RESUMO
Mammalian Hox gene clusters contain a range of CTCF binding sites. In addition to their importance in organizing a TAD border, which isolates the most posterior genes from the rest of the cluster, the positions and orientations of these sites suggest that CTCF may be instrumental in the selection of various subsets of contiguous genes, which are targets of distinct remote enhancers located in the flanking regulatory landscapes. We examined this possibility by producing an allelic series of cumulative in cis mutations in these sites, up to the abrogation of CTCF binding in the five sites located on one side of the TAD border. In the most impactful alleles, the global chromatin architecture of the locus was modified, yet not drastically, illustrating that CTCF sites located on one side of a strong TAD border are sufficient to organize at least part of this insulation. Spatial colinearity in the expression of these genes along the major body axis was nevertheless maintained, despite abnormal expression boundaries. In contrast, strong effects were scored in the selection of target genes responding to particular enhancers, leading to the misregulation of Hoxd genes in specific structures. Altogether, while most enhancer-promoter interactions can occur in the absence of this series of CTCF sites, the binding of CTCF in the Hox cluster is required to properly transform a rather unprecise process into a highly discriminative mechanism of interactions, which is translated into various patterns of transcription accompanied by the distinctive chromatin topology found at this locus. Our allelic series also allowed us to reveal the distinct functional contributions for CTCF sites within this Hox cluster, some acting as insulator elements, others being necessary to anchor or stabilize enhancer-promoter interactions, and some doing both, whereas they all together contribute to the formation of a TAD border. This variety of tasks may explain the amazing evolutionary conservation in the distribution of these sites among paralogous Hox clusters or between various vertebrates.
Assuntos
Cromatina , Elementos Facilitadores Genéticos , Animais , Sítios de Ligação , Fator de Ligação a CCCTC/genética , Fator de Ligação a CCCTC/metabolismo , Cromatina/genética , Elementos Facilitadores Genéticos/genética , Genes Homeobox/genética , Mamíferos/genética , Camundongos , MutagêneseRESUMO
In the course of evolution, pecorans (i.e., higher ruminants) developed a remarkable diversity of osseous cranial appendages, collectively referred to as "headgear," which likely share the same origin and genetic basis. However, the nature and function of the genetic determinants underlying their number and position remain elusive. Jacob and other rare populations of sheep and goats are characterized by polyceraty, the presence of more than two horns. Here, we characterize distinct POLYCERATE alleles in each species, both associated with defective HOXD1 function. We show that haploinsufficiency at this locus results in the splitting of horn bud primordia, likely following the abnormal extension of an initial morphogenetic field. These results highlight the key role played by this gene in headgear patterning and illustrate the evolutionary co-option of a gene involved in the early development of bilateria to properly fix the position and number of these distinctive organs of Bovidae.
Assuntos
Evolução Biológica , Cabras/genética , Proteínas de Homeodomínio/genética , Cornos , Ovinos/genética , Animais , Biometria , Regulação da Expressão Gênica no Desenvolvimento , Cabras/embriologia , Cabras/metabolismo , Proteínas de Homeodomínio/metabolismo , Masculino , Camundongos Transgênicos , Mutação , Ovinos/embriologia , Ovinos/metabolismoRESUMO
In many animal species with a bilateral symmetry, Hox genes are clustered either at one or at several genomic loci. This organization has a functional relevance, as the transcriptional control applied to each gene depends upon its relative position within the gene cluster. It was previously noted that vertebrate Hox clusters display a much higher level of genomic organization than their invertebrate counterparts. The former are always more compact than the latter, they are generally devoid of repeats and of interspersed genes, and all genes are transcribed by the same DNA strand, suggesting that particular factors constrained these clusters toward a tighter structure during the evolution of the vertebrate lineage. Here, we investigate the importance of uniform transcriptional orientation by engineering several alleles within the HoxD cluster, such as to invert one or several transcription units, with or without a neighboring CTCF site. We observe that the association between the tight structure of mammalian Hox clusters and their regulation makes inversions likely detrimental to the proper implementation of this complex genetic system. We propose that the consolidation of Hox clusters in vertebrates, including transcriptional polarity, evolved in conjunction with the emergence of global gene regulation via the flanking regulatory landscapes, to optimize a coordinated response of selected subsets of target genes in cis.
