RESUMO
Xenopus young tadpoles regenerate a limb with the anteroposterior (AP) pattern, but metamorphosed froglets regenerate a hypomorphic limb after amputation. The key gene for AP patterning, shh, is expressed in a regenerating limb of the tadpole but not in that of the froglet. Genomic DNA in the shh limb-specific enhancer, MFCS1 (ZRS), is hypermethylated in froglets but hypomethylated in tadpoles: shh expression may be controlled by epigenetic regulation of MFCS1. Is MFCS1 specifically activated for regenerating the AP-patterned limb? We generated transgenic Xenopus laevis lines that visualize the MFCS1 enhancer activity with a GFP reporter. The transgenic tadpoles showed GFP expression in hoxd13-and shh-expressing domains of developing and regenerating limbs, whereas the froglets showed no GFP expression in the regenerating limbs despite having hoxd13 expression. Genome sequence analysis and co-transfection assays using cultured cells revealed that Hoxd13 can activate Xenopus MFCS1. These results suggest that MFCS1 activation correlates with regeneration of AP-patterned limbs and that re-activation of epigenetically inactivated MFCS1 would be crucial to confer the ability to non-regenerative animals for regenerating a properly patterned limb.
Assuntos
Epigênese Genética , Extremidades , Animais , Xenopus laevis/genética , Animais Geneticamente Modificados , Extremidades/fisiologia , Fatores de Transcrição/genéticaRESUMO
Chronic inflammation is widely recognized as a major risk factor for cancer formation, but the underlying mechanisms are poorly understood. Recently, it was shown that Gasdermin D (GSDMD) protein drives pyroptotic cell death in macrophages on cleavage by inflammatory caspases. Even though the Gsdmd gene is specifically expressed in the intestinal epithelium, the role of Gsdmd in the intestinal tissues remains poorly characterized. In this study, we examined the biological role of Gsdmd in colorectal cancer (CRC) development, employing an azoxymethane/dextran sulfate sodium carcinogenesis model. Results show that GSDMD deficiency enhances CRC development, probably due to decreased apoptosis caused by downregulation of interferon-gamma (IFNγ)-signal transducer and activator 1 (STAT1) signaling. Furthermore, we show that GSDMD protein is diminished in human colorectal cancer, indicating involvement of GSDMD in repression of CRC development in humans. Our findings provide a new insight into functions of Gsdmd/GSDMD in colonic inflammation and human CRC development.
Assuntos
Neoplasias do Colo , Neoplasias Colorretais , Humanos , Gasderminas , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Neoplasias/genética , Apoptose , Inflamação , Neoplasias do Colo/genéticaRESUMO
The tongue is a highly specialised muscular organ with a complex anatomy required for normal function. We have utilised multiple genetic approaches to investigate local temporospatial requirements for sonic hedgehog (SHH) signalling during tongue development. Mice lacking a Shh cis-enhancer, MFCS4 (ShhMFCS4/-), with reduced SHH in dorsal tongue epithelium have perturbed lingual septum tendon formation and disrupted intrinsic muscle patterning, with these defects reproduced following global Shh deletion from E10.5 in pCag-CreERTM; Shhflox/flox embryos. SHH responsiveness was diminished in local cranial neural crest cell (CNCC) populations in both mutants, with SHH targeting these cells through the primary cilium. CNCC-specific deletion of orofaciodigital syndrome 1 (Ofd1), which encodes a ciliary protein, in Wnt1-Cre; Ofdfl/Y mice led to a complete loss of normal myotube arrangement and hypoglossia. In contrast, mesoderm-specific deletion of Ofd1 in Mesp1-Cre; Ofdfl/Y embryos resulted in normal intrinsic muscle arrangement. Collectively, these findings suggest key temporospatial requirements for local SHH signalling in tongue development (specifically, lingual tendon differentiation and intrinsic muscle patterning through signalling to CNCCs) and provide further mechanistic insight into the tongue anomalies seen in patients with disrupted hedgehog signalling.
