The evolution of digits was an essential step in the success of tetrapods. Among the key players, Hoxd genes are coordinately regulated in developing digits, where they help organize growth and patterns. We identified the distal regulatory sites associated with these genes by probing the three-dimensional architecture of this regulatory unit in developing limbs. This approach, combined with in vivo deletions of distinct regulatory regions, revealed that the active part of the gene cluster contacts several enhancer-like sequences. These elements are dispersed throughout the nearby gene desert, and each contributes either quantitatively or qualitatively to Hox gene transcription in presumptive digits. We propose that this genetic system, which we call a "regulatory archipelago," provides an inherent flexibility that may partly underlie the diversity in number and morphology of digits across tetrapods, as well as their resilience to drastic variations.

We previously identified novel human ets-1 transcripts in which the normal order of exons is inverted, and demonstrated that although the order of exons is different than in the genomic DNA, splicing of these exons out of order occurs in pairs using genuine splice sites (1). Here we determine the structure of these novel transcripts, showing that they correspond to circular RNA molecules containing only exons in genomic order. These transcripts are stable molecules, localized in the cytoplasmic component of the cells. To our knowledge, this is the first case of circular transcripts being processed from nuclear pre-mRNA in eukaryotes. This new type of transcript might represent a novel aspect of gene expression and hold some interesting clues about the splicing mechanism.

Hox genes are major determinants of the animal body plan, where they organize structures along both the trunk and appendicular axes. During mouse limb development, Hoxd genes are transcribed in two waves: early on, when the arm and forearm are specified, and later, when digits form. The transition between early and late regulations involves a functional switch between two opposite topological domains. This switch is reflected by a subset of Hoxd genes mapping centrally into the cluster, which initially interact with the telomeric domain and subsequently swing toward the centromeric domain, where they establish new contacts. This transition between independent regulatory landscapes illustrates both the modularity of the limbs and the distinct evolutionary histories of its various pieces. It also allows the formation of an intermediate area of low HOX proteins content, which develops into the wrist, the transition between our arms and our hands. This regulatory strategy accounts for collinear Hox gene regulation in land vertebrate appendages.

Current mouse gene targeting technology is unable to introduce somatic mutations at a chosen time and/or in a given tissue. We report here that conditional site-specific recombination can be achieved in mice using a new version of the Cre/lox system. The Cre recombinase has been fused to a mutated ligand-binding domain of the human estrogen receptor (ER) resulting in a tamoxifen-dependent Cre recombinase, Cre-ERT, which is activated by tamoxifen, but not by estradiol. Transgenic mice were generated expressing Cre-ERT under the control of a cytomegalovirus promoter. We show that excision of a chromosomally integrated gene flanked by loxP sites can be induced by administration of tamoxifen to these transgenic mice, whereas no excision could be detected in untreated animals. This conditional sitespecific recombination system should allow the analysis of knockout phenotypes that cannot be addressed by conventional gene targeting.The study of the genetic control of mammalian development and physiology has been revolutionized by the ability to inactivate (knockout) specific genes by homologous recombination in the mouse (1). However, using current gene targeting technology, interpretations of knockout phenotypes are often limited by several factors. First, the presence of a selection marker may influence the phenotype of the mutation (2, 3). Second, artefacts can arise due to the lack of a gene product for the whole lifetime of the animal. Third, the inactivation of a gene may result in intra-utero lethality, precluding analysis of the possible function(s) of the gene at later stages of development and/or post-natally. A conditional gene targeting method based on the inducible activity of an engineered DNA recombinase could overcome these limitations by allowing the removal of the selection cassette and the timed and tissuespecific inactivation of target genes at will during development and in the adult mouse (4). Furthermore, such an inducible system could help in certain cases to distinguish between anomalies related to a mixed genetic background and those due to mutation of the targeted gene.The bacteriophage P1 Cre recombinase efficiently excises DNA flanked by two directly repeated loxP recognition sites in mammalian cells (5, 6). We have previously reported that fusion of the ligand-binding domain (LBD) of the estrogen receptor (ER) to the Cre recombinase generates a chimeric recombinase whose activity in cultured cells is dependent on the presence of an estrogen (estradiol) or an anti-estrogen (tamoxifen) (7). To achieve conditional gene targeting in mice, where endogenous estradiol is present, we have subsequently fused Cre to a mutated LBD of the human ER (Gly 521 -> Arg, G521R) resulting in the chimeric protein Cre-ERT. Indeed, the corresponding mouse ER LBD mutant (G525R) does not bind 17,B-estradiol (E2), whereas it binds the synthetic ligands tamoxifen and 4-hydroxytamoxifen (OHT) (8). We report here that Cre-ERT is a functional tamoxifen-dependent recombinase in cultured cells and in transgenic m...

