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

PMCID: PMC4482672[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.Grant sponsor: the Spanish Government; Grant number: BFU2011-24972; Grant sponsor: the Canadian Institutes for Health Research; Grant number: MOP-82880; Grant number: 126110. This work was supported by the Spanish Government to M.R. and by the Canadian Institutes for Health Research as well as the Canada Research Chair program to M.K. R.S was supported by a Formación Profesorado Universitario fellowship from the Spanish Ministry of Science and Innovation and currently supported by the Angelo Pizzagalli postdoctoral fellowship.Peer Reviewe

Ectopic nuclear reorganisation driven by a Hoxb1 transgene transposed into Hoxd

The extent to which the nuclear organisation of a gene impacts on its ability to be expressed, or whether nuclear organisation merely reflects gene expression states, remains an important but unresolved issue. A model system that has been instrumental in investigating this question is the murine Hox clusters. Nuclear reorganisation and chromatin decondensation, initiated towards the 3' end of the clusters, accompanies activation of Hox genes in both differentiation and development, and may be linked to mechanisms underlying colinearity. To investigate this, and to delineate the cis-acting elements involved, here we analyse the nuclear behaviour of a 3' Hoxb1 transgene transposed to the 5' end of the Hoxd cluster. We show that this transgene contains the cis-acting elements sufficient to initiate ectopic local nuclear reorganisation and chromatin decondensation, and to break Hoxd colinearity, in the primitive streak region of the early embryo. Significantly, in rhombomere 4 the transgene is able to induce attenuated nuclear reorganisation and decondensation of Hoxd even though there is no detectable expression of the transgene at this site. This shows that chromosome territory reorganisation and chromatin decondensation can be uncoupled from transcription itself, and suggests that they can therefore operate upstream of gene expression

Transcriptional Trajectories in Mouse Limb Buds Reveal the Transition from Anterior-Posterior to Proximal-Distal Patterning at Early Limb Bud Stage

Limb patterning relies in large part on the function of the Hox family of developmental genes. While the differential expression of Hox genes shifts from the anterior–posterior (A–P) to the proximal–distal (P–D) axis around embryonic day 11 (E11), whether this shift coincides with a more global change of A–P to P–D patterning program remains unclear. By performing and analyzing the transcriptome of the developing limb bud from E10.5 to E12.5, at single-cell resolution, we have uncovered transcriptional trajectories that revealed a general switch from A–P to P–D genetic program between E10.5 and E11.5. Interestingly, all the transcriptional trajectories at E10.5 end with cells expressing either proximal or distal markers suggesting a progressive acquisition of P–D identity. Moreover, we identified three categories of genes expressed in the distal limb mesenchyme characterized by distinct temporal expression dynamics. Among these are Hoxa13 and Hoxd13 (Hox13 hereafter), which start to be expressed around E10.5, and importantly the binding of the HOX13 factors was observed within or in the neighborhood of several of the distal limb genes. Our data are consistent with previous evidence suggesting that the transition from the early/proximal to the late/distal transcriptome of the limb mesenchyme largely relies on HOX13 function. Based on these results and the evidence that HOX13 factors restrict Hoxa11 expression to the proximal limb, in progenitor cells of the zeugopod, we propose that HOX13 act as a key determinant of P–D patterning

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 Hoxdependent 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. © 2013. Published by The Company of Biologists Ltd.This work was supported by the Canadian Institute of Health Research [CIHR-82880 to M.K.], the Canada Research Chairs program (to M.K.) and the Spanish Ministry of Science and Innovation [BFU2011-24972 to M.A.R.]. R.S. was supported by the Angelo Pizzagalli post-doctoral fellowship, D.G. was supported by a post-doctoral fellowship from the Fonds de la Recherche en Santé du Québec and M.S. was supported by a pre-doctoral fellowship from the Molecular Biology program of the University of Montreal.Peer Reviewe

De-coupling the Hox-Shh-Fgf interaction reveals multiple inputs of Hox genes on pathways ensuring limb growth

