RESUMO
Gene duplication and divergence is a major driver in the emergence of evolutionary novelties. How variations in amino acid sequences lead to loss of ancestral activity and functional diversification of proteins is poorly understood. We used cross-species functional analysis of Drosophila Labial and its mouse HOX1 orthologs (HOXA1, HOXB1, and HOXD1) as a paradigm to address this issue. Mouse HOX1 proteins display low (30%) sequence similarity with Drosophila Labial. However, substituting endogenous Labial with the mouse proteins revealed that HOXA1 has retained essential ancestral functions of Labial, while HOXB1 and HOXD1 have diverged. Genome-wide analysis demonstrated similar DNA-binding patterns of HOXA1 and Labial in mouse cells, while HOXB1 binds to distinct targets. Compared with HOXB1, HOXA1 shows an enrichment in co-occupancy with PBX proteins on target sites and exists in the same complex with PBX on chromatin. Functional analysis of HOXA1-HOXB1 chimeric proteins uncovered a novel six-amino-acid C-terminal motif (CTM) flanking the homeodomain that serves as a major determinant of ancestral activity. In vitro DNA-binding experiments and structural prediction show that CTM provides an important domain for interaction of HOXA1 proteins with PBX. Our findings show that small changes outside of highly conserved DNA-binding regions can lead to profound changes in protein function.
Assuntos
Motivos de Aminoácidos/genética , Proteínas de Drosophila/genética , Evolução Molecular , Proteínas de Homeodomínio/genética , Animais , Drosophila melanogaster/classificação , Drosophila melanogaster/genética , Estudo de Associação Genômica Ampla , Camundongos , Modelos Moleculares , Ligação Proteica/genética , Domínios Proteicos , Relação Estrutura-AtividadeRESUMO
Signaling pathways regulate the patterns of Hox gene expression that underlie their functions in the specification of axial identity. Little is known about the properties of cis-regulatory elements and underlying transcriptional mechanisms that integrate graded signaling inputs to coordinately control Hox expression. Here, we optimized a single molecule fluorescent in situ hybridization (smFISH) technique with probes spanning introns to evaluate how three shared retinoic acid response element (RARE)-dependent enhancers in the Hoxb cluster regulate patterns of nascent transcription in vivo at the level of single cells in wild-type and mutant embryos. We predominately detect nascent transcription of only a single Hoxb gene in each cell, with no evidence for simultaneous co-transcriptional coupling of all or specific subsets of genes. Single and/or compound RARE mutations indicate that each enhancer differentially impacts global and local patterns of nascent transcription, suggesting that selectivity and competitive interactions between these enhancers is important to robustly maintain the proper levels and patterns of nascent Hoxb transcription. This implies that rapid and dynamic regulatory interactions potentiate transcription of genes through combined inputs from these enhancers in coordinating the retinoic acid response.
Assuntos
Proteínas de Homeodomínio , Tretinoína , Camundongos , Animais , Tretinoína/metabolismo , Proteínas de Homeodomínio/metabolismo , Camundongos Transgênicos , Tubo Neural/metabolismo , Hibridização in Situ Fluorescente , Elementos Facilitadores GenéticosRESUMO
During early development, the hindbrain is sub-divided into rhombomeres that underlie the organisation of neurons and adjacent craniofacial tissues. A gene regulatory network of signals and transcription factors establish and pattern segments with a distinct anteroposterior identity. Initially, the borders of segmental gene expression are imprecise, but then become sharply defined, and specialised boundary cells form. In this Review, we summarise key aspects of the conserved regulatory cascade that underlies the formation of hindbrain segments. We describe how the pattern is sharpened and stabilised through the dynamic regulation of cell identity, acting in parallel with cell segregation. Finally, we discuss evidence that boundary cells have roles in local patterning, and act as a site of neurogenesis within the hindbrain.