Assuntos
Genes Homeobox/genética , Família Multigênica/genética , Alelos , Animais , Fator de Ligação a CCCTC/metabolismo , Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas , Edição de Genes , Regulação da Expressão Gênica/genética , Loci Gênicos/genética , Proteínas de Homeodomínio/genética , Mamíferos/genética , Camundongos , Inversão de Sequência , Fatores de Transcrição/genética , Transcrição Gênica/genéticaRESUMO
During embryonic development, Hox genes participate in the building of a functional digestive system in metazoans, and genetic conditions involving these genes lead to important, sometimes lethal, growth retardation. Recently, this phenotype was obtained after deletion of Haglr, the Hoxd antisense growth-associated long noncoding RNA (lncRNA) located between Hoxd1 and Hoxd3 In this study, we have analyzed the function of Hoxd genes in delayed growth trajectories by looking at several nested targeted deficiencies of the mouse HoxD cluster. Mutant pups were severely stunted during the suckling period, but many recovered after weaning. After comparing seven distinct HoxD alleles, including CRISPR/Cas9 deletions involving Haglr, we identified Hoxd3 as the critical component for the gut to maintain milk-digestive competence. This essential function could be abrogated by the dominant-negative effect of HOXD10 as shown by a genetic rescue approach, thus further illustrating the importance of posterior prevalence in Hox gene function. A role for the lncRNA Haglr in the control of postnatal growth could not be corroborated.
Assuntos
Genes Homeobox/genética , RNA Longo não Codificante/genética , Alelos , Animais , Sistemas CRISPR-Cas/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas de Homeodomínio/genética , Masculino , Camundongos , Fenótipo , Fatores de Transcrição/genéticaRESUMO
Vertebrate Hox genes encode transcription factors operating during the development of multiple organs and structures. However, the evolutionary mechanism underlying this remarkable pleiotropy remains to be fully understood. Here, we show that Hoxd8 and Hoxd9, two genes of the HoxD complex, are transcribed during mammary bud (MB) development. However, unlike in other developmental contexts, their coexpression does not rely on the same regulatory mechanism. Hoxd8 is regulated by the combined activity of closely located sequences and the most distant telomeric gene desert. On the other hand, Hoxd9 is controlled by an enhancer-rich region that is also located within the telomeric gene desert but has no impact on Hoxd8 transcription, thus constituting an exception to the global regulatory logic systematically observed at this locus. The latter DNA region is also involved in Hoxd gene regulation in other contexts and strongly interacts with Hoxd9 in all tissues analyzed thus far, indicating that its regulatory activity was already operational before the appearance of mammary glands. Within this DNA region and neighboring a strong limb enhancer, we identified a short sequence conserved in therian mammals and capable of enhancer activity in the MBs. We propose that Hoxd gene regulation in embryonic MBs evolved by hijacking a preexisting regulatory landscape that was already at work before the emergence of mammals in structures such as the limbs or the intestinal tract.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Animais , Elementos Facilitadores Genéticos , Genes Homeobox , Proteínas de Homeodomínio/metabolismo , Glândulas Mamárias Animais/embriologia , Glândulas Mamárias Animais/crescimento & desenvolvimento , Glândulas Mamárias Animais/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Análise de Sequência de RNA , Transcrição GênicaRESUMO
When positioned into the integrin α-6 gene, an Hoxd9lacZ reporter transgene displayed parental imprinting in mouse embryos. While the expression from the paternal allele was comparable with patterns seen for the same transgene when present at the neighboring HoxD locus, almost no signal was scored at this integration site when the transgene was inherited from the mother, although the Itga6 locus itself is not imprinted. The transgene exhibited maternal allele-specific DNA hypermethylation acquired during oogenesis, and its expression silencing was reversible on passage through the male germ line. Histone modifications also corresponded to profiles described at known imprinted loci. Chromosome conformation analyses revealed distinct chromatin microarchitectures, with a more compact structure characterizing the maternally inherited repressed allele. Such genetic analyses of well-characterized transgene insertions associated with a de novo-induced parental imprint may help us understand the molecular determinants of imprinting.