Assuntos
Padronização Corporal , Proteínas Hedgehog/metabolismo , Crista Neural/citologia , Transdução de Sinais , Língua/embriologia , Alelos , Animais , Proliferação de Células , Elementos Facilitadores Genéticos , Feminino , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Hedgehog/genética , Heterozigoto , Ligantes , Mesoderma/metabolismo , Camundongos , Morfogênese/genética , Fenótipo , Proteínas/metabolismo , Tendões/metabolismo , Fatores de Tempo , Fator de Crescimento Transformador beta/metabolismo , Proteína Wnt1/metabolismoRESUMO
Sonic hedgehog (SHH) signaling plays a pivotal role in 2 different phases during brain development. Early SHH signaling derived from the prechordal plate (PrCP) triggers secondary Shh induction in the forebrain, which overlies the PrCP, and the induced SHH signaling, in turn, directs late neuronal differentiation of the forebrain. Consequently, Shh regulation in the PrCP is crucial for initiation of forebrain development. However, no enhancer that regulates prechordal Shh expression has yet been found. Here, we identified a prechordal enhancer, named SBE7, in the vicinity of a cluster of known forebrain enhancers for Shh This enhancer also directs Shh expression in the ventral midline of the forebrain, which receives the prechordal SHH signal. Thus, the identified enhancer acts not only for the initiation of Shh regulation in the PrCP but also for subsequent Shh induction in the forebrain. Indeed, removal of the enhancer from the mouse genome markedly down-regulated the expression of Shh in the rostral domains of the axial mesoderm and in the ventral midline of the forebrain and hypothalamus in the mouse embryo, and caused a craniofacial abnormality similar to human holoprosencephaly (HPE). These findings demonstrate that SHH signaling mediated by the newly identified enhancer is essential for development and growth of the ventral midline of the forebrain and hypothalamus. Understanding of the Shh regulation governed by this prechordal and brain enhancer provides an insight into the mechanism underlying craniofacial morphogenesis and the etiology of HPE.
Assuntos
Elementos Facilitadores Genéticos , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Hedgehog/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Prosencéfalo/embriologia , Animais , Sistemas CRISPR-Cas , Proteínas do Olho/fisiologia , Técnicas de Inativação de Genes , Genes Reporter , Proteínas Hedgehog/biossíntese , Proteínas Hedgehog/genética , Holoprosencefalia/genética , Proteínas de Homeodomínio/fisiologia , Hipotálamo/anormalidades , Hipotálamo/embriologia , Hipotálamo/metabolismo , Óperon Lac , Mesencéfalo/embriologia , Mesencéfalo/metabolismo , Mesoderma/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Prosencéfalo/anormalidades , Prosencéfalo/metabolismo , Transdução de Sinais , Transgenes , Proteína Homeobox SIX3RESUMO
Acquisition of new cis-regulatory elements (CREs) can cause alteration of developmental gene regulation and may introduce morphological novelty in evolution. Although structural variation in the genome generated by chromosomal rearrangement is one possible source of new CREs, only a few examples are known, except for cases of retrotransposition. In this study, we show the acquisition of novel regulatory sequences as a result of large genomic insertion in the spontaneous mouse mutation Hammer toe (Hm). Hm mice exhibit syndactyly with webbing, due to suppression of interdigital cell death in limb development. We reveal that, in the Hm genome, a 150-kb noncoding DNA fragment from chromosome 14 is inserted into the region upstream of the Sonic hedgehog (Shh) promoter in chromosome 5. Phenotyping of mouse embryos with a series of CRISPR/Cas9-aided partial deletion of the 150-kb insert clearly indicated that two different regions are necessary for the syndactyly phenotype of Hm We found that each of the two regions contains at least one enhancer for interdigital regulation. These results show that a set of enhancers brought by the large genomic insertion elicits the interdigital Shh expression and the Hm phenotype. Transcriptome analysis indicates that ectopic expression of Shh up-regulates Chordin (Chrd) that antagonizes bone morphogenetic protein signaling in the interdigital region. Indeed, Chrd-overexpressing transgenic mice recapitulated syndactyly with webbing. Thus, the Hm mutation provides an insight into enhancer acquisition as a source of creation of novel gene regulation.