Assuntos
Padronização Corporal/fisiologia , Rombencéfalo/crescimento & desenvolvimento , Rombencéfalo/fisiologia , Vertebrados/crescimento & desenvolvimento , Vertebrados/fisiologia , Animais , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Redes Reguladoras de Genes/fisiologia , HumanosRESUMO
Segmentation is an important process that is frequently used during development to segregate groups of cells with distinct features. Segmental compartments provide a mechanism for generating and organizing regional properties along an embryonic axis and within tissues. In vertebrates the development of two major systems, the hindbrain and the paraxial mesoderm, displays overt signs of compartmentalization and depends on the process of segmentation for their functional organization. The hindbrain plays a key role in regulating head development, and it is a complex coordination center for motor activity, breathing rhythms, and many unconscious functions. The paraxial mesoderm generates somites, which give rise to the axial skeleton. The cellular processes of segmentation in these two systems depend on ordered patterns of Hox gene expression as a mechanism for generating a combinatorial code that specifies unique identities of the segments and their derivatives. In this review, we compare and contrast the signaling inputs and transcriptional mechanisms by which Hox gene regulatory networks are established during segmentation in these two different systems.
Assuntos
Osso e Ossos/embriologia , Extremidades/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Genes Homeobox , Rombencéfalo/embriologia , Vertebrados/embriologia , Animais , Padronização Corporal , Mesoderma/metabolismo , Vertebrados/metabolismoRESUMO
Meis genes are known to play important roles in the hindbrain and neural crest cells of jawed vertebrates. To explore the roles of Meis genes in head development during evolution of vertebrates, we have identified four meis genes in the sea lamprey genome and characterized their patterns of expression and regulation, with a focus on the hindbrain and pharynx. Each of the lamprey meis genes displays temporally and spatially dynamic patterns of expression, some of which are coupled to rhombomeric domains in the developing hindbrain and select pharyngeal arches. Studies of Meis loci in mouse and zebrafish have identified enhancers that are bound by Hox and TALE (Meis and Pbx) proteins, implicating these factors in the direct regulation of Meis expression. We examined the lamprey meis loci and identified a series of cis-elements conserved between lamprey and jawed vertebrate meis genes. In transgenic reporter assays we demonstrated that these elements act as neural enhancers in lamprey embryos, directing reporter expression in appropriate domains when compared to expression of their associated endogenous meis gene. Sequence alignments reveal that these conserved elements are in similar relative positions of the meis loci and contain a series of consensus binding motifs for Hox and TALE proteins. This suggests that ancient Hox and TALE-responsive enhancers regulated expression of ancestral vertebrate meis genes in segmental domains in the hindbrain and have been retained in the meis loci during vertebrate evolution. The presence of conserved Meis, Pbx and Hox binding sites in these lamprey enhancers links Hox and TALE factors to regulation of lamprey meis genes in the developing hindbrain, indicating a deep ancestry for these regulatory interactions prior to the divergence of jawed and jawless vertebrates.
Assuntos
Lampreias/genética , Tubo Neural/embriologia , Rombencéfalo/embriologia , Animais , Sítios de Ligação , Padronização Corporal/genética , Sequência Conservada , Elementos Facilitadores Genéticos , Expressão Gênica/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Genes Homeobox/genética , Proteínas de Homeodomínio/metabolismo , Lampreias/metabolismo , Proteína Meis1/genética , Proteína Meis1/metabolismo , Crista Neural/metabolismo , Tubo Neural/metabolismo , Fator de Transcrição 1 de Leucemia de Células Pré-B/genética , Fator de Transcrição 1 de Leucemia de Células Pré-B/metabolismo , Rombencéfalo/metabolismo , Fatores de Transcrição/metabolismoRESUMO
BACKGROUND: The formation of supernumerary teeth is an excellent model for studying the molecular mechanisms that control stem/progenitor cell homeostasis needed to generate a renewable source of replacement cells and tissues. Although multiple growth factors and transcriptional factors have been associated with supernumerary tooth formation, the regulatory inputs of extracellular matrix in this regenerative process remains poorly understood. RESULTS: In this study, we present evidence that disrupting glycosaminoglycans (GAGs) in the dental epithelium of mice by inactivating FAM20B, a xylose kinase essential for GAG assembly, leads to supernumerary tooth formation in a pattern reminiscent of replacement teeth. The dental epithelial GAGs confine murine tooth number by restricting the homeostasis of Sox2(+) dental epithelial stem/progenitor cells in a non-autonomous manner. FAM20B-catalyzed GAGs regulate the cell fate of dental lamina by restricting FGFR2b signaling at the initial stage of tooth development to maintain a subtle balance between the renewal and differentiation of Sox2(+) cells. At the later cap stage, WNT signaling functions as a relay cue to facilitate the supernumerary tooth formation. CONCLUSIONS: The novel mechanism we have characterized through which GAGs control the tooth number in mice may also be more broadly relevant for potentiating signaling interactions in other tissues during development and tissue homeostasis.
