RESUMO
AIMS/HYPOTHESIS: Rodent pancreas development has been described in great detail. On the other hand, there are still gaps in our understanding of the developmental trajectories of pancreatic cells during human ontogenesis. Here, our aim was to map the spatial and chronological dynamics of human pancreatic cell differentiation and proliferation by using 3D imaging of cleared human embryonic and fetal pancreases. METHODS: We combined tissue clearing with light-sheet fluorescence imaging in human embryonic and fetal pancreases during the first trimester of pregnancy. In addition, we validated an explant culture system enabling in vitro proliferation of pancreatic progenitors to determine the mitogenic effect of candidate molecules. RESULTS: We detected the first insulin-positive cells as early as five post-conceptional weeks, two weeks earlier than previously observed. We observed few insulin-positive clusters at five post-conceptional weeks (mean ± SD 9.25±5.65) with a sharp increase to 11 post-conceptional weeks (4307±152.34). We identified a central niche as the location of onset of the earliest insulin cell production and detected extra-pancreatic loci within the adjacent developing gut. Conversely, proliferating pancreatic progenitors were located in the periphery of the epithelium, suggesting the existence of two separated pancreatic niches for differentiation and proliferation. Additionally, we observed that the proliferation ratio of progenitors ranged between 20% and 30%, while for insulin-positive cells it was 1%. We next unveiled a mitogenic effect of the platelet-derived growth factor AA isoform (PDGFAA) in progenitors acting through the pancreatic mesenchyme by increasing threefold the number of proliferating progenitors. CONCLUSIONS/INTERPRETATION: This work presents a first 3D atlas of the human developing pancreas, charting both endocrine and proliferating cells across early development.
Assuntos
Diferenciação Celular , Proliferação de Células , Imageamento Tridimensional , Pâncreas , Humanos , Pâncreas/embriologia , Pâncreas/citologia , Pâncreas/metabolismo , Diferenciação Celular/fisiologia , Feminino , Células-Tronco/citologia , Células-Tronco/metabolismo , Gravidez , Insulina/metabolismoRESUMO
The evolution and development of the head have long captivated researchers due to the crucial role of the head as the gateway for sensory stimuli and the intricate structural complexity of the head. Although significant progress has been made in understanding head development in various vertebrate species, our knowledge of early human head ontogeny remains limited. Here, we used advanced whole-mount immunostaining and 3D imaging techniques to generate a comprehensive 3D cellular atlas of human head embryogenesis. We present detailed developmental series of diverse head tissues and cell types, including muscles, vasculature, cartilage, peripheral nerves, and exocrine glands. These datasets, accessible through a dedicated web interface, provide insights into human embryogenesis. We offer perspectives on the branching morphogenesis of human exocrine glands and unknown features of the development of neurovascular and skeletomuscular structures. These insights into human embryology have important implications for understanding craniofacial defects and neurological disorders and advancing diagnostic and therapeutic strategies.
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Embrião de Mamíferos , Cabeça , Humanos , Morfogênese , Cabeça/crescimento & desenvolvimentoRESUMO
The extraembryonic yolk sac (YS) ensures delivery of nutritional support and oxygen to the developing embryo but remains ill-defined in humans. We therefore assembled a comprehensive multiomic reference of the human YS from 3 to 8 postconception weeks by integrating single-cell protein and gene expression data. Beyond its recognized role as a site of hematopoiesis, we highlight roles in metabolism, coagulation, vascular development, and hematopoietic regulation. We reconstructed the emergence and decline of YS hematopoietic stem and progenitor cells from hemogenic endothelium and revealed a YS-specific accelerated route to macrophage production that seeds developing organs. The multiorgan functions of the YS are superseded as intraembryonic organs develop, effecting a multifaceted relay of vital functions as pregnancy proceeds.