Assuntos
Elementos Facilitadores Genéticos , Proteínas Hedgehog/genética , Sindactilia/genética , Animais , Regulação da Expressão Gênica no Desenvolvimento , Ligação Genética , Glicoproteínas/genética , Proteínas Hedgehog/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Mutantes , Camundongos Transgênicos , Mutagênese Insercional , Mutação , Fenótipo , Sindactilia/embriologia , Sindactilia/metabolismoRESUMO
The organs of vertebrate species display a wide variety of morphology. A remaining challenge in evolutionary developmental biology is to elucidate how vertebrate lineages acquire distinct morphological features. Developmental programs are driven by spatiotemporal regulation of gene expression controlled by hundreds of thousands of cis-regulatory elements. Changes in the regulatory elements caused by the introduction of genetic variants can confer regulatory innovation that may underlie morphological novelties. Recent advances in sequencing technology have revealed a number of potential regulatory variants that can alter gene expression patterns. However, a limited number of studies demonstrate causal dependence between genetic and morphological changes. Regulation of Shh expression is a good model to understand how multiple regulatory elements organize tissue-specific gene expression patterns. This model also provides insights into how evolution of molecular traits, such as gene regulatory networks, lead to phenotypic novelty.
Assuntos
Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Redes Reguladoras de Genes/genética , Proteínas Hedgehog/genética , Animais , Elementos Facilitadores Genéticos/genética , Proteínas Hedgehog/metabolismo , Humanos , FenótipoRESUMO
Coelacanths are known as "living fossils," as they show remarkable morphological resemblance to the fossil record and belong to the most primitive lineage of living Sarcopterygii (lobe-finned fishes and tetrapods). Coelacanths may be key to elucidating the tempo and mode of evolution from fish to tetrapods. Here, we report the genome sequences of five coelacanths, including four Latimeria chalumnae individuals (three specimens from Tanzania and one from Comoros) and one L. menadoensis individual from Indonesia. These sequences cover two African breeding populations and two known extant coelacanth species. The genome is â¼2.74 Gbp and contains a high proportion (â¼60%) of repetitive elements. The genetic diversity among the individuals was extremely low, suggesting a small population size and/or a slow rate of evolution. We found a substantial number of genes that encode olfactory and pheromone receptors with features characteristic of tetrapod receptors for the detection of airborne ligands. We also found that limb enhancers of bmp7 and gli3, both of which are essential for limb formation, are conserved between coelacanth and tetrapods, but not ray-finned fishes. We expect that some tetrapod-like genes may have existed early in the evolution of primitive Sarcopterygii and were later co-opted to adapt to terrestrial environments. These coelacanth genomes will provide a cornerstone for studies to elucidate how ancestral aquatic vertebrates evolved into terrestrial animals.