Assuntos
Cromatina/genética , Metilação de DNA/genética , Impressão Genômica/genética , Integrina alfa6/genética , Transgenes/genética , Animais , Sequência de Bases , Cromatina/ultraestrutura , Imunoprecipitação da Cromatina , Primers do DNA/genética , Hibridização In Situ , Óperon Lac/genética , Masculino , Camundongos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , beta-GalactosidaseRESUMO
BACKGROUND: Four posterior Hoxd genes, from Hoxd13 to Hoxd10, are collectively regulated during the development of tetrapod digits. Besides the well-documented role of Hoxd13, the function of the neighboring genes has been difficult to evaluate due to the close genetic linkage and potential regulatory interferences. We used a combination of five small nested deletions in cis, involving from two to four consecutive genes of the Hoxd13 to Hoxd9 loci, in mice, to evaluate their combined functional importance. RESULTS: We show that deletions leading to a gain of function of Hoxd13, via regulatory re-allocation, generate abnormal phenotypes, in agreement with the dominant negative role of this gene. We also show that Hoxd10, Hoxd11, and Hoxd12 all seem to play a genuine role in digit development, though less compelling than that of Hoxd13. In contrast, the nearby Hoxd9 contributed no measurable function in digits. CONCLUSIONS: We conclude that a slight and transient deregulation of Hoxd13 expression can readily affect the relative lengths of limb segments and that all posterior Hoxd genes likely contribute to the final limb morphology. We discuss the difficulty to clearly assess the functional share of individual genes within such a gene family, where closely located neighbors, coding for homologous proteins, are regulated by a unique circuitry and all contribute to shape the distal parts of our appendages.
Assuntos
Extremidades/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Fatores de Transcrição/genética , Animais , Padronização Corporal/genética , Camundongos , Deleção de SequênciaRESUMO
During development of the vertebrate body axis, Hox genes are transcribed sequentially, in both time and space, following their relative positions within their genomic clusters. Analyses of animal genomes support the idea that Hox gene clustering is essential for coordinating the various times of gene activations. However, the eventual collinear ordering of the gene specific transcript domains in space does not always require genomic clustering. We analyzed these complex regulatory relationships by using mutant alleles at the mouse HoxD locus, including one that splits the cluster into two pieces. We show that both positive and negative regulatory influences, located on either side of the cluster, control an early phase of collinear expression in the trunk. Interestingly, this early phase does not systematically impact upon the subsequent expression patterns along the main body axis, indicating that the mechanism underlying temporal collinearity is distinct from those acting during the second phase. We discuss the potential functions and evolutionary origins of these mechanisms, as well as their relationship with similar processes at work during limb development.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Camundongos/crescimento & desenvolvimento , Família Multigênica , Animais , Padronização Corporal , Centrômero/genética , Proteínas de Homeodomínio/metabolismo , Camundongos/embriologia , Camundongos/genética , Camundongos/metabolismo , Deleção de SequênciaRESUMO
The potential role of Hox genes during vertebrate limb development was brought into focus by gene expression analyses in mice (P Dolle, JC Izpisua-Belmonte, H Falkenstein, A Renucci, D Duboule, Nature 1989, 342:767-772), at a time when limb growth and patterning were thought to depend upon two distinct and rather independent systems of coordinates; one for the anterior-to-posterior axis and the other for the proximal-to-distal axis (see D Duboule, P Dolle, EMBO J 1989, 8:1497-1505). Over the past years, the function and regulation of these genes have been addressed using both gain-of-function and loss-of-function approaches in chick and mice. The use of multiple mutations either in cis-configuration in trans-configuration or in cis/trans configurations, has confirmed that Hox genes are essential for proper limb development, where they participate in both the growth and organization of the structures. Even though their molecular mechanisms of action remain somewhat elusive, the results of these extensive genetic analyses confirm that, during the development of the limbs, the various axes cannot be considered in isolation from each other and that a more holistic view of limb development should prevail over a simple cartesian, chess grid-like approach of these complex structures. With this in mind, the functional input of Hox genes during limb growth and development can now be re-assessed.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Morfogênese/genética , Vertebrados/genética , Animais , Padronização Corporal/genética , Padronização Corporal/fisiologia , Extremidades/embriologia , Extremidades/crescimento & desenvolvimento , Proteínas de Homeodomínio/fisiologia , Humanos , Modelos Genéticos , Morfogênese/fisiologia , Família Multigênica , Vertebrados/embriologia , Vertebrados/crescimento & desenvolvimentoRESUMO
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.