Assuntos
Desenvolvimento Embrionário , Saco Vitelino , Feminino , Humanos , Gravidez , Coagulação Sanguínea/genética , Macrófagos , Saco Vitelino/citologia , Saco Vitelino/metabolismo , Desenvolvimento Embrionário/genética , Atlas como Assunto , Expressão Gênica , Perfilação da Expressão Gênica , Hematopoese/genética , Fígado/embriologiaRESUMO
Anterolateral system neurons relay pain, itch, and temperature information from the spinal cord to pain-related brain regions, but the differentiation of these neurons and their specific contribution to pain perception remain poorly defined. Here, we show that most mouse spinal neurons that embryonically express the autonomic-system-associated Paired-like homeobox 2A (Phox2a) transcription factor innervate nociceptive brain targets, including the parabrachial nucleus and the thalamus. We define the Phox2a anterolateral system neuron birth order, migration, and differentiation and uncover an essential role for Phox2a in the development of relay of nociceptive signals from the spinal cord to the brain. Finally, we also demonstrate that the molecular identity of Phox2a neurons is conserved in the human fetal spinal cord, arguing that the developmental expression of Phox2a is a prominent feature of anterolateral system neurons.
Assuntos
Proteínas de Homeodomínio/metabolismo , Vias Neurais/metabolismo , Animais , Humanos , CamundongosRESUMO
We provide the first detailed ontogenetic analysis of human limb muscles using whole-mount immunostaining. We compare our observations with the few earlier studies that have focused on the development of these muscles, and with data available on limb evolution, variations and pathologies. Our study confirms the transient presence of several atavistic muscles - present in our ancestors but normally absent from the adult human - during normal embryonic human development, and reveals the existence of others not previously described in human embryos. These atavistic muscles are found both as rare variations in the adult population and as anomalies in human congenital malformations, reinforcing the idea that such variations/anomalies can be related to delayed or arrested development. We further show that there is a striking difference in the developmental order of muscle appearance in the upper versus lower limbs, reinforcing the idea that the similarity between various distal upper versus lower limb muscles of tetrapod adults may be derived.
Assuntos
Evolução Biológica , Extremidades/embriologia , Músculo Esquelético/embriologia , Animais , Membro Anterior/embriologia , Humanos , Extremidade Inferior/embriologia , FilogeniaRESUMO
Definitive haematopoiesis in the fetal liver supports self-renewal and differentiation of haematopoietic stem cells and multipotent progenitors (HSC/MPPs) but remains poorly defined in humans. Here, using single-cell transcriptome profiling of approximately 140,000 liver and 74,000 skin, kidney and yolk sac cells, we identify the repertoire of human blood and immune cells during development. We infer differentiation trajectories from HSC/MPPs and evaluate the influence of the tissue microenvironment on blood and immune cell development. We reveal physiological erythropoiesis in fetal skin and the presence of mast cells, natural killer and innate lymphoid cell precursors in the yolk sac. We demonstrate a shift in the haemopoietic composition of fetal liver during gestation away from being predominantly erythroid, accompanied by a parallel change in differentiation potential of HSC/MPPs, which we functionally validate. Our integrated map of fetal liver haematopoiesis provides a blueprint for the study of paediatric blood and immune disorders, and a reference for harnessing the therapeutic potential of HSC/MPPs.
