RESUMO
Limb patterning by Sonic hedgehog (Shh), via either graded spatial or temporal signal integration, is a paradigm for "morphogen" function, yet how Shh instructs distinct digit identities remains controversial. Here, we bypass the Shh requirement in cell survival during outgrowth and demonstrate that a transient, early Shh pulse is both necessary and sufficient for normal mouse limb development. Shh response is only short range and is limited to the Shh-expressing zone during this time window. Shh patterns digits 1-3, anterior to this zone, by an indirect mechanism rather than direct spatial or temporal signal integration. Using a genetic relay-signaling assay, we discover that Shh also specifies digit 1/thumb (thought to be exclusively Shh independent) indirectly, and this finding implicates Shh in a unique regulatory hierarchy for digit 1 evolutionary adaptations such as opposable thumbs. This study illuminates Shh as a trigger for an indirect downstream network that becomes rapidly self-sustaining, with mechanistic relevance for limb development, regeneration, and evolution.
Assuntos
Padronização Corporal , Proteínas Hedgehog , Animais , Padronização Corporal/genética , Extremidades , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Camundongos , Transdução de SinaisRESUMO
BMP signaling is crucial for differentiation of secretory ameloblasts, the cells that secrete enamel matrix. However, whether BMP signaling is required for differentiation of maturation-stage ameloblasts (MA), which are instrumental for enamel maturation into hard tissue, is hitherto unknown. To address this, we used an in vivo genetic approach which revealed that combined deactivation of the Bmp2 and Bmp4 genes in the murine dental epithelium causes development of dysmorphic and dysfunctional MA. These fail to exhibit a ruffled apical plasma membrane and to reabsorb enamel matrix proteins, leading to enamel defects mimicking hypomaturation amelogenesis imperfecta. Furthermore, subsets of mutant MA underwent pathological single or collective cell migration away from the ameloblast layer, forming cysts and/or exuberant tumor-like and gland-like structures. Massive apoptosis in the adjacent stratum intermedium and the abnormal cell-cell contacts and cell-matrix adhesion of MA may contribute to this aberrant behavior. The mutant MA also exhibited severely diminished tissue non-specific alkaline phosphatase activity, revealing that this enzyme's activity in MA crucially depends on BMP2 and BMP4 inputs. Our findings show that combined BMP2 and BMP4 signaling is crucial for survival of the stratum intermedium and for proper development and function of MA to ensure normal enamel maturation.
Assuntos
Ameloblastos , Amelogênese , Amelogênese/genética , Animais , Proteína Morfogenética Óssea 2/genética , Proteína Morfogenética Óssea 2/metabolismo , Proteína Morfogenética Óssea 4/genética , Proteína Morfogenética Óssea 4/metabolismo , Diferenciação Celular , Epitélio , Camundongos , Transdução de SinaisRESUMO
The vertebrate notochord plays an essential role in patterning multiple structures during embryonic development. In the early 2000s, descendants of notochord cells were demonstrated to form the entire nucleus pulposus of the intervertebral disc in addition to their key role in embryonic patterning. The nucleus pulposus undergoes degeneration during postnatal life, which can lead to back pain. Recently, gene and protein profiles of notochord and nucleus pulposus cells have been identified. These datasets, coupled with the ability to differentiate human induced pluripotent stem cells (iPSCs) into cells that resemble nucleus pulposus cells, provide the possibility of generating a cell-based therapy to halt and/or reverse disc degeneration.