Assuntos
Glicosaminoglicanos/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Transdução de Sinais , Dente Supranumerário/genética , Animais , Diferenciação Celular , Camundongos , Odontogênese , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Células-Tronco/metabolismoRESUMO
BACKGROUND: The basic ground plan of vertebrate hindbrain is established through a process of segmentation, which generates eight transient lineage-restricted cellular compartments called rhombomeres (r). The segments adopt distinct individual identities in response to axial patterning signals. It is unclear whether signaling between rhombomeres plays a conserved role in regulating segmental patterning during hindbrain development. RESULTS: Using tissue manipulations of rhombomeres in chicken embryos, we have uncovered roles for r2 and r4 in regulating the expression of EphA4 in r3 and r5. Perturbations of signaling pathways reveal that these regulatory inputs from r2 and r4 into EphA4 expression are mediated independent of inputs from Krox20 through cues involving fibroblast growth factor (FGF) signaling. These interactions are stage dependent and are set up in embryos with <10 somites. CONCLUSIONS: We show that r2 and r4 function as temporally dynamic signaling centers in the early patterning of adjacent hindbrain segments and this activity is dependent upon the FGF pathway. These results reveal that inter-rhombomeric signaling is a conserved feature of the regulatory networks that control the specification of individual rhombomere identities in vertebrate hindbrain segmentation. However, the timing of when restricted domains of FGF signaling are coupled to formation of r4 may vary between the species.
Assuntos
Fatores de Crescimento de Fibroblastos/metabolismo , Receptor EphA4/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Rombencéfalo/metabolismo , Animais , Embrião de Galinha , Fatores de Crescimento de Fibroblastos/genética , Hibridização In Situ , Receptor EphA4/genética , Receptores da Família Eph/genética , Receptores da Família Eph/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/genética , Rombencéfalo/embriologiaRESUMO
In the hindbrain and the adjacent cranial neural crest (NC) cells of jawed vertebrates (gnathostomes), nested and segmentally-restricted domains of Hox gene expression provide a combinatorial Hox-code for specifying regional properties during head development. Extant jawless vertebrates, such as the sea lamprey (Petromyzon marinus), can provide insights into the evolution and diversification of this Hox-code in vertebrates. There is evidence for gnathostome-like spatial patterns of Hox expression in lamprey; however, the expression domains of the majority of lamprey hox genes from paralogy groups (PG) 1-4 are yet to be characterized, so it is unknown whether they are coupled to hindbrain segments (rhombomeres) and NC. In this study, we systematically describe the spatiotemporal expression of all 14 sea lamprey hox genes from PG1-PG4 in the developing hindbrain and pharynx to investigate the extent to which their expression conforms to the archetypal gnathostome hindbrain and pharyngeal hox-codes. We find many similarities in Hox expression between lamprey and gnathostome species, particularly in rhombomeric domains during hindbrain segmentation and in the cranial neural crest, enabling inference of aspects of Hox expression in the ancestral vertebrate embryonic head. These data are consistent with the idea that a Hox regulatory network underlying hindbrain segmentation is a pan vertebrate trait. We also reveal differences in hindbrain domains at later stages, as well as expression in the endostyle and in pharyngeal arch (PA) 1 mesoderm. Our analysis suggests that many Hox expression domains that are observed in extant gnathostomes were present in ancestral vertebrates but have been partitioned differently across Hox clusters in gnathostome and cyclostome lineages after duplication.