Assuntos
Feto/citologia , Hematopoese , Fígado/citologia , Fígado/embriologia , Células Sanguíneas/citologia , Microambiente Celular , Feminino , Feto/metabolismo , Citometria de Fluxo , Perfilação da Expressão Gênica , Humanos , Fígado/metabolismo , Tecido Linfoide/citologia , Análise de Célula Única , Células-Tronco/metabolismoRESUMO
Gnathostome jaws derive from the first pharyngeal arch (PA1), a complex structure constituted by Neural Crest Cells (NCCs), mesodermal, ectodermal and endodermal cells. Here, to determine the regionalized morphogenetic impact of Dlx5/6 expression, we specifically target their inactivation or overexpression to NCCs. NCC-specific Dlx5/6 inactivation (NCC∆Dlx5/6) generates severely hypomorphic lower jaws that present typical maxillary traits. Therefore, differently from Dlx5/6 null-embryos, the upper and the lower jaws of NCC∆Dlx5/6 mice present a different size. Reciprocally, forced Dlx5 expression in maxillary NCCs provokes the appearance of distinct mandibular characters in the upper jaw. We conclude that: (1) Dlx5/6 activation in NCCs invariably determines lower jaw identity; (2) the morphogenetic processes that generate functional matching jaws depend on the harmonization of Dlx5/6 expression in NCCs and in distinct ectodermal territories. The co-evolution of synergistic opposing jaws requires the coordination of distinct regulatory pathways involving the same transcription factors in distant embryonic territories.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/biossíntese , Mandíbula/embriologia , Maxila/embriologia , Crista Neural/embriologia , Animais , Proteínas de Homeodomínio/genética , Mandíbula/citologia , Maxila/citologia , Camundongos , Camundongos Mutantes , Crista Neural/citologiaRESUMO
FOXP2 encodes a transcription factor involved in speech and language acquisition. Growing evidence now suggests that dysregulated FOXP2 activity may also be instrumental in human oncogenesis, along the lines of other cardinal developmental transcription factors such as DLX5 and DLX6 [1-4]. Several FOXP familymembers are directly involved during cancer initiation, maintenance and progression in the adult [5-8]. This may comprise either a pro-oncogenic activity or a deficient tumor-suppressor role, depending upon cell types and associated signaling pathways. While FOXP2 is expressed in numerous cell types, its expression has been found to be down-regulated in breast cancer [9], hepatocellular carcinoma [8] and gastric cancer biopsies [10]. Conversely, overexpressed FOXP2 has been reported in multiple myelomas, MGUS (Monoclonal Gammopathy of Undetermined Significance), several subtypes of lymphomas [5,11], as well as in neuroblastomas [12] and ERG fusion-negative prostate cancers [13]. According to functional evidences reported in breast cancer [9] and survey of recent transcriptomic and proteomic analyses of different tumor biopsies, we postulate that FOXP2 dysregulation may play a main role throughout cancer initiation and progression. In some cancer conditions, FOXP2 levels are now considered as a critical diagnostic marker of neoplastic cells, and in many situations, they even bear strong prognostic value [5]. Whether FOXP2 may further become a therapeutic target is an actively explored lead. Knowledge reviewed here may help improve our understanding of FOXP2 roles during oncogenesis and provide cues for diagnostic, prognostic and therapeutic analyses.
RESUMO
During neuronal development and maturation, microRNAs (miRs) play diverse functions ranging from early patterning, proliferation and commitment to differentiation, survival, homeostasis, activity and plasticity of more mature and adult neurons. The role of miRs in the differentiation of olfactory receptor neurons (ORNs) is emerging from the conditional inactivation of Dicer in immature ORN, and the depletion of all mature miRs in this system. Here, we identify specific miRs involved in olfactory development, by focusing on mice null for Dlx5, a homeogene essential for both ORN differentiation and axon guidance and connectivity. Analysis of miR expression in Dlx5(-/-) olfactory epithelium pointed to reduced levels of miR-9, miR-376a and four miRs of the -200 class in the absence of Dlx5. To functionally examine the role of these miRs, we depleted miR-9 and miR-200 class in reporter zebrafish embryos and observed delayed ORN differentiation, altered axonal trajectory/targeting, and altered genesis and position of olfactory-associated GnRH neurons, i.e. a phenotype known as Kallmann syndrome in humans. miR-9 and miR-200-class negatively control Foxg1 mRNA, a fork-head transcription factor essential for development of the olfactory epithelium and of the forebrain, known to maintain progenitors in a stem state. Increased levels of z-foxg1 mRNA resulted in delayed ORN differentiation and altered axon trajectory, in zebrafish embryos. This work describes for the first time the role of specific miR (-9 and -200) in olfactory/GnRH development, and uncovers a Dlx5-Foxg1 regulation whose alteration affects receptor neuron differentiation, axonal targeting, GnRH neuron development, the hallmarks of the Kallmann syndrome.