Assuntos
Células-Tronco Pluripotentes Induzidas , Degeneração do Disco Intervertebral , Disco Intervertebral , Desenvolvimento Embrionário , Humanos , Degeneração do Disco Intervertebral/terapia , Notocorda/metabolismoRESUMO
Glial-like supporting (or sustentacular) cells are important constituents of the olfactory epithelium that are involved in several physiological processes such as production of endocannabinoids, insulin, and ATP and regulation of the ionic composition of the mucus layer that covers the apical surface of the olfactory epithelium. Supporting cells express metabotropic P2Y purinergic receptors that generate ATP-induced Ca2+ signaling through the activation of a PLC-mediated cascade. Recently, we reported that a subpopulation of supporting cells expresses also the Ca2+-activated Cl- channel TMEM16A. Here, we sought to extend our understanding of a possible physiological role of this channel in the olfactory system by asking whether Ca2+ can activate Cl- currents mediated by TMEM16A. We use whole-cell patch-clamp analysis in slices of the olfactory epithelium to measure dose-response relations in the presence of various intracellular Ca2+ concentrations, ion selectivity, and blockage. We find that knockout of TMEM16A abolishes Ca2+-activated Cl- currents, demonstrating that TMEM16A is essential for these currents in supporting cells. Also, by using extracellular ATP as physiological stimuli, we found that the stimulation of purinergic receptors activates a large TMEM16A-dependent Cl- current, indicating a possible role of TMEM16A in ATP-mediated signaling. Altogether, our results establish that TMEM16A-mediated currents are functional in olfactory supporting cells and provide a foundation for future work investigating the precise physiological role of TMEM16A in the olfactory system.
Assuntos
Potenciais de Ação , Anoctamina-1/metabolismo , Mucosa Olfatória/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Cálcio/metabolismo , Cloretos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mucosa Olfatória/fisiologia , Receptores Purinérgicos/metabolismoRESUMO
Deciphering how signaling pathways interact during development is necessary for understanding the etiopathogenesis of congenital malformations and disease. In several embryonic structures, components of the Hedgehog and retinoic acid pathways, two potent players in development and disease are expressed and operate in the same or adjacent tissues and cells. Yet whether and, if so, how these pathways interact during organogenesis is, to a large extent, unclear. Using genetic and experimental approaches in the mouse, we show that during development of ontogenetically different organs, including the tail, genital tubercle, and secondary palate, Sonic hedgehog (SHH) loss-of-function causes anomalies phenocopying those induced by enhanced retinoic acid signaling and that SHH is required to prevent supraphysiological activation of retinoic signaling through maintenance and reinforcement of expression of the Cyp26 genes. Furthermore, in other tissues and organs, disruptions of the Hedgehog or the retinoic acid pathways during development generate similar phenotypes. These findings reveal that rigidly calibrated Hedgehog and retinoic acid activities are required for normal organogenesis and tissue patterning.
Assuntos
Família 26 do Citocromo P450/genética , Desenvolvimento Embrionário/genética , Proteínas Hedgehog/genética , Ácido Retinoico 4 Hidroxilase/genética , Animais , Apoptose/genética , Diferenciação Celular/genética , Embrião de Mamíferos , Células Epiteliais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Camundongos , Organogênese/genética , Transdução de Sinais/genética , Dente/crescimento & desenvolvimento , Dente/metabolismo , Tretinoína/metabolismoRESUMO
Recapitulation of developmental signals represents a promising strategy for treating intervertebral disc degeneration. During development, embryonic notochord-derived cells (NDCs) are the direct progenitors of cells that populate the adult nucleus pulposus (NP) and are an important source of secreted signaling molecules. The objective of this study was to define global gene expression profiles of NDCs at key stages of embryonic disc formation. NDCs were isolated from Shh-cre;ROSA:YFP mice at embryonic day 12.5 and postnatal day 0, representing opposite ends of the notochord to NP transformation. Differences in global mRNA abundance across this developmental window were established using RNA-Seq. Protein expression of selected molecules was confirmed using immunohistochemistry. Principal component analysis revealed clustering of gene expression at each developmental stage with more than 5000 genes significantly differentially expressed between E12.5 and P0. There was significantly lower mRNA abundance of sonic hedgehog pathway elements at P0 vs E12.5, while abundance of elements of the transforming growth factor-beta and insulin-like growth factors pathways, and extracellular matrix components including collagen 6 and aggrecan, were significantly higher at P0. This study represents the first transcriptome-wide analysis of embryonic NDCs. Results suggest signaling and biosynthesis of NDCs change dramatically as a function of developmental stage.