Assuntos
Embrião não Mamífero/metabolismo , Evolução Molecular , Regulação da Expressão Gênica no Desenvolvimento , Genes Homeobox , Cabeça/embriologia , Petromyzon/embriologia , Petromyzon/genética , Animais , Faringe/embriologia , Rombencéfalo/embriologiaRESUMO
Hoxa1 has diverse functional roles in differentiation and development. We identify and characterize properties of regions bound by HOXA1 on a genome-wide basis in differentiating mouse ES cells. HOXA1-bound regions are enriched for clusters of consensus binding motifs for HOX, PBX, and MEIS, and many display co-occupancy of PBX and MEIS. PBX and MEIS are members of the TALE family and genome-wide analysis of multiple TALE members (PBX, MEIS, TGIF, PREP1, and PREP2) shows that nearly all HOXA1 targets display occupancy of one or more TALE members. The combinatorial binding patterns of TALE proteins define distinct classes of HOXA1 targets, which may create functional diversity. Transgenic reporter assays in zebrafish confirm enhancer activities for many HOXA1-bound regions and the importance of HOX-PBX and TGIF motifs for their regulation. Proteomic analyses show that HOXA1 physically interacts on chromatin with PBX, MEIS, and PREP family members, but not with TGIF, suggesting that TGIF may have an independent input into HOXA1-bound regions. Therefore, TALE proteins appear to represent a wide repertoire of HOX cofactors, which may coregulate enhancers through distinct mechanisms. We also discover extensive auto- and cross-regulatory interactions among the Hoxa1 and TALE genes, indicating that the specificity of HOXA1 during development may be regulated though a complex cross-regulatory network of HOXA1 and TALE proteins. This study provides new insight into a regulatory network involving combinatorial interactions between HOXA1 and TALE proteins.
Assuntos
Proteínas de Homeodomínio/genética , Mapas de Interação de Proteínas/genética , Proteínas Repressoras/genética , Fatores de Transcrição/genética , Transcrição Gênica , Animais , Cromatina/genética , Genoma/genética , Camundongos , Células-Tronco Embrionárias Murinas , Ligação Proteica/genética , ProteômicaRESUMO
During development and homeostasis, precise control of Wnt/ß-catenin signaling is in part achieved by secreted and membrane proteins that negatively control activity of the Wnt co-receptors Lrp5 and Lrp6. Lrp4 is related to Lrp5/6 and is implicated in modulation of Wnt/ß-catenin signaling, presumably through its ability to bind to the Wise (Sostdc1)/sclerostin (Sost) family of Wnt antagonists. To gain insights into the molecular mechanisms of Lrp4 function in modulating Wnt signaling, we performed an array of genetic analyses in murine tooth development, where Lrp4 and Wise play important roles. We provide genetic evidence that Lrp4 mediates the Wnt inhibitory function of Wise and also modulates Wnt/ß-catenin signaling independently of Wise. Chimeric receptor analyses raise the possibility that the Lrp4 extracellular domain interacts with Wnt ligands, as well as the Wnt antagonists. Diverse modes of Lrp4 function are supported by severe tooth phenotypes of mice carrying a human mutation known to abolish Lrp4 binding to Sost. Our data suggest a model whereby Lrp4 modulates Wnt/ß-catenin signaling via interaction with Wnt ligands and antagonists in a context-dependent manner.