Assuntos
Membro Anterior/embriologia , Deleção de Genes , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Animais , Padronização Corporal , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Hedgehog , Camundongos , Família Multigênica/genética , Transdução de Sinais , Transativadores/deficiência , Transativadores/genética , Transativadores/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
OBJECTIVE: We had previously reported a mouse line carrying the Atypical female courtship (HoxD Afc ) allele, where an ectopic accumulation of Hoxd10 transcripts was observed in a sparse population of cells in the adult isocortex, as a result of a partial deletion of the HoxD gene cluster. Female mice carrying this allele displayed an exacerbated paracopulatory behavior, culminating in a severe mutilation of the studs' external genitals. To unequivocally demonstrate that this intriguing phenotype was indeed caused by an illegitimate function of the HOXD10 protein, we use CRISPR/Cas9 technology to induce a microdeletion into the homeobox of the Hoxd10 gene in cis with the HoxD Afc allele. RESULTS: Females carrying this novel HoxDDel(1-9)d10hd allele no longer mutilate males. We conclude that a brain malfunction leading to a severe pathological behavior can be caused by the mere binding to DNA of a transcription factor expressed ectopically. We also show that in HoxD Afc mice, Hoxd10 was expressed in cells containing glutamate decarboxylase (Gad1) and Cholecystokinin (Cck) transcripts, corroborating our proposal that a small fraction of GABAergic neurons in adult hippocampus may participate to some aspects of female courtship.
Assuntos
Sistemas CRISPR-Cas , Corte , Proteínas de Homeodomínio/genética , Mutação , Deleção de Sequência , Animais , Colecistocinina/genética , Feminino , Expressão Gênica , Glutamato Descarboxilase/genética , Hibridização in Situ Fluorescente , Masculino , Camundongos , Neocórtex/citologia , Neocórtex/metabolismo , Neocórtex/fisiologia , Fatores de Transcrição/genéticaRESUMO
Genes belonging to both HoxA and HoxD clusters are required for proper vertebrate limb development. Mice lacking all, or parts of, Hoxa and Hoxd functions in forelimbs, as well as mice with a gain of function of these genes in the early limb bud, have helped us to understand functional and regulatory issues associated with these genes, such that, for example, the tight mechanistic interdependency that exists between the production of the limb and its anterior to posterior (AP) polarity. Our studies suggest that the evolutionary recruitment of Hox gene function into growing appendages was crucial to implement hedgehog signalling, subsequently leading to the distal extension of tetrapod appendages, with an already built-in AP polarity. We propose that this process results from the evolutionary co-option, in the developing limbs, of a particular regulatory mechanism (collinearity), which is necessary to pattern the developing trunk. This major regulatory constraint imposed a polarity to our limbs as the most parsimonious solution to grow appendages.
Assuntos
Evolução Biológica , Extremidades/embriologia , Extremidades/fisiologia , Animais , Padronização Corporal , Evolução Molecular , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Hedgehog/metabolismo , Humanos , Botões de Extremidades , Camundongos , Modelos Biológicos , VertebradosRESUMO
The number of phalanges and joints are key features of digit 'identity' and are central to limb functionality and evolutionary adaptation. Prior chick work indicated that digit phalanges and their associated joints arise in a different manner than the more sparsely jointed long bones, and their identity is regulated by differential signalling from adjacent interdigits. Currently, there is no genetic evidence for this model, and the molecular mechanisms governing digit joint specification remain poorly understood. Using genetic approaches in mouse, here we show that functional 5'Hoxd-Gli3 antagonism acts indirectly, through Bmp signalling from the interdigital mesenchyme, to regulate specification of joint progenitors, which arise in conjunction with phalangeal precursors at the digit tip. Phalanx number, although co-regulated, can be uncoupled from joint specification. We propose that 5'Hoxd genes and Gli3 are part of an interdigital signalling centre that sets net Bmp signalling levels from different interdigits to coordinately regulate phalanx and joint formation.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Extremidades/embriologia , Proteínas de Homeodomínio/fisiologia , Articulações/embriologia , Proteínas do Tecido Nervoso/fisiologia , Proteína Gli3 com Dedos de Zinco/fisiologia , Animais , Proteínas de Transporte/metabolismo , Dosagem de Genes , Técnicas de Introdução de Genes , Articulações/metabolismo , Camundongos , FenótipoRESUMO
Although neural substrates of mammalian female mating behavior have been described, the association between complex courtship activity and specific underlying mechanisms remains elusive. We have isolated a mouse line that unexpectedly shows altered female social behavior with increased investigation of males and increased genital biting. We investigated adult individuals by behavioral observation and genetic and molecular neuroanatomy methods. We report exacerbated inverse pursuits and incapacitating bites directed at the genitals of stud males. This extreme deviation from wild-type female courtship segregates with a deletion of the Hoxd1 to Hoxd9 genomic region. This dominant Atypical female courtship allele (HoxD(Afc)) induces ectopic Hoxd10 gene expression in several regions in newborn forebrain transitorily and stably in a sparse subpopulation of cells in the cornu ammonis fields of adult hippocampus, which may thus lead to an abnormal modulation in the sexual behavior of mutant females. The resulting compulsive sexual solicitation behavior displayed by the most affected individuals suggests new avenues to study the genetic and molecular bases of normal and pathological mammalian affect and raises the potential involvement of the hippocampus in the control of female courtship behavior. The potential relevance to human 2q.31.1 microdeletion syndrome is discussed.