Assuntos
Perfilação da Expressão Gênica , Notocorda/citologia , Núcleo Pulposo/embriologia , Núcleo Pulposo/metabolismo , Transcriptoma , Animais , Embrião de Mamíferos , Matriz Extracelular/metabolismo , Regulação da Expressão Gênica , Imuno-Histoquímica , Camundongos , Camundongos Transgênicos , RNA Mensageiro , Reprodutibilidade dos Testes , Transdução de Sinais , Coluna Vertebral/embriologia , Coluna Vertebral/metabolismo , Fator de Crescimento Transformador beta/metabolismoRESUMO
The interaction between signaling pathways is a central question in the study of organogenesis. Using the developing murine tongue as a model, we uncovered unknown relationships between Sonic hedgehog (SHH) and retinoic acid (RA) signaling. Genetic loss of SHH signaling leads to enhanced RA activity subsequent to loss of SHH-dependent expression of Cyp26a1 and Cyp26c1. This causes a cell identity switch, prompting the epithelium of the tongue to form heterotopic minor salivary glands and to overproduce oversized taste buds. At developmental stages during which Wnt10b expression normally ceases and Shh becomes confined to taste bud cells, loss of SHH inputs causes the lingual epithelium to undergo an ectopic and anachronic expression of Shh and Wnt10b in the basal layer, specifying de novo taste placode induction. Surprisingly, in the absence of SHH signaling, lingual epithelial cells adopted a Merkel cell fate, but this was not caused by enhanced RA signaling. We show that RA promotes, whereas SHH, acting strictly within the lingual epithelium, inhibits taste placode and lingual gland formation by thwarting RA activity. These findings reveal key functions for SHH and RA in cell fate specification in the lingual epithelium and aid in deciphering the molecular mechanisms that assign cell identity.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Epitélio/efeitos dos fármacos , Proteínas Hedgehog/metabolismo , Tretinoína/farmacologia , Alelos , Animais , Linhagem Celular , Família 26 do Citocromo P450/genética , Família 26 do Citocromo P450/metabolismo , Células Epiteliais/metabolismo , Epitélio/crescimento & desenvolvimento , Feminino , Proteínas Hedgehog/genética , Masculino , Células de Merkel/efeitos dos fármacos , Células de Merkel/metabolismo , Camundongos , Ácido Retinoico 4 Hidroxilase/genética , Ácido Retinoico 4 Hidroxilase/metabolismo , Transdução de Sinais , Papilas Gustativas/metabolismo , Língua/crescimento & desenvolvimento , Proteínas Wnt/genética , Proteínas Wnt/metabolismoRESUMO
The vertebral column consists of repeating units of ossified vertebrae that are adjoined by fibrocartilagenous intervertebral discs. These structures form from the embryonic notochord and somitic mesoderm. In humans, congenital malformations of the vertebral column include scoliosis, kyphosis, spina bifida, and Klippel Feil syndrome. In adulthood, a common malady affecting the vertebral column includes disc degeneration and associated back pain. Indeed, recent reports estimate that low back pain is the number one cause of disability worldwide. Our review provides an overview of the molecular mechanisms underlying vertebral column morphogenesis and intervertebral disc development and maintenance, with an emphasis on what has been gleaned from recent genetic studies in mice. The aim of this review is to provide a developmental framework through which vertebral column formation can be understood so that ultimately, research scientists and clinicians alike can restore disc health with appropriately designed gene and cell-based therapies. WIREs Dev Biol 2017, 6:e283. doi: 10.1002/wdev.283 For further resources related to this article, please visit the WIREs website.
Assuntos
Disco Intervertebral/citologia , Animais , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Disco Intervertebral/metabolismo , Notocorda/citologia , Notocorda/metabolismoRESUMO
mRNA variance has been proposed to play key roles in normal development, population fitness, adaptability, and disease. While variance in gene expression levels may be beneficial for certain cellular processes, for example in a cell's ability to respond to external stimuli, variance may be detrimental for the development of some organs. In the bilaterally symmetric vertebrate limb buds, the amount of Sonic Hedgehog (SHH) protein present at specific stages of development is essential to ensure proper patterning of this structure. To our surprise, we found that SHH protein variance is present during the first 10 hr of limb development. The variance is virtually eliminated after the first 10 hr of limb development. By examining mutant animals, we determined that the ability of the limb bud apical ectodermal ridge (AER) to respond to SHH protein was required for reducing SHH variance during limb formation. One consequence of the failure to eliminate variance in SHH protein was the presence of polydactyly and an increase in digit length. These data suggest a potential novel mechanism in which alterations in SHH variance during evolution may have driven changes in limb patterning and digit length.