Resumen del trabajo presentado a los Congresos: 17th International Congress of Developmental Biology, 72nd Annual Meeting of the Society for Developmental Biology, VII Latin American Society of Developmental Biology Meeting y XI Congreso de la Sociedad Mexicana de Biologia del Desarrollo, celebrados en Cancún (México) del 16 al 20 de junio de 2013.-- et al.One of the most intriguing questions in developmental biology is how organ growth and patterning are coordinated during embryogenesis. Limb development relies on an exquisite coordination between growth and patterning but the underlying mechanisms remain elusive. Previous studies showed that A-P and P-D limb bud growth and patterning relies on a positive feedback loop between Sonic Hedgehog (Shh), the BMP antagonist Gremlin1 (Grem1), both expressed in mesenchymal cells, and Fibroblast growth factors (Fgfs) produced in the Apical Ectodermal Ridge (AER). In addition, the collinear expression of HoxA and HoxD genes has a key role in A-P and P-D patterning by establishing positional identity along these axes. Here, we show that HoxA and HoxD genes are required at early stages for Grem1 activation and proper Fgf10 and Fgf8 expression and therefore are mandatory for the establishment of the Shh-Grem1-Fgf feedback loop. Our results provide evidence that, in addition to this early function, HoxA and HoxD genes remain indispensable for proper limb growth at later stages. Together, our results reveal a dual role of Hox genes in controlling limb growth and establishing the limb architecture and we propose that the intricate interactions between Hox function and growth pathways act as the molecular network coordinating limb bud growth and patterning.Peer reviewe

Homeobox genes in the ribbonworm Lineus sanguineus: Evolutionary implications

From our current understanding of the genetic basis of development and pattern formation in Drosophila and vertebrates it is commonly thought that clusters of Hox genes sculpt the morphology of animals in specific body regions. Based on Hox gene conservation throughout the animal kingdom it is proposed that these genes and their role in pattern formation evolved early during the evolution of metazoans. Knowledge of the history of Hox genes will lead to a better understanding of the role of Hox genes in the evolution of animal body plans. To infer Hox gene evolution, reliable data on lower chordates and invertebrates are crucial. Among the lower triploblasts, the body plan of the ribbonworm Lineus (nemertini) appears to be close to the common ancestral condition of protostomes and deuterostomes. In this paper we present the isolation and identification of Hox genes in Lineus sanguineus. We find that the Lineus genome contains a single cluster of at least six Hox genes: two anterior-class genes, three middle-class genes, and one posterior-class gene. Each of the genes can be definitely assigned to an ortholog group on the basis of its homeobox and its flanking sequences. The most closely related homeodomain sequences are invariably found among the mouse or Amphioxus orthologs, rather than Drosophila and other invertebrates. This suggests that the ribbonworms have diverged relatively little from the last common ancestors of protostomes and deuterostomes, the urbilateria

A nested deletion approach to generate Cre deleter mice with progressive Hox profiles

In mice, the loxP/Cre recombinase-dependent system of recombination offers powerful possibilities for engineering genetic configurations of interest. This system can also be advantageously used for conditional mutagenesis in vivo, whenever such an approach is required due to deleterious effects of either one mutation, or a combination thereof. Here, we report on the production of an allelic series of insertions of a Hoxd11/Cre fusion transgene at different positions within the HoxD complex, in order to produce the CRE recombinase with a 'Hox profile' progressively more extended. We used the R26R (R26R) reporter mouse line to functionally assess the distribution and efficiency of the CRE enzyme and discuss the usefulness of these various lines as deleter strains

Mechanisms of Hox gene colinearity: transposition of the anterior Hoxb1 gene into the posterior HoxD complex

Transposition of Hoxd genes to a more posterior (5′) location within the HoxD complex suggested that colinearity in the expression of these genes was due, in part, to the existence of a silencing mechanism originating at the 5′ end of the cluster and extending towards the 3′ direction. To assess the strength and specificity of this repression, as well as to challenge available models on colinearity, we inserted a Hoxb1/lacZ transgene within the posterior HoxD complex, thereby reconstructing a cluster with a copy of the most anterior gene inserted at the most posterior position. Analysis of Hoxb1 expression after ectopic relocation revealed that Hoxb1-specific activity in the fourth rhombomere was totally abolished. Treatment with retinoic acid, or subsequent relocations toward more 3′ positions in the HoxD complex, did not release this silencing in hindbrain cells. In contrast, however, early and anterior transgene expression in the mesoderm was unexpectedly not suppressed. Furthermore, the transgene induced a transient ectopic activation of the neighboring Hoxd13 gene, without affecting other genes of the complex. Such a local and transient break in colinearity was also observed after transposition of the Hoxd9/lacZ reporter gene, indicating that it may be a general property of these transgenes when transposed at an ectopic location. These results are discussed in the context of existing models, which account for colinear activation of vertebrate Hox genes