Assuntos
Receptores de LDL/metabolismo , Dente/embriologia , Dente/metabolismo , beta Catenina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas Morfogenéticas Ósseas/deficiência , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas Relacionadas a Receptor de LDL , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Camundongos , Camundongos Mutantes , Receptores de LDL/deficiência , Receptores de LDL/genética , Dente/patologia , Via de Sinalização Wnt/genética , Via de Sinalização Wnt/fisiologia , beta Catenina/genéticaRESUMO
The patterning of repeated structures is a major theme in developmental biology, and the inter-relationship between spacing and size of such structures is an unresolved issue. Fungiform papillae are repeated epithelial structures that house taste buds on the anterior tongue. Here, we report that FGF signaling is a crucial regulator of fungiform papillae development. We found that mesenchymal FGF10 controls the size of the papillary area, while overall patterning remains unchanged. Our results show that FGF signaling negatively affects the extent of canonical Wnt signaling, which is the main activation pathway during fungiform papillae development; however, this effect does not occur at the level of gene transcription. Rather, our experimental data, together with computational modeling, indicate that FGF10 modulates the range of Wnt effects, likely via induction of Sostdc1 expression. We suggest that modification of the reach of Wnt signaling could be due to local changes in morphogen diffusion, representing a novel mechanism in this tissue context, and we propose that this phenomenon might be involved in a broader array of mammalian developmental processes.
Assuntos
Fator 10 de Crescimento de Fibroblastos/metabolismo , Papilas Gustativas/embriologia , Papilas Gustativas/metabolismo , Via de Sinalização Wnt , Proteínas Adaptadoras de Transdução de Sinal , Animais , Padronização Corporal/genética , Padronização Corporal/fisiologia , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Simulação por Computador , Feminino , Fator 10 de Crescimento de Fibroblastos/deficiência , Fator 10 de Crescimento de Fibroblastos/genética , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Modelos Biológicos , Gravidez , Proteínas Serina-Treonina QuinasesRESUMO
A defining feature governing head patterning of jawed vertebrates is a highly conserved gene regulatory network that integrates hindbrain segmentation with segmentally restricted domains of Hox gene expression. Although non-vertebrate chordates display nested domains of axial Hox expression, they lack hindbrain segmentation. The sea lamprey, a jawless fish, can provide unique insights into vertebrate origins owing to its phylogenetic position at the base of the vertebrate tree. It has been suggested that lamprey may represent an intermediate state where nested Hox expression has not been coupled to the process of hindbrain segmentation. However, little is known about the regulatory network underlying Hox expression in lamprey or its relationship to hindbrain segmentation. Here, using a novel tool that allows cross-species comparisons of regulatory elements between jawed and jawless vertebrates, we report deep conservation of both upstream regulators and segmental activity of enhancer elements across these distant species. Regulatory regions from diverse gnathostomes drive segmental reporter expression in the lamprey hindbrain and require the same transcriptional inputs (for example, Kreisler (also known as Mafba), Krox20 (also known as Egr2a)) in both lamprey and zebrafish. We find that lamprey hox genes display dynamic segmentally restricted domains of expression; we also isolated a conserved exonic hox2 enhancer from lamprey that drives segmental expression in rhombomeres 2 and 4. Our results show that coupling of Hox gene expression to segmentation of the hindbrain is an ancient trait with origin at the base of vertebrates that probably led to the formation of rhombomeric compartments with an underlying Hox code.
Assuntos
Sequência Conservada/genética , Evolução Molecular , Redes Reguladoras de Genes/genética , Genes Homeobox/genética , Rombencéfalo/embriologia , Rombencéfalo/metabolismo , Vertebrados/embriologia , Animais , Sequência de Bases , Padronização Corporal/genética , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica no Desenvolvimento , Lampreias/embriologia , Lampreias/genética , Dados de Sequência Molecular , Filogenia , Vertebrados/genética , Peixe-Zebra/embriologia , Peixe-Zebra/genéticaRESUMO
Homeobox a1 (Hoxa1) is one of the most rapidly induced genes in ES cell differentiation and it is the earliest expressed Hox gene in the mouse embryo. In this study, we used genomic approaches to identify Hoxa1-bound regions during early stages of ES cell differentiation into the neuro-ectoderm. Within 2 h of retinoic acid treatment, Hoxa1 is rapidly recruited to target sites that are associated with genes involved in regulation of pluripotency, and these genes display early changes in expression. The pattern of occupancy of Hoxa1 is dynamic and changes over time. At 12 h of differentiation, many sites bound at 2 h are lost and a new cohort of bound regions appears. At both time points the genome-wide mapping reveals that there is significant co-occupancy of Nanog (Nanog homeobox) and Hoxa1 on many common target sites, and these are linked to genes in the pluripotential regulatory network. In addition to shared target genes, Hoxa1 binds to regulatory regions of Nanog, and conversely Nanog binds to a 3' enhancer of Hoxa1 This finding provides evidence for direct cross-regulatory feedback between Hoxa1 and Nanog through a mechanism of mutual repression. Hoxa1 also binds to regulatory regions of Sox2 (sex-determining region Y box 2), Esrrb (estrogen-related receptor beta), and Myc, which underscores its key input into core components of the pluripotential regulatory network. We propose a model whereby direct inputs of Nanog and Hoxa1 on shared targets and mutual repression between Hoxa1 and the core pluripotency network provides a molecular mechanism that modulates the fine balance between the alternate states of pluripotency and differentiation.