Assuntos
Padronização Corporal/genética , Retroalimentação Fisiológica , Proteínas Hedgehog/metabolismo , Botões de Extremidades/metabolismo , Animais , Proteínas Hedgehog/genética , Botões de Extremidades/embriologia , Camundongos , Modelos Biológicos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Somitos/embriologia , Somitos/metabolismoRESUMO
During vertebrate embryogenesis the interdigital mesenchyme is removed by programmed cell death (PCD), except in species with webbed limbs. Although bone morphogenetic proteins (BMPs) have long been known to be players in this process, it is unclear if they play a direct role in the interdigital mesenchyme or if they only act indirectly, by affecting fibroblast growth factor (FGF) signaling. A series of genetic studies have shown that BMPs act indirectly by regulating the withdrawal of FGF activity from the apical ectodermal ridge (AER); this FGF activity acts as a cell survival factor for the underlying mesenchyme. Other studies using exogenous factors to inhibit BMP activity in explanted mouse limbs suggest that BMPs do not act directly in the mesenchyme. To address the question of whether BMPs act directly, we used an interdigit-specific Cre line to inactivate several genes that encode components of the BMP signaling pathway, without perturbing the normal downregulation of AER-FGF activity. Of three Bmps expressed in the interdigital mesenchyme, Bmp7 is necessary for PCD, but Bmp2 and Bmp4 both have redundant roles, with Bmp2 being the more prominent player. Removing BMP signals to the interdigit by deleting the receptor gene, Bmpr1a, causes a loss of PCD and syndactyly, thereby unequivocally proving that BMPs are direct triggers of PCD in this tissue. We present a model in which two events must occur for normal interdigital PCD: the presence of a BMP death trigger and the absence of an FGF survival activity. We demonstrate that neither event is required for formation of the interdigital vasculature, which is necessary for PCD. However, both events converge on the production of reactive oxygen species that activate PCD.
Assuntos
Apoptose , Proteína Morfogenética Óssea 2/fisiologia , Proteína Morfogenética Óssea 4/fisiologia , Proteína Morfogenética Óssea 7/fisiologia , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/genética , Regulação da Expressão Gênica no Desenvolvimento , Animais , Cruzamentos Genéticos , Extremidades/embriologia , Feminino , Fatores de Crescimento de Fibroblastos/metabolismo , Membro Anterior/patologia , Integrases/metabolismo , Masculino , Mesoderma/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Confocal , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Sindactilia/genética , Fatores de Tempo , Dedos do Pé/patologia , beta-Galactosidase/metabolismoRESUMO
A tissue that commonly deteriorates in older vertebrates is the intervertebral disc, which is located between the vertebrae. Age-related changes in the intervertebral discs are thought to cause most cases of back pain. Back pain affects more than half of people over the age of 65, and the treatment of back pain costs 50-100 billion dollars per year in the USA. The normal intervertebral disc is composed of three distinct regions: a thick outer ring of fibrous cartilage called the annulus fibrosus, a gel-like material that is surrounded by the annulus fibrosus called the nucleus pulposus, and superior and inferior cartilaginous end plates. The nucleus pulposus has been shown to be critical for disc health and function. Damage to this structure often leads to disc disease. Recent reports have demonstrated that the embryonic notochord, a rod-like structure present in the midline of vertebrate embryos, gives rise to all cell types found in adult nuclei pulposi. The mechanism responsible for the transformation of the notochord into nuclei pulposi is unknown. In this review, we discuss potential molecular and physical mechanisms that may be responsible for the notochord to nuclei pulposi transition.