Assuntos
Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Hormônio Liberador de Gonadotropina/metabolismo , Proteínas de Homeodomínio/metabolismo , MicroRNAs/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/fisiologia , Condutos Olfatórios/fisiologia , Animais , Animais Geneticamente Modificados , Diferenciação Celular/genética , Células Cultivadas , Embrião de Mamíferos , Feminino , Fatores de Transcrição Forkhead/genética , Hormônio Liberador de Gonadotropina/genética , Proteínas de Homeodomínio/genética , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Mutação/genética , Proteínas do Tecido Nervoso/genética , Mucosa Olfatória/citologia , Mucosa Olfatória/embriologia , Condutos Olfatórios/citologia , Gravidez , Peixe-ZebraRESUMO
Asymmetric, articulated jaws support active predation in vertebrates; they derive from the first pharyngeal arch (PA1) which generates both maxillary and mandibular components. PA1 is colonized by cranial neural crest cells (CNCCs) which give rise to most bones and tendons of the jaws. The elements formed by different CNCCs contingents are specified by the combinatorial expression of Dlx genes. Dlx5 and Dlx6 are predominantly expressed by mandibular CNCCs. Analysis of the phenotype of Dlx5 and Dlx6 double mutant mice has suggested that they are necessary and sufficient to specify mandibular identity. Here, using 3D reconstruction, we show that inactivation of Dlx5 and Dlx6 does not only affect the mandibular arch, but results in the simultaneous transformation of mandibular and maxillary skeletal elements which assume a similar morphology with gain of symmetry. As Dlx5- and Dlx6-expressing cells are not found in the maxillary bud, we have examined the lineage of Dlx5-expressing progenitors using an in vivo genetic approach. We find that a contingent of cells deriving from precursors transiently expressing Dlx5 participate in the formation of the maxillary arch. These cells are mostly located in the distal part of the maxillary arch and might derive from its lambdoidal junction with the olfactory pit. Our findings extend current models of jaw morphogenesis and provide an explanation for the maxillary defects of Dlx5 and Dlx6 mutants. Our results imply that Dlx5 and Dlx6 model the upper and the lower PA1 components through different morphogenetic mechanisms which are, however, coordinated as they give rise to functional, articulated jaws.
RESUMO
Morphogenesis of the vertebrate facial skeleton depends upon inductive interactions between cephalic neural crest cells (CNCCs) and cephalic epithelia. The nasal capsule is a CNCC-derived cartilaginous structure comprising a ventral midline bar (mesethmoid) overlaid by a dorsal capsule (ectethmoid). Although Shh signalling from the anterior-most region of the endoderm (EZ-I) patterns the mesethmoid, the cues involved in ectethmoid induction are still undefined. Here, we show that ectethmoid formation depends upon Dlx5 and Dlx6 expression in a restricted ectodermal territory of the anterior neural folds, which we name NF-ZA. In both chick and mouse neurulas, Dlx5 and Dlx6 expression is mostly restricted to NF-ZA. Simultaneous Dlx5 and Dlx6 inactivation in the mouse precludes ectethmoid formation, while the mesethmoid is still present. Consistently, siRNA-mediated downregulation of Dlx5 and Dlx6 in the cephalic region of the early avian neurula specifically prevents ectethmoid formation, whereas other CNCC-derived structures, including the mesethmoid, are not affected. Similarly, NF-ZA surgical removal in chick neurulas averts ectethmoid development, whereas grafting a supernumerary NF-ZA results in an ectopic ectethmoid. Simultaneous ablation or grafting of both NF-ZA and EZ-I result, respectively, in the absence or duplication of both dorsal and ventral nasal capsule components. The present work shows that early ectodermal and endodermal signals instruct different contingents of CNCCs to form the ectethmoid and the mesethmoid, which then assemble to form a complete nasal capsule.
