RESUMO
Human families with chromosomal rearrangements at 2q31, where the human HOXD locus maps, display mesomelic dysplasia, a severe shortening and bending of the limb. In mice, the dominant Ulnaless inversion of the HoxD cluster produces a similar phenotype suggesting the same origin for these malformations in humans and mice. Here we engineer 1 Mb inversion including the HoxD gene cluster, which positioned Hoxd13 close to proximal limb enhancers. Using this model, we show that these enhancers contact and activate Hoxd13 in proximal cells, inducing the formation of mesomelic dysplasia. We show that a secondary Hoxd13 null mutation in-cis with the inversion completely rescues the alterations, demonstrating that ectopic HOXD13 is directly responsible for this bone anomaly. Single-cell expression analysis and evaluation of HOXD13 binding sites suggests that the phenotype arises primarily by acting through genes normally controlled by HOXD13 in distal limb cells. Altogether, these results provide a conceptual and mechanistic framework to understand and unify the molecular origins of human mesomelic dysplasia associated with 2q31.
Assuntos
Anormalidades Múltiplas/genética , Doenças do Desenvolvimento Ósseo/genética , Proteínas de Homeodomínio/genética , Deformidades Congênitas dos Membros/genética , Fatores de Transcrição/genética , Anormalidades Múltiplas/embriologia , Anormalidades Múltiplas/metabolismo , Animais , Doenças do Desenvolvimento Ósseo/embriologia , Doenças do Desenvolvimento Ósseo/metabolismo , Modelos Animais de Doenças , Feminino , Deleção de Genes , Proteínas de Homeodomínio/metabolismo , Humanos , Deformidades Congênitas dos Membros/embriologia , Deformidades Congênitas dos Membros/metabolismo , Mutação com Perda de Função , Masculino , Camundongos Endogâmicos C57BL , Família Multigênica , Fatores de Transcrição/metabolismoRESUMO
Vertebrate Hox genes are critical for the establishment of structures during the development of the main body axis. Subsequently, they play important roles either in organizing secondary axial structures such as the appendages, or during homeostasis in postnatal stages and adulthood. Here, we set up to analyze their elusive function in the ectodermal compartment, using the mouse limb bud as a model. We report that the HoxC gene cluster was co-opted to be transcribed in the distal limb ectoderm, where it is activated following the rule of temporal colinearity. These ectodermal cells subsequently produce various keratinized organs such as nails or claws. Accordingly, deletion of the HoxC cluster led to mice lacking nails (anonychia), a condition stronger than the previously reported loss of function of Hoxc13, which is the causative gene of the ectodermal dysplasia 9 (ECTD9) in human patients. We further identified two mammalian-specific ectodermal enhancers located upstream of the HoxC gene cluster, which together regulate Hoxc gene expression in the hair and nail ectodermal organs. Deletion of these regulatory elements alone or in combination revealed a strong quantitative component in the regulation of Hoxc genes in the ectoderm, suggesting that these two enhancers may have evolved along with the mammalian taxon to provide the level of HOXC proteins necessary for the full development of hair and nail.
Assuntos
Ectoderma/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Genes Homeobox , Folículo Piloso/metabolismo , Unhas/metabolismo , Animais , Biomarcadores , Ectoderma/embriologia , Folículo Piloso/embriologia , Humanos , Camundongos , Camundongos Knockout , Unhas/embriologiaRESUMO
The HoxD cluster is critical for vertebrate limb development. Enhancers located in both the telomeric and centromeric gene deserts flanking the cluster regulate the transcription of HoxD genes. In rare patients, duplications, balanced translocations or inversions misregulating HOXD genes are responsible for mesomelic dysplasia of the upper and lower limbs. By aCGH, whole-genome mate-pair sequencing, long-range PCR and fiber fluorescent in situ hybridization, we studied patients from two families displaying mesomelic dysplasia limited to the upper limbs. We identified microduplications including the HOXD cluster and showed that microduplications were in an inverted orientation and inserted between the HOXD cluster and the telomeric enhancers. Our results highlight the existence of an autosomal dominant condition consisting of isolated ulnar dysplasia caused by microduplications inserted between the HOXD cluster and the telomeric enhancers. The duplications likely disconnect the HOXD9 to HOXD11 genes from their regulatory sequences. This presumptive loss-of-function may have contributed to the phenotype. In both cases, however, these rearrangements brought HOXD13 closer to telomeric enhancers, suggesting that the alterations derive from the dominant-negative effect of this digit-specific protein when ectopically expressed during the early development of forearms, through the disruption of topologically associating domain structure at the HOXD locus.