Assuntos
Cordoma/etiologia , Disco Intervertebral/embriologia , Notocorda/fisiologia , HumanosRESUMO
TMEM16A/ANO1 is a calcium-activated chloride channel expressed in several types of epithelia and involved in various physiological processes, including proliferation and development. During mouse embryonic development, the expression of TMEM16A in the olfactory epithelium is dynamic. TMEM16A is expressed at the apical surface of the entire olfactory epithelium at embryonic day E12.5 while from E16.5 its expression is restricted to a region near the transition zone with the respiratory epithelium. To investigate whether TMEM16A plays a role in the development of the mouse olfactory epithelium, we obtained the first immunohistochemistry study comparing the morphological properties of the olfactory epithelium and nasal glands in TMEM16A-/- and TMEM16A+/+ littermate mice. A comparison between the expression of the olfactory marker protein and adenylyl cyclase III shows that genetic ablation of TMEM16A did not seem to affect the maturation of olfactory sensory neurons and their ciliary layer. As TMEM16A is expressed at the apical part of supporting cells and in their microvilli, we used ezrin and cytokeratin 8 as markers of microvilli and cell body of supporting cells, respectively, and found that morphology and development of supporting cells were similar in TMEM16A-/- and TMEM16A+/+ littermate mice. The average number of supporting cells, olfactory sensory neurons, horizontal and globose basal cells were not significantly different in the two types of mice. Moreover, we also observed that the morphology of Bowman's glands, nasal septal glands and lateral nasal glands did not change in the absence of TMEM16A. Our results indicate that the development of mouse olfactory epithelium and nasal glands does not seem to be affected by the genetic ablation of TMEM16A.
Assuntos
Canais de Cloreto/metabolismo , Mucosa Nasal/metabolismo , Mucosa Olfatória/metabolismo , Animais , Anoctamina-1 , Canais de Cloreto/deficiência , Canais de Cloreto/genética , Embrião de Mamíferos/metabolismo , Feminino , Imuno-Histoquímica , Queratina-8/metabolismo , Antígeno Ki-67/metabolismo , Camundongos , Camundongos Knockout , Microscopia Confocal , Microvilosidades/metabolismo , Neurônios Receptores Olfatórios/metabolismo , GravidezRESUMO
The long tendons of the limb extend from muscles that reside in the zeugopod (arm/leg) to their skeletal insertions in the autopod (paw). How these connections are established along the length of the limb remains unknown. Here, we show that mouse limb tendons are formed in modular units that combine to form a functional contiguous structure; in muscle-less limbs, tendons develop in the autopod but do not extend into the zeugopod, and in the absence of limb cartilage the zeugopod segments of tendons develop despite the absence of tendons in the autopod. Analyses of cell lineage and proliferation indicate that distinct mechanisms govern the growth of autopod and zeugopod tendon segments. To elucidate the integration of these autopod and zeugopod developmental programs, we re-examined early tendon development. At E12.5, muscles extend across the full length of a very short zeugopod and connect through short anlagen of tendon progenitors at the presumptive wrist to their respective autopod tendon segment, thereby initiating musculoskeletal integration. Zeugopod tendon segments are subsequently generated by proximal elongation of the wrist tendon anlagen, in parallel with skeletal growth, underscoring the dependence of zeugopod tendon development on muscles for tendon anchoring. Moreover, a subset of extensor tendons initially form as fused structures due to initial attachment of their respective wrist tendon anlage to multiple muscles. Subsequent individuation of these tendons depends on muscle activity. These results establish an integrated model for limb tendon development that provides a framework for future analyses of tendon and musculoskeletal phenotypes.
Assuntos
Extremidades/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Músculo Esquelético/embriologia , Tendões/embriologia , Animais , Apoptose , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Cartilagem/metabolismo , Diferenciação Celular , Linhagem da Célula , Proliferação de Células , Deleção de Genes , Proteínas de Fluorescência Verde/metabolismo , Articulação Metacarpofalângica/patologia , Camundongos , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Músculo Esquelético/metabolismo , Fenótipo , Fatores de Transcrição SOX9/genética , Tendões/metabolismoRESUMO
Interstitial cells of Cajal (ICC) are pacemaker cells that generate electrical activity to drive contractility in the gastrointestinal tract via ion channels. Ano1 (Tmem16a), a Ca(2+)-activated Cl(-) channel, is an ion channel expressed in ICC. Genetic deletion of Ano1 in mice resulted in loss of slow waves in smooth muscle of small intestine. In this study, we show that Ano1 is required to maintain coordinated Ca(2+) transients between myenteric ICC (ICC-MY) of small intestine. First, we found spontaneous Ca(2+) transients in ICC-MY in both Ano1 WT and knockout (KO) mice. However, Ca(2+) transients within the ICC-MY network in Ano1 KO mice were uncoordinated, while ICC-MY Ca(2+) transients in Ano1 WT mice were rhythmic and coordinated. To confirm the role of Ano1 in the loss of Ca(2+) transient coordination, we used pharmacological inhibitors of Ano1 activity and shRNA-mediated knock down of Ano1 expression in organotypic cultures of Ano1 WT small intestine. Coordinated Ca(2+) transients became uncoordinated using both these approaches, supporting the conclusion that Ano1 is required to maintain coordination/rhythmicity of Ca(2+) transients. We next determined the effect on smooth muscle contractility using spatiotemporal maps of contractile activity in Ano1 KO and WT tissues. Significantly decreased contractility that appeared to be non-rhythmic and uncoordinated was observed in Ano1 KO jejunum. In conclusion, Ano1 has a previously unidentified role in the regulation of coordinated gastrointestinal smooth muscle function through coordination of Ca(2+) transients in ICC-MY.