Assuntos
Adaptação Biológica , Evolução Molecular , Peixes/classificação , Peixes/genética , Genoma , África , Animais , Organismos Aquáticos/genética , Sequência de Bases , Biodiversidade , Proteína Morfogenética Óssea 7/genética , Extremidades/crescimento & desenvolvimento , Especiação Genética , Variação Genética , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/genética , Filogenia , Receptores Odorantes/genética , Receptores de Feromônios/genética , Análise de Sequência de DNA , Vertebrados/classificação , Vertebrados/genética , ÁguaRESUMO
Partial trisomy distal 4q (denoted 4q+) is a human chromosomal disorder caused by duplication of the distal end of the long arm of chromosome 4 (Chr4). This disorder manifests typical phenotypes, including craniofacial, renal, heart and thumb developmental defects. Although these clinical features are likely caused by a dosage imbalance in the gene network involving the trisomic region, the causative gene or genes and the molecular bases are largely unknown. Here, we report mouse Recombination-induced mutation 4 (Rim4) as a model animal of 4q+. The Rim4 genome contains an insertion of a 6.5 Mb fragment from mouse chromosome 8 into chromosome 6. This insertion fragment contains 17 genes, including Hand2, that encode the basic helix-loop-helix transcription factor and is syntenic to the distal end of human Chr4, 4q32.3 to 4q34.1, which is responsible for 4q+. A comparison of phenotypes between patients with Rim4 and 4q+ revealed that Rim4 shows direct parallels with many phenotypes of 4q+ such as craniofacial, heart, cervical vertebra and limb deformities. Rebalancing the gene dosage by a genetic cross with Hand2 knockout mice ameliorated symptoms of the heart and limb deformities of Rim4. Conversely, an increase in copy number of Hand2 in wild-type mice recaptures the heart and limb deformities of Rim4. Our results collectively demonstrate that overdosage of Hand2 is a major cause for at least the limb and heart phenotypes of 4q+ and that mouse Rim4 provides a unique animal model for understanding the molecular bases underlying the complex phenotypes of 4q+.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Dosagem de Genes , Cardiopatias Congênitas/genética , Deformidades Congênitas dos Membros/genética , Trissomia/genética , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Transtornos Cromossômicos/genética , Transtornos Cromossômicos/metabolismo , Cromossomos Humanos Par 4/genética , Modelos Animais de Doenças , Extremidades/crescimento & desenvolvimento , Feminino , Coração/crescimento & desenvolvimento , Cardiopatias Congênitas/metabolismo , Humanos , Deformidades Congênitas dos Membros/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Allele-specific monoallelic gene expression is a unique phenomenon and a great resource for analyzing gene regulation. To study this phenomenon, we established new embryonic stem (ES) cell lines derived from F1 hybrid blastocysts from crosses between four mouse subspecies (Mus musculus domesticus, C57BL/6; M. musculus molossinus, MSM/Ms; M. musculus musculus, PWK; M. musculus castaneus, HMI/Ms) and analyzed the expression levels of undifferentiated pluripotent stem cell markers and karyotypes of each line. To demonstrate the utility of our cell lines, we analyzed the allele-specific expression pattern of the Inpp5d gene as an example. The allelic expression depended on the parental alleles; this dependence could be a consequence of differences in compatibility between cis- and trans-elements of the Inpp5d gene from different subspecies. The use of parental mice from four subspecies greatly enhanced genetic polymorphism. The F1 hybrid ES cells retained this polymorphism not only in the Inpp5d gene, but also at a genome-wide level. As we demonstrated for the Inpp5d gene, the established cell lines can contribute to the analysis of allelic expression imbalance based on the incompatibility between cis- and trans-elements and of phenotypes related to this incompatibility.
Assuntos
Desequilíbrio Alélico , Animais , Camundongos , Desequilíbrio Alélico/genética , Camundongos Endogâmicos C57BL , Alelos , Expressão Gênica/genética , Linhagem Celular , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/genética , Polimorfismo Genético , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Células Híbridas , Células-Tronco Embrionárias , Feminino , Especificidade da Espécie , MasculinoRESUMO
Developing vertebrate limbs are often utilized as a model for studying pattern formation and morphogenetic cell death. Herein, we report that conditional deletion of Rac1, a member of the Rho family of proteins, in mouse limb bud mesenchyme led to skeletal deformities in the autopod and soft tissue syndactyly, with the latter caused by a complete absence of interdigital programmed cell death. Furthermore, the lack of interdigital programmed cell death and associated syndactyly was related to down-regulated gene expression of Bmp2, Bmp7, Msx1, and Msx2, which are known to promote apoptosis in the interdigital mesenchyme. Our findings from Rac1 conditional mutants indicate crucial roles for Rac1 in limb bud morphogenesis, especially interdigital programmed cell death.