Assuntos
Doenças do Desenvolvimento Ósseo/genética , Duplicação Gênica , Proteínas de Homeodomínio/genética , Deformidades Congênitas das Extremidades Superiores/genética , Doenças do Desenvolvimento Ósseo/patologia , Células Cultivadas , Feminino , Humanos , Lactente , Mutação com Perda de Função , Masculino , Família Multigênica , Fenótipo , Deformidades Congênitas das Extremidades Superiores/patologiaRESUMO
The molecular mechanisms that govern bone and joint formation are complex, involving an integrated network of signaling pathways and gene regulators. We investigated the role of Hox genes, which are known to specify individual segments of the skeleton, in the formation of autopod limb bones (i.e., the hands and feet) using the mouse mutant synpolydactyly homolog (spdh), which encodes a polyalanine expansion in Hoxd13. We found that no cortical bone was formed in the autopod in spdh/spdh mice; instead, these bones underwent trabecular ossification after birth. Spdh/spdh metacarpals acquired an ovoid shape and developed ectopic joints, indicating a loss of long bone characteristics and thus a transformation of metacarpals into carpal bones. The perichondrium of spdh/spdh mice showed abnormal morphology and decreased expression of Runt-related transcription factor 2 (Runx2), which was identified as a direct Hoxd13 transcriptional target. Hoxd11-/-Hoxd12-/-Hoxd13-/- triple-knockout mice and Hoxd13-/-Hoxa13+/- mice exhibited similar but less severe defects, suggesting that these Hox genes have similar and complementary functions and that the spdh allele acts as a dominant negative. This effect was shown to be due to sequestration of other polyalanine-containing transcription factors by the mutant Hoxd13 in the cytoplasm, leading to their degradation. These data indicate that Hox genes not only regulate patterning but also directly influence bone formation and the ossification pattern of bones, in part via Runx2.
Assuntos
Osso e Ossos/metabolismo , Cartilagem/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Alelos , Animais , Extremidades , Camundongos , Camundongos Knockout , Peptídeos , Transdução de Sinais/genética , Fatores de Transcrição/biossínteseRESUMO
Hox genes encode transcription factors that play a central role in the specification of regional identities along the anterior to posterior body axis. In the developing mouse embryo, Hox genes from all four genomic clusters are involved in range of developmental processes, including the patterning of skeletal structures and the formation of several organs. However, the functional redundancy observed either between paralogous genes, or among neighboring genes from the same cluster, has hampered functional analyses, in particular when synergistic, cluster-specific functions are considered. Here, we report that mutant mice lacking the entire HoxA cluster in mesodermal lineages display the expected spectrum of postnatal respiratory, cardiac and urogenital defects, previously reported for single gene mutations. Likewise, mild phenotypes are observed in both appendicular and axial skeleton. However, a striking effect was uncovered in the hematopoietic system, much stronger than that seen for Hoxa9 inactivation alone, which involves stem cells (HSCs) as well as the erythroid lineage, indicating that several Hoxa genes are necessary for normal hematopoiesis to occur. Finally, the combined deletions of Hoxa and Hoxd genes reveal abnormalities in axial elongation as well as skin morphogenesis that are likely the results of defects in epithelial-mesenchymal interactions.
Assuntos
Proteínas de Homeodomínio/metabolismo , Mesoderma/metabolismo , Animais , Osso e Ossos/embriologia , Osso e Ossos/metabolismo , Hematopoese , Proteínas de Homeodomínio/genética , Camundongos , Morfogênese , Pele/embriologia , Pele/metabolismoRESUMO
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.