Assuntos
Sinalização do Cálcio , Canais de Cloreto/metabolismo , Células Intersticiais de Cajal/metabolismo , Jejuno/metabolismo , Contração Muscular , Animais , Anoctamina-1 , Cálcio/metabolismo , Canais de Cloreto/genética , Células Intersticiais de Cajal/fisiologia , Jejuno/fisiologia , CamundongosRESUMO
Mutations in the Bone Morphogenetic Protein (BMP) pathway are associated with a range of defects in skeletal formation. Genetic analysis of BMP signaling requirements is complicated by the presence of three partially redundant BMPs that are required for multiple stages of limb development. We generated an inducible allele of a BMP inhibitor, Gremlin, which reduces BMP signaling. We show that BMPs act in a dose and time dependent manner in which early reduction of BMPs result in digit loss, while inhibiting overall BMP signaling between E10.5 and E11.5 allows polydactylous digit formation. During this period, inhibiting BMPs extends the duration of FGF signaling. Sox9 is initially expressed in normal digit ray domains but at reduced levels that correlate with the reduction in BMP signaling. The persistence of elevated FGF signaling likely promotes cell proliferation and survival, inhibiting the activation of Sox9 and secondarily, inhibiting the differentiation of Sox9-expressing chondrocytes. Our results provide new insights into the timing and clarify the mechanisms underlying BMP signaling during digit morphogenesis.
Assuntos
Proteína Morfogenética Óssea 2/genética , Proteína Morfogenética Óssea 4/genética , Proteína Morfogenética Óssea 7/genética , Botões de Extremidades/embriologia , Polidactilia/genética , Animais , Apoptose , Proteína Morfogenética Óssea 2/antagonistas & inibidores , Proteína Morfogenética Óssea 4/antagonistas & inibidores , Proteína Morfogenética Óssea 7/antagonistas & inibidores , Proteínas Morfogenéticas Ósseas/antagonistas & inibidores , Proteínas Morfogenéticas Ósseas/genética , Diferenciação Celular/genética , Proliferação de Células , Condrogênese/genética , Citocinas , Fatores de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Membro Posterior/embriologia , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Peptídeos e Proteínas de Sinalização Intercelular/genética , Mesoderma/embriologia , Camundongos , Camundongos Transgênicos , Mutação , Polidactilia/embriologia , Fatores de Transcrição SOX9/biossíntese , Transdução de Sinais/genéticaRESUMO
The role of the habenular nuclei in modulating fear and reward pathways has sparked a renewed interest in this conserved forebrain region. The bilaterally paired habenular nuclei, each consisting of a medial/dorsal and lateral/ventral nucleus, can be further divided into discrete subdomains whose neuronal populations, precise connectivity, and specific functions are not well understood. An added complexity is that the left and right habenulae show pronounced morphological differences in many non-mammalian species. Notably, the dorsal habenulae of larval zebrafish provide a vertebrate genetic model to probe the development and functional significance of brain asymmetry. Previous reports have described a number of genes that are expressed in the zebrafish habenulae, either in bilaterally symmetric patterns or more extensively on one side of the brain than the other. The goal of our study was to generate a comprehensive map of the zebrafish dorsal habenular nuclei, by delineating the relationship between gene expression domains, comparing the extent of left-right asymmetry at larval and adult stages, and identifying potentially functional subnuclear regions as defined by neurotransmitter phenotype. Although many aspects of habenular organization appear conserved with rodents, the zebrafish habenulae also possess unique properties that may underlie lateralization of their functions.