Assuntos
Padronização Corporal/genética , Proteínas de Homeodomínio/genética , Crista Neural/metabolismo , Nariz/embriologia , Animais , Galinhas , Ectoderma , Embrião de Mamíferos , Embrião não Mamífero , Camundongos , Morfogênese , Nariz/crescimento & desenvolvimentoRESUMO
BACKGROUND: The DLX gene family encodes for homeobox transcription factors involved in the control of morphogenesis and tissue homeostasis. Their expression can be regulated by Endothelin1 (ET1), a peptide associated with breast cancer invasive phenotype. Deregulation of DLX gene expression was found in human solid tumors and hematologic malignancies. In particular, DLX4 overexpression represents a possible prognostic marker in ovarian cancer. We have investigated the role of DLX genes in human breast cancer progression. METHODS: MDA-MB-231 human breast carcinoma cells were grown in vitro or injected in nude mice, either subcutaneously, to mimic primary tumor growth, or intravenously, to mimic metastatic spreading. Expression of DLX2, DLX5 and DLX6 was assessed in cultured cells, either treated or not with ET1, tumors and metastases by RT-PCR. In situ hybridization was used to confirm DLX gene expression in primary tumors and in lung and bone metastases. The expression of DLX2 and DLX5 was evaluated in 408 primary human breast cancers examining the GSE1456 and GSE3494 microarray datasets. Kaplan-Meier estimates for disease-free survival were calculated for the patients grouped on the basis of DLX2/DLX5 expression. RESULTS: Before injection, or after subcutaneous growth, MDA-MB-231 cells expressed DLX2 but neither DLX5 nor DLX6. Instead, in bone and lung metastases resulting from intravenous injection we detected expression of DLX5/6 but not of DLX2, suggesting that DLX5/6 are activated during metastasis formation, and that their expression is alternative to that of DLX2. The in vitro treatment of MDA-MB-231 cells with ET1, resulted in switch from DLX2 to DLX5 expression. By data mining in microarray datasets we found that expression of DLX2 occurred in 21.6% of patients, and was significantly correlated with prolonged disease-free survival and reduced incidence of relapse. Instead, DLX5 was expressed in a small subset of cases, 2.2% of total, displaying reduced disease-free survival and high incidence of relapse which was, however, non-significantly different from the other groups due to the small size of the DLX+ cohort. In all cases, we found mutually exclusive expression of DLX2 and DLX5. CONCLUSIONS: Our studies indicate that DLX genes are involved in human breast cancer progression, and that DLX2 and DLX5 genes might serve as prognostic markers.
Assuntos
Biomarcadores Tumorais/genética , Neoplasias Ósseas/genética , Neoplasias da Mama/genética , Movimento Celular/genética , Proteínas de Homeodomínio/genética , Neoplasias Pulmonares/genética , Fatores de Transcrição/genética , Animais , Neoplasias Ósseas/secundário , Neoplasias da Mama/metabolismo , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Intervalo Livre de Doença , Endotelina-1/metabolismo , Feminino , Perfilação da Expressão Gênica , Humanos , Hibridização In Situ/métodos , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/secundário , Camundongos , Camundongos Nus , Invasividade Neoplásica , Análise de Sequência com Séries de Oligonucleotídeos , Prognóstico , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de TempoRESUMO
The shaping of the vertebrate head results from highly dynamic integrated processes involving the growth and exchange of signals between the ectoderm, the endoderm, the mesoderm and Cephalic Neural Crest Cells (CNCCs). During embryonic development, these tissues change their shape and relative position rapidly and come transiently in contact with each other. Molecular signals exchanged in restricted regions of tissue interaction are crucial in providing positional identity to the mesenchymes which will form the different skeletal and muscular components of the head. Slight spatio-temporal modifications of these signalling maps can result in profound changes in craniofacial development and might have contributed to the evolution of facial diversity. Abnormal signalling patterns could also be at the origin of congenital craniofacial malformations. This review brings into perspective recent work on spatial and temporal aspects of facial morphogenesis with particular focus on the molecular mechanisms of jaw specification.