Assuntos
Apoptose/fisiologia , Botões de Extremidades/embriologia , Mesoderma/enzimologia , Proteínas rac1 de Ligação ao GTP/fisiologia , Animais , Sequência de Bases , Primers do DNA , Marcadores Genéticos , Imuno-Histoquímica , Camundongos , Camundongos Transgênicos , Reação em Cadeia da Polimerase , Proteínas Wnt/metabolismoRESUMO
In tetrapod limbs, an anteriormost digit has common traits of small, short and less-phalange morphology. In this study, we focused on three genes, Mkp3, Sef and Tsukushi (TSK), which have anterior-specific or anterior-prominent expression patterns in the developing limb bud at the autopod-forming stage. The anterior expression is not fixed in the period of limb development, but the expression domains of Mkp3, Sef and TSK change considerably from the distal domain to the anterior domain. This change in expression domains, anterior shift, of these genes involves maintenance of gene expression in the anterior side and downregulation in the posterior side. Manipulated overdose of fibroblast growth factor (FGF) in the presumptive digit 2 region of chick forelimb bud results in elongation of cartilage elements of digit 2, suggesting that attenuated FGF signaling, which Mkp3, Sef, and TSK negatively regulate, provides digit 2-specific traits of morphology. The anterior expression of Mkp3 and Sef but not TSK is conserved also in limb buds of the mouse and gecko, and the anterior shift of these genes, accumulation of their transcripts in the anterior side and appropriate regulation of strength of FGF signaling may control species-specific morphology of the anteriormost digit.
Assuntos
Fosfatase 6 de Especificidade Dupla/genética , Extremidades/anatomia & histologia , Extremidades/embriologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas de Membrana/genética , Proteínas Tirosina Quinases/genética , Animais , Embrião de Galinha , Fatores de Crescimento de Fibroblastos/metabolismo , Mesoderma/citologia , Mesoderma/embriologia , Mesoderma/metabolismo , Camundongos , Camundongos Endogâmicos , Transdução de Sinais/genéticaRESUMO
Accumulation of ubiquitinated protein aggregates is a common pathology associated with a number of neurodegenerative diseases and selective autophagy plays a critical role in their elimination. Although aging-related decreases in protein degradation properties may enhance protein aggregation, it remains unclear whether proteasome dysfunction is indispensable for ubiquitinated-protein aggregation in neurodegenerative diseases. Here, we show that N-oleoyl-dopamine and N-arachidonyl-dopamine, which are endogenous brain substances and belong to the N-acyldopamine (AcylDA) family, generate cellular inclusions through aggresome formation without proteasome inhibition. Although AcylDA itself does not inhibit proteasome activity in vitro, it activates the rearrangement of vimentin distribution to form a vimentin cage surrounding aggresomes and sequesters ubiquitinated proteins in aggresomes. The gene transcription of p62/SQSTM1 was significantly increased by AcylDAs, whereas the transcription of other ubiquitin-dependent autophagy receptors was unaffected. Genetic depletion of p62 resulted in the loss of ubiquitinated-protein sequestration in aggresomes, indicating that p62 is a critical component of aggresomes. Furthermore, AcylDAs accelerate the aggregation of mutant huntingtin exon 1 proteins. These results suggest that aggresome formation does not require proteasome dysfunction and AcylDA-induced aggresome formation may participate in forming cytoplasmic protein inclusions.