Assuntos
Habenula/embriologia , Neurotransmissores/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados , Expressão Gênica , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter , Habenula/metabolismo , Imuno-Histoquímica , Neurônios/metabolismo , Neurotransmissores/genética , Especificidade de Órgãos/genética , Fenótipo , Peixe-Zebra/genéticaRESUMO
MicroRNAs regulate gene expression in diverse physiological scenarios. Their role in the control of morphogen related signaling pathways has been less studied, particularly in the context of embryonic Central Nervous System (CNS) development. Here, we uncover a role for microRNAs in limiting the spatiotemporal range of morphogen expression and function. Wnt1 is a key morphogen in the embryonic midbrain, and directs proliferation, survival, patterning and neurogenesis. We reveal an autoregulatory negative feedback loop between the transcription factor Lmx1b and a newly characterized microRNA, miR135a2, which modulates the extent of Wnt1/Wnt signaling and the size of the dopamine progenitor domain. Conditional gain of function studies reveal that Lmx1b promotes Wnt1/Wnt signaling, and thereby increases midbrain size and dopamine progenitor allocation. Conditional removal of Lmx1b has the opposite effect, in that expansion of the dopamine progenitor domain is severely compromised. Next, we provide evidence that microRNAs are involved in restricting dopamine progenitor allocation. Conditional loss of Dicer1 in embryonic stem cells (ESCs) results in expanded Lmx1a/b+ progenitors. In contrast, forced elevation of miR135a2 during an early window in vivo phenocopies the Lmx1b conditional knockout. When En1::Cre, but not Shh::Cre or Nes::Cre, is used for recombination, the expansion of Lmx1a/b+ progenitors is selectively reduced. Bioinformatics and luciferase assay data suggests that miR135a2 targets Lmx1b and many genes in the Wnt signaling pathway, including Ccnd1, Gsk3b, and Tcf7l2. Consistent with this, we demonstrate that this mutant displays reductions in the size of the Lmx1b/Wnt1 domain and range of canonical Wnt signaling. We posit that microRNA modulation of the Lmx1b/Wnt axis in the early midbrain/isthmus could determine midbrain size and allocation of dopamine progenitors. Since canonical Wnt activity has recently been recognized as a key ingredient for programming ESCs towards a dopaminergic fate in vitro, these studies could impact the rational design of such protocols.
Assuntos
Proteínas com Homeodomínio LIM/genética , MicroRNAs/metabolismo , Neurogênese/genética , Doença de Parkinson/genética , Fatores de Transcrição/genética , Proteína Wnt1/genética , Animais , Diferenciação Celular/genética , RNA Helicases DEAD-box/metabolismo , Dopamina/metabolismo , Neurônios Dopaminérgicos/citologia , Neurônios Dopaminérgicos/metabolismo , Embrião de Mamíferos , Células-Tronco Embrionárias , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Proteínas com Homeodomínio LIM/metabolismo , Mesencéfalo/crescimento & desenvolvimento , Mesencéfalo/metabolismo , Camundongos , MicroRNAs/genética , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Ribonuclease III/metabolismo , Fatores de Transcrição/metabolismo , Via de Sinalização Wnt/genéticaRESUMO
Ca(2+)-activated Cl(-) channels (CaCCs) are critical to processes such as epithelial transport, membrane excitability, and signal transduction. Anoctamin, or TMEM16, is a family of 10 mammalian transmembrane proteins, 2 of which were recently shown to function as CaCCs. The functions of other family members have not been firmly established, and almost nothing is known about anoctamins in invertebrates. Therefore, we performed a phylogenetic analysis of anoctamins across the animal kingdom and examined the expression and function of anoctamins in the genetically tractable nematode Caenorhabditis elegans. Phylogenetic analyses support five anoctamin clades that are at least as old as the deuterostome/protosome ancestor. This includes a branch containing two Drosophila paralogs that group with mammalian ANO1 and ANO2, the two best characterized CaCCs. We identify two anoctamins in C. elegans (ANOH-1 and ANOH-2) that are also present in basal metazoans. The anoh-1 promoter is active in amphid sensory neurons that detect external chemical and nociceptive cues. Within amphid neurons, ANOH-1::GFP fusion protein is enriched within sensory cilia. RNA interference silencing of anoh-1 reduced avoidance of steep osmotic gradients without disrupting amphid cilia development, chemotaxis, or withdrawal from noxious stimuli, suggesting that ANOH-1 functions in a sensory mode-specific manner. The anoh-2 promoter is active in mechanoreceptive neurons and the spermatheca, but loss of anoh-2 had no effect on motility or brood size. Our study indicates that at least five anoctamin duplicates are evolutionarily ancient and suggests that sensory signaling may be a basal function of the anoctamin protein family.