Assuntos
Anormalidades Craniofaciais/genética , Regulação da Expressão Gênica no Desenvolvimento , Crista Neural/citologia , Animais , Padronização Corporal , Anormalidades Craniofaciais/embriologia , Ectoderma/metabolismo , Endoderma/citologia , Endotelina-1/genética , Humanos , Arcada Osseodentária/embriologia , Mesoderma/citologia , Mesoderma/metabolismo , Modelos Biológicos , Transdução de Sinais , Fatores de Tempo , Tretinoína/química , Tretinoína/metabolismoRESUMO
BACKGROUND: Understanding and manipulating gene function in physiological conditions is a major objective for both fundamental and applied research. In contrast to other experimental settings, which use either purely genetic or gene delivery (viral or non-viral) strategies, we report here a strategy based on direct protein delivery to central nervous system (CNS) tissues. We fused Cre recombinase with cell-penetrating peptides and analyzed the intracellular biological activity of the resulting chimerical proteins when delivered into cells endowed with Cre-mediated reporter gene expression. RESULTS: We show that active Cre enzymatic conjugates are readily internalized and exert their enzymatic activity in the nucleus of adherent cultured cells. We then evaluated this strategy in organotypic cultures of neural tissue explants derived from reporter mice carrying reporter "floxed" alleles. The efficacy of two protocols was compared on explants, either by direct addition of an overlying drop of protein conjugate or by implantation of conjugate-coated beads. In both cases, delivery of Cre recombinase resulted in genomic recombination that, with the bead protocol, was restricted to discrete areas of embryonic and adult neural tissues. Furthermore, delivery to adult brain tissue resulted in the transduction of mature postmitotic populations of neurons. CONCLUSION: We provide tools for the spatially restricted genetic modification of cells in explant culture. This strategy allows to study lineage, migration, differentiation and death of neural cells. As a proof-of-concept applied to CNS tissue, direct delivery of Cre recombinase enabled the selective elimination of an interneuron subpopulation of the spinal cord, thereby providing a model to study early events of neurodegenerative processes. Thus our work opens new perspectives for both fundamental and applied cell targeting protocols using proteic cargoes which need to retain full bioactivity upon internalisation, as illustrated here with the oligomeric Cre recombinase.
Assuntos
Sistema Nervoso Central/metabolismo , Técnicas de Transferência de Genes , Integrases/metabolismo , Neurônios/citologia , Animais , Células Cultivadas , Sistema Nervoso Central/embriologia , Embrião de Mamíferos/inervação , Vetores Genéticos , Camundongos , Camundongos Transgênicos , Neurônios/metabolismo , Plasmídeos , Proteínas Recombinantes de Fusão/metabolismo , Recombinação Genética , Transdução GenéticaRESUMO
Morphogenesis of the facial skeleton depends on inductive interactions between cephalic neural crest cells and cephalic epithelia, including the foregut endoderm. We show that Shh expression in the most rostral zone of the endoderm, endoderm zone I (EZ-I), is necessary to induce the formation of the ventral component of the avian nasal capsule: the mesethmoid cartilage. Surgical removal of EZ-I specifically prevented mesethmoid formation, whereas grafting a supernumerary EZ-I resulted in an ectopic mesethmoid. EZ-I ablation was rescued by Shh-loaded beads, whereas inhibition of Shh signalling suppressed mesethmoid formation. This interaction between the endoderm and cephalic neural crest cells was reproduced in vitro, as evidenced by Gli1 induction. Our work bolsters the hypothesis that early endodermal regionalisation provides the blueprint for facial morphogenesis and that its disruption might cause foetal craniofacial defects, including those of the nasal region.
Assuntos
Endoderma/embriologia , Endoderma/metabolismo , Trato Gastrointestinal/embriologia , Trato Gastrointestinal/metabolismo , Proteínas Hedgehog/metabolismo , Mucosa Nasal/metabolismo , Nariz/enzimologia , Animais , Padronização Corporal , Galinhas , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Hedgehog/genética , Crista Neural/embriologia , Crista Neural/metabolismo , Proteínas Oncogênicas/metabolismo , Codorniz , Transdução de Sinais , Transativadores/metabolismo , Proteína GLI1 em Dedos de ZincoRESUMO
BACKGROUND: Intake of retinoic acid (RA) or of its precursor, vitamin A, during early pregnancy is associated with increased incidence of craniofacial lesions. The origin of these teratogenic effects remains enigmatic as in cranial neural crest cells (CNCCs), which largely contribute to craniofacial structures, the RA-transduction pathway is not active. Recent results suggest that RA could act on the endoderm of the first pharyngeal arch (1stPA), through a RARbeta-dependent mechanism. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that RA provokes dramatically different craniofacial malformations when administered at slightly different developmental times within a narrow temporal interval corresponding to the colonization of the 1(st) PA by CNCCs. We provide evidence showing that RA acts on the signalling epithelium of the 1(st) PA, gradually reducing the expression of endothelin-1 and Fgf8. These two molecular signals are instrumental in activating Dlx genes in incoming CNCCs, thereby triggering the morphogenetic programs, which specify different jaw elements. CONCLUSIONS/SIGNIFICANCE: The anatomical series induced by RA-treatments at different developmental times parallels, at least in some instances, the supposed origin of modern jaws (e.g., the fate of the incus). Our results might provide a conceptual framework for the rise of jaw morphotypes characteristic of gnathostomes.