Assuntos
Padronização Corporal , Região Branquial/embriologia , Crista Neural/embriologia , Animais , Animais Recém-Nascidos , Região Branquial/citologia , Região Branquial/metabolismo , Cartilagem/citologia , Cartilagem/embriologia , Cartilagem/metabolismo , Coristoma/metabolismo , Embrião de Mamíferos/anormalidades , Embrião de Mamíferos/patologia , Deleção de Genes , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Osso Hioide/citologia , Osso Hioide/embriologia , Osso Hioide/metabolismo , Camundongos , Camundongos Mutantes , Modelos Biológicos , Família Multigênica , Crista Neural/citologia , Crista Neural/metabolismoRESUMO
Hox genes encode homeodomain-containing proteins that control embryonic development in multiple contexts. Up to 30 Hox genes, distributed among all four clusters, are expressed during mammalian kidney morphogenesis, but functional redundancy between them has made a detailed functional account difficult to achieve. We have investigated the role of the HoxD cluster through comparative molecular embryological analysis of a set of mouse strains carrying targeted genomic rearrangements such as deletions, duplications, and inversions. This analysis allowed us to uncover and genetically dissect the complex role of the HoxD cluster. Regulation of metanephric mesenchyme-ureteric bud interactions and maintenance of structural integrity of tubular epithelia are differentially controlled by some Hoxd genes during renal development, consistent with their specific expression profiles. We also provide evidence for a kidney-specific form of colinearity that underlies the differential expression of two distinct sets of genes located on both sides and overlapping at the Hoxd9 locus. These insights further our knowledge of the genetic control of kidney morphogenesis and may contribute to understanding certain congenital kidney malformations, including polycystic kidney disease and renal hypoplasia.
Assuntos
Genes Homeobox , Rim/embriologia , Família Multigênica , Animais , Animais Recém-Nascidos , Apoptose , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Feminino , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Marcação de Genes , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/fisiologia , Rim/anormalidades , Rim/metabolismo , Túbulos Renais/metabolismo , Túbulos Renais/patologia , Óperon Lac , Mesoderma/anormalidades , Mesoderma/embriologia , Mesoderma/metabolismo , Camundongos , Camundongos Knockout , Camundongos Mutantes , Camundongos Transgênicos , Proteínas de Neoplasias/deficiência , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiologia , Fenótipo , Doenças Renais Policísticas/embriologia , Doenças Renais Policísticas/genética , Doenças Renais Policísticas/patologia , Gravidez , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , Ureter/anormalidades , Ureter/embriologia , Ureter/metabolismoRESUMO
The development of the vertebrate limb is dependent upon two signaling centers, the apical ectodermal ridge (AER), which provides the underlying mesenchyme with essential growth factors, and the zone of polarizing activity (ZPA), the source of the Sonic hedgehog (SHH) product. Recent work involving gain and loss of function of Hox genes has emphasized their impact both on AER maintenance and Shh transcriptional activation. Here, we describe antagonistic interactions between posterior Hoxd genes and Gli3, suggesting that the latter product protects the AER from the deleterious effect of the formers, and we present evidence that Fgf10 is the mediator of HOX-dependent AER expansion. Furthermore, the striking similarity between some of the hereby observed Hox/Gli3-dependent morphogenetic defects and those displayed by fetuses with severely altered retinoic acid metabolism suggests a tight connection between these various pathways. The nature of these potential interactions is discussed in the context of proximal-distal growth and patterning.
