of additional digits (polydactyly) during limb bud development. The analysis of mouse embryos constitutively lacking Gli3 has revealed the essential GLI3 functions in specifying the anteroposterior (AP) limb axis and digit identities. We conditionally inactivated Gli3 during mouse hand plate development, which uncoupled the resulting preaxial polydactyly from known GLI3 functions in establishing AP and digit identities. Our analysis revealed that GLI3 directly restricts the expression of regulators of the G(1)-S cell-cycle transition such as Cdk6 and constrains S phase entry of digit progenitors in the anterior hand plate. Furthermore, GLI3 promotes the exit of proliferating progenitors toward BMP-dependent chondrogenic differentiation by spatiotemporally restricting and terminating the expression of the BMP antagonist Gremlin1. Thus, Gli3 is a negative regulator of the proliferative expansion of digit progenitors and acts as a gatekeeper for the exit to chondrogenic differentiation.

Rostral and caudal pharyngeal arches share a common neural crest ground pattern

In vertebrates, face and throat structures, such as jaw, hyoid and thyroid cartilages develop from a rostrocaudal metameric series of pharyngeal arches, colonized by cranial neural crest cells (NCCs). Colinear Hox gene expression patterns underlie arch specific morphologies, with the exception of the first (mandibular) arch, which is devoid of any Hox gene activity. We have previously shown that the first and second (hyoid) arches share a common, Hox-free, patterning program. However, whether or not more posterior pharyngeal arch neural crest derivatives are also patterned on the top of the same ground-state remained an unanswered question. Here, we show that the simultaneous inactivation of all Hoxa cluster genes in NCCs leads to multiple jaw and first arch-like structures, partially replacing second, third and fourth arch derivatives, suggesting that rostral and caudal arches share the same mandibular arch-like ground patterning program. The additional inactivation of the Hoxd cluster did not significantly enhance such a homeotic phenotype, thus indicating a preponderant role of Hoxa genes in patterning skeletogenic NCCs. Moreover, we found that Hoxa2 and Hoxa3 act synergistically to pattern third and fourth arch derivatives. These results provide insights into how facial and throat structures are assembled during development, and have implications for the evolution of the pharyngeal region of the vertebrate head.

Sall genes regulate region-specific morphogenesis in the mouse limb by modulating Hox activities.

The genetic mechanisms that regulate the complex morphogenesis of generating cartilage elements in correct positions with precise shapes during organogenesis, fundamental issues in developmental biology, are still not well understood. By focusing on the developing mouse limb, we confirm the importance of transcription factors encoded by the Sall gene family in proper limb morphogenesis, and further show that they have overlapping activities in regulating regional morphogenesis in the autopod. Sall1/Sall3 double null mutants exhibit a loss of digit1 as well as a loss or fusion of digit2 and digit3, metacarpals and carpals in the autopod. We show that Sall activity affects different pathways, including the Shh signaling pathway, as well as the Hox network. Shh signaling in the mesenchyme is partially impaired in the Sall mutant limbs. Additionally, our data suggest an antagonism between Sall1-Sall3 and Hoxa13-Hoxd13. We demonstrate that expression of Epha3 and Epha4 is downregulated in the Sall1/Sall3 double null mutants, and, conversely, is upregulated in Hoxa13 and Hoxd13 mutants. Moreover, the expression of Sall1 and Sall3 is upregulated in Hoxa13 and Hoxd13 mutants. Furthermore, by using DNA-binding assays, we show that Sall and Hox compete for a target sequence in the Epha4 upstream region. In conjunction with the Shh pathway, the antagonistic interaction between Hoxa13-Hoxd13 and Sall1-Sall3 in the developing limb may contribute to the fine-tuning of local Hox activity that leads to proper morphogenesis of each cartilage element of the vertebrate autopod.