Assuntos
Ácidos Araquidônicos/metabolismo , Dopamina/análogos & derivados , Regulação da Expressão Gênica/efeitos dos fármacos , Agregados Proteicos/efeitos dos fármacos , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/metabolismo , Ácidos Araquidônicos/farmacologia , Autofagia/efeitos dos fármacos , Linhagem Celular , Dopamina/metabolismo , Dopamina/farmacologia , Avaliação Pré-Clínica de Medicamentos , Humanos , Proteína Huntingtina/química , Proteína Huntingtina/genética , Leupeptinas/farmacologia , Mutação , Fosforilação/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
An enhancer named MFCS1 regulates Sonic hedgehog (Shh) expression in the posterior mesenchyme of limb buds. Several mutations in MFCS1 induce ectopic Shh expression in the anterior limb bud, and these result in preaxial polydactyly (PPD). However, the molecular basis of ectopic Shh expression remains elusive, although some mutations are known to disrupt the negative regulation of Shh expression in the anterior limb bud. Here, we analyzed the molecular mechanism of ectopic Shh expression in PPD including in a mouse mutation-hemimelic extra toes (Hx)-and in other MFCS1 mutations in different species. First, we generated transgenic mouse lines with a LacZ reporter cassette flanked with tandem repeats of 40 bp MFCS1 fragments harboring a mutation. The transgenic mouse line with the Hx-type fragment showed reporter expression exclusively in the anterior, but not in the posterior margins of limb buds. In contrast, no specific LacZ expression was observed in lines carrying the MFCS1 fragment with other mutations. Yeast one-hybrid assays revealed that the msh-like homeodomain protein, MSX1, bound specifically to the Hx sequence of MFCS1. Thus, PPD caused by mutations in MFCS1 has two major types of molecular etiology: loss of a cis-motif for negative regulation of Shh, and acquisition of a new cis-motif binding to a preexisting transcription factor, as represented by the Hx mutation.
Assuntos
Padronização Corporal/genética , Elementos Facilitadores Genéticos , Extremidades/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Hedgehog/genética , Mutação , Animais , Sequência de Bases , Sítios de Ligação , Expressão Ectópica do Gene , Expressão Gênica , Genes Reporter , Camundongos , Camundongos Transgênicos , Especificidade de Órgãos/genética , Fenótipo , Mutação Puntual , Ligação ProteicaRESUMO
Interaction between the epithelium and mesenchyme coordinates patterning and differentiation of oral cavity structures including teeth, palatal rugae and tongue papillae. SHH is one of the key signaling molecules for this interaction. Epithelial expression of Shh in the tooth buds and tongue papillae is regulated by at least two enhancers, MRCS1 and MFCS4. However, it is unclear how the two enhancers cooperate to regulate Shh. Here, we found that simultaneous deletion of MRCS1 and MFCS4 results in the formation of a supernumerary tooth in front of the first molar. Since deletion of either single enhancer barely affects tooth development, MRCS1 and MFCS4 evidently act in a redundant fashion. Binding motifs for WNT signaling mediators are shared by MRCS1 and MFCS4, and play a central role in regulating Shh expression, indicating that the two redundant enhancers additively exert their Shh regulation by responding to WNT signal input.
Assuntos
Elementos Facilitadores Genéticos/genética , Epitélio/embriologia , Epitélio/metabolismo , Proteínas Hedgehog/metabolismo , Transdução de Sinais , Dente/embriologia , Dente/metabolismo , Animais , Sequência de Bases , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Humanos , Camundongos Knockout , Motivos de Nucleotídeos/genética , Deleção de Sequência , Dente Supranumerário/genética , Xenopus/genéticaRESUMO
Shh signalling plays a crucial role for endoderm development. A Shh endoderm enhancer, MACS1, is well conserved across terrestrial animals with lungs. Here, we first show that eliminating mouse MACS1 causes severe defects in laryngeal development, indicating that MACS1-directed Shh signalling is indispensable for respiratory organogenesis. Extensive phylogenetic analyses revealed that MACS1 emerged prior to the divergence of cartilaginous and bony fishes, and even euteleost fishes have a MACS1 orthologue. Meanwhile, ray-finned fishes evolved a novel conserved non-coding sequence in the neighbouring region. Transgenic assays showed that MACS1 drives reporter expression ventrally in laryngeal epithelium. This activity has been lost in the euteleost lineage, and instead, the conserved non-coding sequence of euteleosts acquired an enhancer activity to elicit dorsal epithelial expression in the posterior pharynx and oesophagus. These results implicate that evolution of these two enhancers is relevant to the morphological transition from ventral lungs to dorsal gas bladder.