During vertebrate limb development, Hoxd genes are regulated following a bimodal strategy involving two topologically associating domains (TADs) located on either side of the gene cluster. These regulatory landscapes alternatively control different subsets of Hoxd targets, first into the arm and subsequently into the digits. We studied the transition between these two global regulations, a switch that correlates with the positioning of the wrist, which articulates these two main limb segments. We show that the HOX13 proteins themselves help switch off the telomeric TAD, likely through a global repressive mechanism. At the same time, they directly interact with distal enhancers to sustain the activity of the centromeric TAD, thus explaining both the sequential and exclusive operating processes of these two regulatory domains. We propose a model in which the activation of Hox13 gene expression in distal limb cells both interrupts the proximal Hox gene regulation and re-enforces the distal regulation. In the absence of HOX13 proteins, a proximal limb structure grows without any sign of wrist articulation, likely related to an ancestral fish-like condition.

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Despite the crucial importance of Hox genes functions during animal development, the mechanisms that control their transcription in time and space are not yet fully understood. In this context, it was proposed that Hotair, a lncRNA transcribed from within the HoxC cluster regulates Hoxd gene expression in trans, through the targeting of Polycomb and consecutive transcriptional repression. This activity was recently supported by the skeletal phenotype of mice lacking Hotair function. However, other loss of function alleles at this locus did not elicit the same effects. Here, we re-analyze the molecular and phenotypic consequences of deleting the Hotair locus in vivo. In contrast with previous findings, we show that deleting Hotair has no detectable effect on Hoxd genes expression in vivo. In addition, we were unable to observe any significant morphological alteration in mice lacking the Hotair transcript. However, we find a subtle impact of deleting the Hotair locus upon the expression of the neighboring Hoxc11 and Hoxc12 genes in cis. Our results do not support any substantial role for Hotair during mammalian development in vivo. Instead, they argue in favor of a DNA-dependent effect of the Hotair deletion upon the transcriptional landscape in cis.

Using genetic and pharmacological approaches, we demonstrate that both RAR␥/RXR␣ heterodimers involved in repression events, as well as PPAR␤(␦)/RXR␣ heterodimers involved in activation events, are cell-autonomously required in suprabasal keratinocytes for the generation of lamellar granules (LG), the organelles instrumental to the formation of the skin permeability barrier. In activating PPAR␤(␦)/RXR␣ heterodimers, RXR␣ is transcriptionally active as its AF-2 activation function is required and can be inhibited by an RXR-selective antagonist. Within repressing RAR␥/RXR␣ heterodimers, induction of the transcriptional activity of RXR␣ is subordinated to the addition of an agonistic ligand for RAR␥. Thus, the ligand that possibly binds and activates RXR␣ heterodimerized with PPAR␤(␦) cannot be a retinoic acid, as it would also bind RAR␥ and relieve the RAR␥-mediated repression, thereby yielding abnormal LGs. Our data also demonstrate for the first time that subordination of RXR transcriptional activity to that of its RAR partner plays a crucial role in vivo, because it allows RXRs to act concomitantly, within the same cell, as heterodimerization partners for repression, as well as for activation events in which they are transcriptionally active.[Keywords: Conditional somatic mutagenesis; RAR␥; PPAR␤ (␦); skin permeability barrier; transcriptional subordination; ichthyosis] Supplemental material is available at http://www.genesdev.org.