Assuntos
Anormalidades Craniofaciais/induzido quimicamente , Anormalidades Craniofaciais/metabolismo , Embrião de Mamíferos/efeitos dos fármacos , Arcada Osseodentária/anatomia & histologia , Ceratolíticos/farmacologia , Tretinoína/farmacologia , Animais , Anormalidades Craniofaciais/patologia , Proteína 2 de Resposta de Crescimento Precoce/fisiologia , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Endotelina-1/fisiologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/fisiologia , Hibridização In Situ , Camundongos , Camundongos Transgênicos , Simulação de Dinâmica Molecular , Crista Neural/citologia , Crista Neural/efeitos dos fármacos , Crista Neural/metabolismo , Gravidez , Receptores do Ácido Retinoico/genética , Receptores do Ácido Retinoico/metabolismo , Transdução de SinaisRESUMO
Heat-shock factors (HSFs) are associated with multiple developmental processes, but their mechanisms of action in these processes remain largely enigmatic. Hsf2-null mice display gametogenesis defects and brain abnormalities characterized by enlarged ventricles. Here, we show that Hsf2-/- cerebral cortex displays mispositioning of neurons of superficial layers. HSF2 deficiency resulted in a reduced number of radial glia fibers, the architectural guides for migrating neurons, and of Cajal-Retzius cells, which secrete the positioning signal Reelin. Therefore, we focused on the radial migration signaling pathways. The levels of Reelin and Dab1 tyrosine phosphorylation were reduced, suggesting that the Reelin cascade is affected in Hsf2-/- cortices. The expression of p35, an activator of cyclin-dependent kinase 5 (Cdk5), essential for radial migration, was dependent on the amount of HSF2 in gain- and loss-of-function systems. p39, another Cdk5 activator, displayed reduced mRNA levels in Hsf2-/- cortices, which, together with the lowered p35 levels, decreased Cdk5 activity. We demonstrate in vivo binding of HSF2 to the p35 promoter and thereby identify p35 as the first target gene for HSF2 in cortical development. In conclusion, HSF2 affects cellular populations that assist in radial migration and directly regulates the expression of p35, a crucial actor of radial neuronal migration.
Assuntos
Córtex Cerebral/crescimento & desenvolvimento , Proteínas de Ligação a DNA/fisiologia , Proteínas de Choque Térmico/fisiologia , Fosfotransferases/genética , Fatores de Transcrição/fisiologia , Animais , Sítios de Ligação/genética , Movimento Celular , Córtex Cerebral/embriologia , Córtex Cerebral/metabolismo , DNA/genética , DNA/metabolismo , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Transcrição de Choque Térmico , Proteínas de Choque Térmico/deficiência , Proteínas de Choque Térmico/genética , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neuroglia/citologia , Neuroglia/fisiologia , Neurônios/citologia , Neurônios/fisiologia , Gravidez , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteína Reelina , Transdução de Sinais , Fatores de Transcrição/deficiência , Fatores de Transcrição/genéticaRESUMO
Sonic hedgehog (Shh) signaling controls many aspects of ontogeny, orchestrating congruent growth and patterning. During brain development, Shh regulates early ventral patterning while later on it is critical for the regulation of precursor proliferation in the dorsal brain, namely in the neocortex, tectum and cerebellum. We have recently shown that Shh also controls the behavior of cells with stem cell properties in the mouse embryonic neocortex, and additional studies have implicated it in the control of cell proliferation in the adult ventral forebrain and in the hippocampus. However, it remains unclear whether it regulates adult stem cell lineages in an equivalent manner. Similarly, it is not known which cells respond to Shh signaling in stem cell niches. Here we demonstrate that Shh is required for cell proliferation in the mouse forebrain's subventricular zone (SVZ) stem cell niche and for the production of new olfactory interneurons in vivo. We identify two populations of Gli1+ Shh signaling responding cells: GFAP+ SVZ stem cells and GFAP- precursors. Consistently, we show that Shh regulates the self-renewal of neurosphere-forming stem cells and that it modulates proliferation of SVZ lineages by acting as a mitogen in cooperation with epidermal growth factor (EGF). Together, our data demonstrate a critical and conserved role of Shh signaling in the regulation of stem cell lineages in the adult mammalian brain, highlight the subventricular stem cell astrocytes and their more abundant derived precursors as in vivo targets of Shh signaling, and demonstrate the requirement for Shh signaling in postnatal and adult neurogenesis.