Assuntos
Sacos Aéreos/embriologia , Elementos Facilitadores Genéticos , Evolução Molecular , Proteínas Hedgehog/genética , Pulmão/embriologia , Animais , Animais Geneticamente Modificados , Sítios de Ligação , Coenzima A Ligases/genética , Peixes/embriologia , Peixes/genética , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter , Íntrons , Laringe/embriologia , Laringe/metabolismo , Camundongos , Camundongos Knockout , Proteínas Mitocondriais/genética , Oryzias , Filogenia , Sequências Reguladoras de Ácido Nucleico , Transdução de SinaisRESUMO
In a differential display screening for genes regulated by retinoic acid in the developing chick limb bud, we have isolated a novel gene, termed rigf, retinoic-acid induced growth factor, that encodes a protein belonging to the vascular endothelial growth factor (VEGF) family. Rigf transcripts were found in the posterior region of the limb bud in a region-specific manner as well as in other embryonic tissues and regions, including the notochord, head and trunk mesenchyme, retinal pigment epithelium, and branchial arches. Several manipulations revealed that retinoic acid and sonic hedgehog signaling pathways regulate rigf expression in the limb bud. VEGF family members, which promote the migration, differentiation and proliferation of endothelial cells in both blood and lymphatic vessels, are important factors for the formation of blood and lymphatic vasculatures during development. We demonstrated that the anterior border of the rigf expression domain in the limb bud corresponds with the position of the primary central artery (the subclavian artery in the forelimb), which is a main artery for supplying blood to the limb. These observations taken together with results from some experimental manipulations suggest that the limb tissue attracts blood vessels into the limb bud and that rigf is involved in the pattern formation of blood vessels in the limb.
Assuntos
Vasos Sanguíneos/embriologia , Padronização Corporal/genética , Fatores de Crescimento Endotelial/genética , Fatores de Crescimento Endotelial/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Botões de Extremidades/irrigação sanguínea , Sequência de Aminoácidos , Animais , Artérias/embriologia , Embrião de Galinha , Clonagem Molecular , Embrião não Mamífero , Proteínas Hedgehog , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Botões de Extremidades/embriologia , Linfocinas/genética , Dados de Sequência Molecular , Família Multigênica , Codorniz/embriologia , Homologia de Sequência de Aminoácidos , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Tretinoína/metabolismo , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Heart- and neural crest derivatives-expressed (Hand) proteins belong to the Twist family of the basic helix-loop-helix (bHLH) transcription factors, and play crucial roles in the development of several organs. They form heterodimers with Twist1 via their HLH domain. Disruption of the expression balance between Hand2 and Twist1 causes limb malformation, indicating that the expression level of Hand2 relative to Twist1 is essential for limb development. Mutations of the TWIST1 and TWIST2 genes are involved in human diseases. Although, the functions of the Hand proteins are indispensable for limb, heart, and craniofacial development, mutations of the Hand genes that are causative of human diseases remain elusive. Recently, comparative analyses of a human chromosomal disorder, partial trisomy distal 4q, and its mouse model, which is a spontaneously occurring mutant, clearly demonstrated that over dosage of Hand2 results in developmental defects of limbs, craniofacial, and lumbar vertebrae, and that trisomy of the Hand2 gene directly causes a human congenital disorder. In this review, we focus on gene dosage effect of Hand2 in limb, heart, and craniofacial development, and discuss its implication in human diseases.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Anormalidades Congênitas/genética , Dosagem de Genes , Regulação da Expressão Gênica no Desenvolvimento , Sequência de Aminoácidos , Animais , Cromossomos Humanos Par 4 , Embrião de Mamíferos/fisiologia , Humanos , Camundongos , Dados de Sequência Molecular , TrissomiaRESUMO
Hedgehog (Hh) signaling plays pivotal roles in morphogenesis of several embryonic tissues, including the primitive gut. In the mouse embryo, Sonic hedgehog (Shh) is expressed in endodermal epithelia from the oral cavity to the intestine, and contributes to cell proliferation in the underlying mesenchyme and subsequent differentiation into the gastrointestinal smooth muscle. Three evolutionary conserved non-coding sequences in the region upstream of the Shh coding sequence contain endoderm-specific enhancers for Shh expression. Although Shh expression in the endodermal epithelial lining is mostly attributed to these three enhancers, none of them regulates gene expression in the gastroesophageal epithelium. Here, we found that a 1.7Kb fragment located 100Kb upstream of the Shh coding sequence contains a functional element for Shh expression in endodermal organs, including the esophagus and stomach. Compared with the three known endodermal enhancers, this novel enhancer shows less evolutionary conservation, even among rodents. In mouse embryonic endodermal tissues, the seamless expression of Shh is achieved by a patchwork of multiple enhancers with different rates of evolution.