Assuntos
Células-Tronco Embrionárias/metabolismo , Redes Reguladoras de Genes , Proteína Homeobox Nanog/genética , Transdução de Sinais , Animais , Linhagem Celular , Células-Tronco Embrionárias/citologia , Camundongos , Modelos Genéticos , Proteína Homeobox Nanog/metabolismoRESUMO
One of the major regulatory challenges of animal development is to precisely coordinate in space and time the formation, specification, and patterning of cells that underlie elaboration of the basic body plan. How does the vertebrate plan for the nervous and hematopoietic systems, heart, limbs, digestive, and reproductive organs derive from seemingly similar population of cells? These systems are initially established and patterned along the anteroposterior axis (AP) by opposing signaling gradients that lead to the activation of gene regulatory networks involved in axial specification, including the Hox genes. The retinoid signaling pathway is one of the key signaling gradients coupled to the establishment of axial patterning. The nested domains of Hox gene expression, which provide a combinatorial code for axial patterning, arise in part through a differential response to retinoic acid (RA) diffusing from anabolic centers established within the embryo during development. Hence, Hox genes are important direct effectors of retinoid signaling in embryogenesis. This review focuses on describing current knowledge on the complex mechanisms and regulatory processes, which govern the response of Hox genes to RA in several tissue contexts including the nervous system during vertebrate development.
Assuntos
Desenvolvimento Embrionário , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Tretinoína/metabolismo , Animais , Encéfalo/embriologia , Encéfalo/metabolismo , Sistema Cardiovascular/embriologia , Sistema Cardiovascular/metabolismo , Sistema Hematopoético/embriologia , Sistema Hematopoético/metabolismo , Proteínas de Homeodomínio/metabolismo , Humanos , Transdução de SinaisRESUMO
The neural crest is a transient population of cells that forms within the developing central nervous system and migrates away to generate a wide range of derivatives throughout the body during vertebrate embryogenesis. These cells are of evolutionary and clinical interest, constituting a key defining trait in the evolution of vertebrates and alterations in their development are implicated in a high proportion of birth defects and craniofacial abnormalities. In the hindbrain and the adjacent cranial neural crest cells (cNCCs), nested domains of Hox gene expression provide a combinatorial'Hox-code' for specifying regional properties in the developing head. Hox genes have been shown to play important roles at multiple stages in cNCC development, including specification, migration, and differentiation. However, relatively little is known about the underlying gene-regulatory mechanisms involved, both upstream and downstream of Hox genes. Furthermore, it is still an open question as to how the genes of the neural crest GRN are linked to Hox-dependent pathways. In this review, we describe Hox gene expression, function and regulation in cNCCs with a view to integrating these genes within the emerging gene regulatory network for cNCC development. We highlight early roles for Hox1 genes in cNCC specification, proposing that this may be achieved, in part, by regulation of the balance between pluripotency and differentiation in precursor cells within the neuro-epithelium. We then describe what is known about the regulation of Hox gene expression in cNCCs and discuss this from the perspective of early vertebrate evolution.