Assuntos
Genes Homeobox , Hipocampo/anormalidades , Proteínas de Homeodomínio/genética , Comportamento Sexual Animal , Animais , Feminino , Genótipo , Proteínas de Homeodomínio/fisiologia , Masculino , Camundongos , Mutação , Deleção de SequênciaRESUMO
Finding sequences that control expression of genes is central to understanding genome function. Previous studies have used evolutionary conservation as an indicator of regulatory potential. Here, we present a method for the unbiased in vivo screen of putative enhancers in large DNA regions, using the mouse as a model. We cloned a library of 142 overlapping fragments from a 200 kb-long murine BAC in a lentiviral vector expressing LacZ from a minimal promoter, and used the resulting vectors to infect fertilized murine oocytes. LacZ staining of E11 embryos obtained by first using the vectors in pools and then testing individual candidates led to the identification of 3 enhancers, only one of which shows significant evolutionary conservation. In situ hybridization and 3C/4C experiments suggest that this enhancer, which is active in the neural tube and posterior diencephalon, influences the expression of the Olig1 and/or Olig2 genes. This work provides a new approach for the large-scale in vivo screening of transcriptional regulatory sequences, and further demonstrates that evolutionary conservation alone seems too limiting a criterion for the identification of enhancers.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Lentivirus/genética , Proteínas do Tecido Nervoso/genética , Animais , Sistema Nervoso Central/metabolismo , Galinhas , Cromossomos Artificiais Bacterianos , Elementos Facilitadores Genéticos , Humanos , Hibridização In Situ , Óperon Lac , Camundongos , Fator de Transcrição 2 de Oligodendrócitos , Regiões Promotoras Genéticas , Análise de Sequência de DNA , TransgenesRESUMO
The digestive tract is made of different subdivisions with various functions. During embryonic development, the developing intestine expresses combinations of Hox genes along its anterior to posterior axis, suggesting a role for these genes in this regionalization process. In particular, the transition from small to large intestine is labelled by the transcription of all Hoxd genes except Hoxd12 and Hoxd13, the latter two genes being transcribed only near the anus. Here, we describe two lines of mice that express Hoxd12 ectopically within this morphological transition. As a consequence, budding of the caecum is impeded, leading to complete agenesis in homozygous individuals. This effect is concurrent with a dramatic reduction of both Fgf10 and Pitx1 expression. Furthermore, the interactions between ;anterior' Hox genes and ectopic Hoxd12 suggest a model whereby anterior and posterior Hox products compete in controlling Fgf10 signalling, which is required for the growth of this organ in mice. These results illuminate components of the genetic cascade necessary for the emergence of this gut segment, crucial for many vertebrates.
Assuntos
Ceco/embriologia , Genes Homeobox/fisiologia , Intestinos/embriologia , Animais , Animais Recém-Nascidos , Ceco/crescimento & desenvolvimento , Ceco/metabolismo , Feminino , Mucosa Intestinal/metabolismo , Intestinos/crescimento & desenvolvimento , Masculino , Camundongos , Camundongos Transgênicos , Família Multigênica , Especificidade de Órgãos , Gravidez , Vertebrados/embriologia , Vertebrados/genéticaRESUMO
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
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.