Assuntos
Diferenciação Celular/genética , Trato Gastrointestinal/crescimento & desenvolvimento , Proteínas Hedgehog/biossíntese , Pulmão/crescimento & desenvolvimento , Elementos Reguladores de Transcrição/genética , Animais , Embrião de Mamíferos/metabolismo , Endoderma/crescimento & desenvolvimento , Endoderma/metabolismo , Trato Gastrointestinal/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Hedgehog/genética , Pulmão/metabolismo , Mesoderma/crescimento & desenvolvimento , Mesoderma/metabolismo , Camundongos , Morfogênese , Transdução de SinaisRESUMO
Despite a strong clinical need for inducing scarless wound healing, the molecular factors required to accomplish it are unknown. Although skin-wound healing in adult mammals often results in scarring, some amphibians can regenerate injured body parts, even an amputated limb, without it. To understand the mechanisms of perfect skin-wound healing in regenerative tetrapods, we studied the healing process in young adult Xenopus "froglets" after experimental skin excision. We found that the excision wound healed completely in Xenopus froglets, without scarring. Mononuclear cells expressing a homeobox gene, prx1, accumulated under the new epidermis of skin wounds on the limb and trunk and at the regenerating limb. In transgenic Xenopus froglets expressing a reporter for the mouse prx1 limb-specific enhancer, activity was seen in the healing skin and in the regenerating limb. Comparable activity did not accompany skin-wound healing in adult mice. Our results suggest that scarless skin-wound healing may require activation of the prx1 limb enhancer, and competence to activate the enhancer is probably a prerequisite for epimorphic regeneration, such as limb regeneration. Finally, the induction of this prx1 enhancer activity may be useful as a reliable marker for therapeutically induced scarless wound healing in mammals.
Assuntos
Proteínas de Homeodomínio/genética , Cicatrização/genética , Proteínas de Xenopus/genética , Animais , Animais Geneticamente Modificados , Cicatriz/genética , Extremidades/fisiologia , Expressão Gênica , Camundongos , Monócitos/metabolismo , Regiões Promotoras Genéticas , Pele/metabolismo , Xenopus laevisRESUMO
The expression of Sonic hedgehog (Shh) in mouse limb buds is regulated by a long-range enhancer 1 Mb upstream of the Shh promoter. We used 3D-FISH and chromosome conformation capture assays to track changes at the Shh locus and found that long-range promoter-enhancer interactions are specific to limb bud tissues competent to express Shh. However, the Shh locus loops out from its chromosome territory only in the posterior limb bud (zone of polarizing activity or ZPA), where Shh expression is active. Notably, while Shh mRNA is detected throughout the ZPA, enhancer-promoter interactions and looping out were only observed in small fractions of ZPA cells. In situ detection of nascent Shh transcripts and unstable EGFP reporters revealed that active Shh transcription is likewise only seen in a small fraction of ZPA cells. These results suggest that chromosome conformation dynamics at the Shh locus allow transient pulses of Shh transcription.