Assuntos
Ectoderma/metabolismo , Extremidades/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/fisiologia , Fatores de Transcrição Kruppel-Like/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Animais , Padronização Corporal , Fator 10 de Crescimento de Fibroblastos/metabolismo , Proteínas de Homeodomínio/genética , Homozigoto , Fatores de Transcrição Kruppel-Like/genética , Botões de Extremidades/metabolismo , Camundongos , Mutação , Proteínas do Tecido Nervoso/genética , Fenótipo , Transdução de Sinais , Ativação Transcricional , Proteína Gli3 com Dedos de ZincoRESUMO
Deficiencies or mutations in the human pseudoautosomal SHOX gene are associated with a series of short-stature conditions, including Turner syndrome, Leri-Weill dyschondrosteosis, and Langer mesomelic dysplasia. Although this gene is absent from the mouse genome, the closely related paralogous gene Shox2 displays a similar expression pattern in developing limbs. Here, we report that the conditional inactivation of Shox2 in developing appendages leads to a strong phenotype, similar to the human conditions, although it affects a different proximodistal limb segment. Furthermore, using this mouse model, we establish the cellular etiology of these defects and show that Shox2 acts upstream the Runx2 gene, a key regulator of chondrogenesis.
Assuntos
Condrogênese/genética , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Modelos Animais de Doenças , Regulação da Expressão Gênica no Desenvolvimento , Transtornos do Crescimento/genética , Proteínas de Homeodomínio/metabolismo , Camundongos/genética , Animais , Desenvolvimento Ósseo/genética , Osso e Ossos/anormalidades , Condrócitos/química , Condrócitos/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/análise , Transtornos do Crescimento/patologia , Proteínas de Homeodomínio/genética , Humanos , Deformidades Congênitas dos Membros/genética , Deformidades Congênitas dos Membros/patologia , Camundongos Mutantes , Fenótipo , Deleção de Sequência , SíndromeRESUMO
Mammalian Hox genes encode transcription factors that are crucial for proper morphogenesis along the various body axes. Despite their extensive structural and functional characterization, the nature of their target genes remains elusive. We have addressed this question by using DNA microarrays to screen for genes whose expression in developing distal forelimbs and genital eminences was significantly modified in the absence of the full Hoxd gene complement. This comparative approach not only identified specific candidate genes, but also allowed the examination of whether a similar Hox expression pattern in distinct tissues leads to the modulation of the same or different downstream genes. We report here a set of potential target genes, most of which were not previously known to play a role in the early stages of either limb or genital bud development. Interestingly, we find that the majority of these candidate genes are differentially expressed in both structures, although often at different times. This supports the idea that both appendices involve similar genetic controls, both upstream and downstream of the Hox gene family. These results highlight the surprising mechanistic relationship between these rather different body parts and suggest a common developmental strategy to build up the most distal appendicular structures of the body, i.e. the digits and the penis/clitoris.
Assuntos
Extremidades/embriologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Genitália Feminina/embriologia , Genitália Masculina/embriologia , Proteínas de Homeodomínio/genética , Família Multigênica , Animais , Feminino , Proteínas de Homeodomínio/metabolismo , Proteínas Imediatamente Precoces , Hibridização In Situ , Masculino , Proteínas de Membrana , Camundongos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-ret , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Receptor EphA3/genética , Receptor EphA3/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/genéticaRESUMO
Anterior-to-posterior patterning, the process whereby our digits are differently shaped, is a key aspect of limb development. It depends on the localized expression in posterior limb bud of Sonic hedgehog (Shh) and the morphogenetic potential of its diffusing product. By using an inversion of and a large deficiency in the mouse HoxD cluster, we found that a perturbation in the early collinear expression of Hoxd11, Hoxd12, and Hoxd13 in limb buds led to a loss of asymmetry. Ectopic Hox gene expression triggered abnormal Shh transcription, which in turn induced symmetrical expression of Hox genes in digits, thereby generating double posterior limbs. We conclude that early posterior restriction of Hox gene products sets up an anterior-posterior prepattern, which determines the localized activation of Shh. This signal is subsequently translated into digit morphological asymmetry by promoting the late expression of Hoxd genes, two collinear processes relying on opposite genomic topographies, upstream and downstream Shh signaling.