Assuntos
Caenorhabditis elegans/metabolismo , Canais de Cloreto/metabolismo , Proteínas de Membrana/metabolismo , Filogenia , Animais , Transporte Biológico/genética , Caenorhabditis elegans/genética , Cálcio/metabolismo , Canais de Cloreto/genética , Proteínas de Membrana/genética , Neurônios Aferentes/metabolismo , Transdução de Sinais/genéticaRESUMO
Low back pain is a significant socioeconomic burden and intervertebral disc degeneration has been implicated as a cause. A reliable animal model of disc degeneration is necessary to evaluate therapeutics, and functional metrics are essential to quantify their benefit. To this end, needle puncture injuries were created in the caudal intervertebral discs of mice to induce disc degeneration. Compression, torsion, and creep mechanics were assessed both immediately and after eight weeks to distinguish between the effects of injury and the subsequent reparative or degenerative response. Two needle sizes (29 and 26 gauge) were used to determine injury size-dependence. Compressive stiffness (62%), torsional stiffness (60%), and early damping stiffness (84%) decreased immediately after injury with the large needle (26G). These mechanical properties did not change over time despite structural and compositional changes. At 8 weeks following large needle injury, disc height decreased (37%), nucleus pulposus (NP) glycosaminoglycan content decreased (41%), and NP collagen content increased (45%). The small needle size had no significant effect on mechanics and did not initiate degenerative changes in structure and composition. Thus, the injection of therapeutics into the NP with a minimal needle size may limit damage due to the needle insertion. These findings, along with the wide commercial availability of mouse-specific biological probes, indicate that the mouse caudal disc model can be a powerful tool for investigating disc degeneration and therapy.
Assuntos
Modelos Animais de Doenças , Degeneração do Disco Intervertebral/patologia , Degeneração do Disco Intervertebral/fisiopatologia , Disco Intervertebral/patologia , Ferimentos Penetrantes Produzidos por Agulha/patologia , Doença Aguda , Animais , Fenômenos Biomecânicos , Colágeno/metabolismo , Força Compressiva , Elasticidade , Feminino , Glicosaminoglicanos/metabolismo , Disco Intervertebral/lesões , Disco Intervertebral/metabolismo , Degeneração do Disco Intervertebral/etiologia , Lacerações , Camundongos , Camundongos Endogâmicos C57BL , Ferimentos Penetrantes Produzidos por Agulha/complicações , Estresse Mecânico , Fatores de Tempo , Torção MecânicaRESUMO
The intervertebral disc (IVD) is composed of 3 main structures, the collagenous annulus fibrosus (AF), which surrounds the gel-like nucleus pulposus (NP), and hyaline cartilage endplates, which are attached to the vertebral bodies. An IVD is located between each vertebral body. Degeneration of the IVD is thought to be a major cause of back pain, a potentially chronic condition for which there exist few effective treatments. The NP forms from the embryonic notochord. Foxa1 and Foxa2, transcription factors in the forkhead box family, are expressed early during notochord development. However, embryonic lethality and the absence of the notochord in Foxa2 null mice have precluded the study of potential roles these genes may play during IVD formation. Using a conditional Foxa2 allele in conjunction with a tamoxifen-inducible Cre allele (ShhcreER(T2)), we removed Foxa2 from the notochord of E7.5 mice null for Foxa1. Foxa1(-/-);Foxa2(c/c);ShhcreER(T2) double mutant animals had a severely deformed nucleus pulposus, an increase in cell death in the tail, decreased hedgehog signaling, defects in the notochord sheath, and aberrant dorsal-ventral patterning of the neural tube. Embryos lacking only Foxa1 or Foxa2 from the notochord were indistinguishable from control animals, demonstrating a functional redundancy for these genes in IVD formation. In addition, we provide in vivo genetic evidence that Foxa genes are required for activation of Shh in the notochord.