Assuntos
Genes Homeobox/fisiologia , Cabeça/embriologia , Crista Neural/metabolismo , Animais , Evolução Biológica , Padronização Corporal/fisiologia , Diferenciação Celular , Movimento Celular , Sistema Nervoso Central/embriologia , Sequência Conservada , Nervos Cranianos/embriologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Redes Reguladoras de Genes/genética , Genes Homeobox/genética , Humanos , Crista Neural/citologia , Crista Neural/embriologia , Tubo Neural , Neurônios , Rombencéfalo/metabolismo , Crânio , Vertebrados/embriologia , Vertebrados/genéticaRESUMO
Transcriptional regulation of developmentally controlled genes is at the heart of differentiation and organogenesis. In this study, we performed global genomic analyses in murine embryonic stem (ES) cells and in human cells in response to activation signals. We identified an essential role for the ELL (eleven-nineteen lysine-rich leukemia gene)/P-TEFb (positive transcription elongation factor)-containing super elongation complex (SEC) in the regulation of gene expression, including several genes bearing paused RNA polymerase II (Pol II). Paused Pol II has been proposed to be associated with loci that respond rapidly to environmental stimuli. However, our studies in ES cells also identified a requirement for SEC at genes without paused Pol II, which also respond dynamically to differentiation signals. Our findings suggest that SEC is a major class of active P-TEFb-containing complexes required for transcriptional activation in response to environmental cues such as differentiation signals.
Assuntos
Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Elongação da Transcrição/metabolismo , Animais , DNA Polimerase II/metabolismo , Células-Tronco Embrionárias/enzimologia , Células HCT116 , Proteínas de Homeodomínio/metabolismo , Humanos , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , Camundongos , Fatores de Elongação da Transcrição/genéticaRESUMO
Hoxa1 has important functional roles in neural crest specification, hindbrain patterning and heart and ear development, yet the enhancers and genes that are targeted by Hoxa1 are largely unknown. In this study, we performed a comprehensive analysis of Hoxa1 target genes using genome-wide Hoxa1 binding data in mouse ES cells differentiated with retinoic acid (RA) into neural fates in combination with differential gene expression analysis in Hoxa1 gain- and loss-of-function mouse and zebrafish embryos. Our analyses reveal that Hoxa1-bound regions show epigenetic marks of enhancers, occupancy of Hox cofactors and differential expression of nearby genes, suggesting that these regions are enriched for enhancers. In support of this, 80 of them mapped to regions with known reporter activity in transgenic mouse embryos based on the Vista enhancer database. Two additional enhancers in Dok5 and Wls1 were shown to mediate neural expression in developing mouse and zebrafish. Overall, our analysis of the putative target genes indicate that Hoxa1 has input to components of major signaling pathways, including Wnt, TGF-ß, Hedgehog and Hippo, and frequently does so by targeting multiple components of a pathway such as secreted inhibitors, ligands, receptors and down-stream components. We also identified genes implicated in heart and ear development, neural crest migration and neuronal patterning and differentiation, which may underlie major Hoxa1 mutant phenotypes. Finally, we found evidence for a high degree of evolutionary conservation of many binding regions and downstream targets of Hoxa1 between mouse and zebrafish. Our genome-wide analyses in ES cells suggests that we have enriched for in vivo relevant target genes and pathways associated with functional roles of Hoxa1 in mouse development.