Assuntos
Padronização Corporal , Membro Anterior/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Genes Homeobox , Botões de Extremidades/embriologia , Proteínas do Tecido Nervoso , Transativadores/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Inversão Cromossômica , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Membro Anterior/anormalidades , Marcação de Genes , Proteínas Hedgehog , Heterozigoto , Membro Posterior/anormalidades , Membro Posterior/embriologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Homozigoto , Fatores de Transcrição Kruppel-Like , Botões de Extremidades/metabolismo , Camundongos , Morfogênese , Recombinação Genética , Transdução de Sinais , Dedos do Pé/anormalidades , Dedos do Pé/embriologia , Transativadores/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica , Proteínas de Peixe-Zebra , Proteína Gli3 com Dedos de ZincoRESUMO
DBP (albumin D-site-binding protein), HLF (hepatic leukemia factor), and TEF (thyrotroph embryonic factor) are the three members of the PAR bZip (proline and acidic amino acid-rich basic leucine zipper) transcription factor family. All three of these transcriptional regulatory proteins accumulate with robust circadian rhythms in tissues with high amplitudes of clock gene expression, such as the suprachiasmatic nucleus (SCN) and the liver. However, they are expressed at nearly invariable levels in most brain regions, in which clock gene expression only cycles with low amplitude. Here we show that mice deficient for all three PAR bZip proteins are highly susceptible to generalized spontaneous and audiogenic epilepsies that frequently are lethal. Transcriptome profiling revealed pyridoxal kinase (Pdxk) as a target gene of PAR bZip proteins in both liver and brain. Pyridoxal kinase converts vitamin B6 derivatives into pyridoxal phosphate (PLP), the coenzyme of many enzymes involved in amino acid and neurotransmitter metabolism. PAR bZip-deficient mice show decreased brain levels of PLP, serotonin, and dopamine, and such changes have previously been reported to cause epilepsies in other systems. Hence, the expression of some clock-controlled genes, such as Pdxk, may have to remain within narrow limits in the brain. This could explain why the circadian oscillator has evolved to generate only low-amplitude cycles in most brain regions.
Assuntos
Ritmo Circadiano , Epilepsia/etiologia , Proteínas da Matriz Extracelular/deficiência , Proteínas da Matriz Extracelular/fisiologia , Glicoproteínas/deficiência , Glicoproteínas/fisiologia , Animais , Fatores de Transcrição de Zíper de Leucina Básica , Encéfalo/metabolismo , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Eletroencefalografia , Eletromiografia , Proteínas da Matriz Extracelular/análise , Glicoproteínas/análise , Fígado/metabolismo , Camundongos , Camundongos Knockout , Neurotransmissores/metabolismo , Piridoxal Quinase/genética , RNA Mensageiro/análise , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologiaRESUMO
Mesomelic dysplasia is a severe shortening of forearms and forelegs, and is found in several distinct human syndromes. Here, we report the cloning of the breakpoints of a human t(2;8)(q31;p21) balanced translocation associated with mesomelic dysplasia of the upper limbs, as well as with vertebral defects. We show that this translocation does not disrupt any gene, hence it most likely exerts its deleterious effect by modifying gene regulation. The HOXD complex lies approximately 60 kb from the translocation breakpoint on chromosome 2. This cluster of genes has an important role in the development of both the vertebral column and the limbs. Only a few cases of mutations of these homeotic genes have been described so far in humans. However, gain- and loss-of-function of Hoxd genes in mice can induce mesomelic dysplasia-like phenotypes, suggesting that misexpression of HOXD genes may indeed be at the origin of this hereditary phenotype.
Assuntos
Doenças do Desenvolvimento Ósseo/genética , Genes Homeobox , Coluna Vertebral/anormalidades , Translocação Genética , Braço/anormalidades , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Doenças do Desenvolvimento Ósseo/patologia , Mapeamento Cromossômico , Cromossomos Humanos Par 2/genética , Cromossomos Humanos Par 8/genética , Proteínas de Ligação a DNA/genética , Evolução Molecular , Feminino , Proteínas de Homeodomínio/genética , Humanos , Hibridização in Situ Fluorescente , Perna (Membro)/anormalidades , Masculino , Análise de Sequência de DNA , Fatores de Transcrição/genética , Proteínas de Peixe-ZebraRESUMO
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.