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica , Células-Tronco/citologia , Transativadores/fisiologia , Animais , Padronização Corporal , Linhagem da Célula , Proliferação de Células , Cerebelo/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Proteínas Hedgehog , Hipocampo/metabolismo , Imuno-Histoquímica , Hibridização In Situ , Camundongos , Camundongos Endogâmicos C57BL , Mitógenos/metabolismo , Modelos Biológicos , Neurônios/metabolismo , Bulbo Olfatório/metabolismo , Prosencéfalo/embriologia , Prosencéfalo/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Fatores de Tempo , Transativadores/metabolismo , Alcaloides de Veratrum/farmacologiaRESUMO
The DNA sequence of human chromosome 21 (HSA21) has opened the route for a systematic molecular characterization of all of its genes. Trisomy 21 is associated with Down's syndrome, the most common genetic cause of mental retardation in humans. The phenotype includes various organ dysmorphies, stereotypic craniofacial anomalies and brain malformations. Molecular analysis of congenital aneuploidies poses a particular challenge because the aneuploid region contains many protein-coding genes whose function is unknown. One essential step towards understanding their function is to analyse mRNA expression patterns at key stages of organism development. Seminal works in flies, frogs and mice showed that genes whose expression is restricted spatially and/or temporally are often linked with specific ontogenic processes. Here we describe expression profiles of mouse orthologues to HSA21 genes by a combination of large-scale mRNA in situ hybridization at critical stages of embryonic and brain development and in silico (computed) mining of expressed sequence tags. This chromosome-scale expression annotation associates many of the genes tested with a potential biological role and suggests candidates for the pathogenesis of Down's syndrome.
Assuntos
Cromossomos Humanos Par 21/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Camundongos/embriologia , Camundongos/genética , Homologia de Sequência do Ácido Nucleico , Animais , Encéfalo/embriologia , Encéfalo/metabolismo , Síndrome de Down/genética , Etiquetas de Sequências Expressas , Biblioteca Gênica , Humanos , Hibridização In Situ , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Heat shock factor 2, one of the four vertebrate HSFs, transcriptional regulators of heat shock gene expression, is active during embryogenesis and spermatogenesis, with unknown functions and targets. By disrupting the Hsf2 gene, we show that, although the lack of HSF2 is not embryonic lethal, Hsf2(-/-) mice suffer from brain abnormalities, and meiotic and gameto genesis defects in both genders. The disturbances in brain are characterized by the enlargement of lateral and third ventricles and the reduction of hippocampus and striatum, in correlation with HSF2 expression in proliferative cells of the neuroepithelium and in some ependymal cells in adults. Many developing spermatocytes are eliminated via apoptosis in a stage-specific manner in Hsf2(-/-) males, and pachytene spermatocytes also display structural defects in the synaptonemal complexes between homologous chromosomes. Hsf2(-/-) females suffer from multiple fertility defects: the production of abnormal eggs, the reduction in ovarian follicle number and the presence of hemorrhagic cystic follicles are consistent with meiotic defects. Hsf2(-/-) females also display hormone response defects, that can be rescued by superovulation treatment, and exhibit abnormal rates of luteinizing hormone receptor mRNAs.