Assuntos
Células-Tronco Embrionárias/fisiologia , Proteínas de Homeodomínio/genética , Neurônios/fisiologia , Fatores de Transcrição/genética , Animais , Diferenciação Celular/fisiologia , Desenvolvimento Embrionário/efeitos dos fármacos , Desenvolvimento Embrionário/genética , Desenvolvimento Embrionário/fisiologia , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Feminino , Redes Reguladoras de Genes , Genes Homeobox , Proteínas de Homeodomínio/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Crista Neural/citologia , Neurônios/citologia , Neurônios/metabolismo , Gravidez , Rombencéfalo/citologia , Transdução de Sinais , Fatores de Transcrição/metabolismo , Tretinoína/metabolismo , Peixe-ZebraRESUMO
The clustered Hox genes, which are highly conserved across metazoans, encode homeodomain-containing transcription factors that provide a blueprint for segmental identity along the body axis. Recent studies have underscored that in addition to encoding Hox genes, the homeotic clusters contain key noncoding RNA genes that play a central role in development. In this study, we have taken advantage of genome-wide approaches to provide a detailed analysis of retinoic acid (RA)-induced transcriptional and epigenetic changes within the homeotic clusters of mouse embryonic stem cells. Although there is a general colinear response, our analyses suggest a lack of strict colinearity for several genes in the HoxA and HoxB clusters. We have identified transcribed novel noncoding RNAs (ncRNAs) and their cis-regulatory elements that function in response to RA and demonstrated that the expression of these ncRNAs from both strands represent some of the most rapidly induced transcripts in ES cells. Finally, we have provided dynamic analyses of chromatin modifications for the coding and noncoding genes expressed upon activation and suggest that active transcription can occur in the presence of chromatin modifications and machineries associated with repressed transcription state over the clusters. Overall, our data provide a resource for a better understanding of the dynamic nature of the coding and noncoding transcripts and their associated chromatin marks in the regulation of homeotic gene transcription during development.
Assuntos
Epigênese Genética/efeitos dos fármacos , Proteínas de Homeodomínio/genética , RNA não Traduzido/genética , Transcrição Gênica/efeitos dos fármacos , Tretinoína/farmacologia , Animais , Linhagem Celular , Cromatina/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Análise de Sequência com Séries de Oligonucleotídeos , Elementos Reguladores de Transcrição/efeitos dos fármacosRESUMO
Hindbrain development is orchestrated by a vertebrate gene regulatory network that generates segmental patterning along the anterior-posterior axis via Hox genes. Here, we review analyses of vertebrate and invertebrate chordate models that inform upon the evolutionary origin and diversification of this network. Evidence from the sea lamprey reveals that the hindbrain regulatory network generates rhombomeric compartments with segmental Hox expression and an underlying Hox code. We infer that this basal feature was present in ancestral vertebrates and, as an evolutionarily constrained developmental state, is fundamentally important for patterning of the vertebrate hindbrain across diverse lineages. Despite the common ground plan, vertebrates exhibit neuroanatomical diversity in lineage-specific patterns, with different vertebrates revealing variations of Hox expression in the hindbrain that could underlie this diversification. Invertebrate chordates lack hindbrain segmentation but exhibit some conserved aspects of this network, with retinoic acid signaling playing a role in establishing nested domains of Hox expression.
Assuntos
Evolução Biológica , Padronização Corporal , Cordados/metabolismo , Redes Reguladoras de Genes , Genes Homeobox/genética , Rombencéfalo/metabolismo , Animais , Cordados/embriologia , Cordados/genética , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Rombencéfalo/embriologiaRESUMO
Hoxa2 gene is a primary player in regulation of craniofacial programs of head development in vertebrates. Here we investigate the evolution of a Hoxa2 neural crest enhancer identified originally in mouse by comparing and contrasting the fugu hoxa2a and hoxa2b genes with their orthologous teleost and mammalian sequences. Using sequence analyses in combination with transgenic regulatory assays in zebrafish and mouse embryos we demonstrate subfunctionalization of regulatory activity for expression in hindbrain segments and neural crest cells between these two fugu co-orthologs. hoxa2a regulatory sequences have retained the ability to mediate expression in neural crest cells while those of hoxa2b include cis-elements that direct expression in rhombomeres. Functional dissection of the neural crest regulatory potential of the fugu hoxa2a and hoxa2b genes identify the previously unknown cis-element NC5, which is implicated in generating the differential activity of the enhancers from these genes. The NC5 region plays a similar role in the ability of this enhancer to mediate reporter expression in mice, suggesting it is a conserved component involved in control of neural crest expression of Hoxa2 in vertebrate craniofacial development.