RESUMO
Significant progress has been made in elucidating the basic principles that govern neuronal specification in the developing central nervous system. In contrast, much less is known about the origin of astrocytic diversity. Here, we demonstrate that a restricted pool of progenitors in the mouse spinal cord, expressing the transcription factor Dbx1, produces a subset of astrocytes, in addition to interneurons. Ventral p0-derived astrocytes (vA0 cells) exclusively populate intermediate regions of spinal cord with extraordinary precision. The postnatal vA0 population comprises gray matter protoplasmic and white matter fibrous astrocytes and a group of cells with strict radial morphology contacting the pia. We identified that vA0 cells in the lateral funiculus are distinguished by the expression of reelin and Kcnmb4. We show that Dbx1 mutants have an increased number of vA0 cells at the expense of p0-derived interneurons. Manipulation of the Notch pathway, together with the alteration in their ligands seen in Dbx1 knockouts, suggest that Dbx1 controls neuron-glial balance by modulating Notch-dependent cell interactions. In summary, this study highlights that restricted progenitors in the dorsal-ventral neural tube produce region-specific astrocytic subgroups and that progenitor transcriptional programs highly influence glial fate and are instrumental in creating astrocyte diversity.
Assuntos
Astrócitos , Medula Espinal , Animais , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Interneurônios/metabolismo , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Camundongos , Neuroglia/metabolismo , Transdução de Sinais/genética , Medula Espinal/metabolismoRESUMO
Generation of neuronal types at the right time, location, and number is essential for building a functional nervous system. Significant progress has been reached in understanding the mechanisms that govern neuronal diversity. Cerebrospinal fluid-contacting neurons (CSF-cNs), an intriguing spinal cord central canal population, are produced during advanced developmental stages, simultaneous with glial and ependymal cells. It is unknown how CSF-cNs are specified after the neurogenesis-to-gliogenesis switch. Here, we identify delayed Ascl1 expression in mouse spinal progenitors during the gliogenic phase as key in CSF-cN differentiation. With fate mappings and time-controlled deletions, we demonstrate that CSF-cNs derive from Ascl1-expressing cells and that Ascl1 triggers late neurogenesis in the amniote spinal cord. Ascl1 abrogation transforms prospective CSF-cN progenitors into ependymocytes. These results demonstrate that late spinal progenitors have the potential to produce neurons and that Ascl1 initiates CSF-cN differentiation, controlling the precise neuronal and nonneuronal composition of the spinal central canal.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Epêndima/metabolismo , Neurogênese , Neurônios/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Epêndima/citologia , Camundongos , Neurônios/citologia , Medula Espinal/citologia , Medula Espinal/embriologia , Medula Espinal/metabolismo , Peixe-ZebraRESUMO
Considerable progress has been made in understanding the mechanisms that control the production of specialized neuronal types. However, how the timing of differentiation contributes to neuronal diversity in the developing spinal cord is still a pending question. In this study, we show that cerebrospinal fluid-contacting neurons (CSF-cNs), an anatomically discrete cell type of the ependymal area, originate from surprisingly late neurogenic events in the ventral spinal cord. CSF-cNs are identified by the expression of the transcription factors Gata2 and Gata3, and the ionic channels Pkd2l1 and Pkd1l2. Contrasting with Gata2/3(+) V2b interneurons, differentiation of CSF-cNs is independent of Foxn4 and takes place during advanced developmental stages previously assumed to be exclusively gliogenic. CSF-cNs are produced from two distinct dorsoventral regions of the mouse spinal cord. Most CSF-cNs derive from progenitors circumscribed to the late-p2 and the oligodendrogenic (pOL) domains, whereas a second subset of CSF-cNs arises from cells bordering the floor plate. The development of these two subgroups of CSF-cNs is differentially controlled by Pax6, they adopt separate locations around the postnatal central canal and they display electrophysiological differences. Our results highlight that spatiotemporal mechanisms are instrumental in creating neural cell diversity in the ventral spinal cord to produce distinct classes of interneurons, motoneurons, CSF-cNs, glial cells and ependymal cells.
Assuntos
Líquido Cefalorraquidiano/metabolismo , Neurônios/citologia , Medula Espinal/embriologia , Medula Espinal/fisiologia , Animais , Diferenciação Celular , Linhagem da Célula , Eletrofisiologia , Proteínas do Olho/genética , Feminino , Fatores de Transcrição Forkhead/genética , Fator de Transcrição GATA2/genética , Genótipo , Imuno-Histoquímica , Hibridização In Situ , Interneurônios/citologia , Camundongos , Neurônios Motores/citologia , Células-Tronco/citologiaRESUMO
Developing granule cells (GCs) of the adult dentate gyrus undergo a critical period of enhanced activity and synaptic plasticity before becoming mature. The impact of developing GCs on the activity of preexisting dentate circuits remains unknown. Here we combine optogenetics, acute slice electrophysiology, and in vivo chemogenetics to activate GCs at different stages of maturation to study the recruitment of local target networks. We show that immature (4-week-old) GCs can efficiently drive distal CA3 targets but poorly activate proximal interneurons responsible for feedback inhibition (FBI). As new GCs transition toward maturity, they reliably recruit GABAergic feedback loops that restrict spiking of neighbor GCs, a mechanism that would promote sparse coding. Such inhibitory loop impinges only weakly in new cohorts of young GCs. A computational model reveals that the delayed coupling of new GCs to FBI could be crucial to achieve a fine-grain representation of novel inputs in the dentate gyrus.
Assuntos
Região CA3 Hipocampal/metabolismo , Giro Denteado/metabolismo , Retroalimentação Fisiológica/fisiologia , Interneurônios/metabolismo , Plasticidade Neuronal/fisiologia , Neurônios/metabolismo , Animais , Giro Denteado/citologia , Giro Denteado/crescimento & desenvolvimento , Neurônios GABAérgicos/metabolismo , Camundongos , Neurônios/citologia , Optogenética , Parvalbuminas/metabolismo , Técnicas de Patch-ClampRESUMO
The development of the nervous system is critically dependent on the production of functionally diverse neuronal cell types at their correct locations. In the embryonic neural tube, dorsoventral signaling has emerged as a fundamental mechanism for generating neuronal diversity. In contrast, far less is known about how different neuronal cell types are organized along the rostrocaudal axis. In the developing mouse and chick neural tube, hindbrain serotonergic neurons and spinal glutamatergic V3 interneurons are produced from ventral p3 progenitors, which possess a common transcriptional identity but are confined to distinct anterior-posterior territories. In this study, we show that the expression of the transcription factor Neurogenin3 (Neurog3) in the spinal cord controls the correct specification of p3-derived neurons. Gain- and loss-of-function manipulations in the chick and mouse embryo show that Neurog3 switches ventral progenitors from a serotonergic to V3 differentiation program by repressing Ascl1 in spinal p3 progenitors through a mechanism dependent on Hes proteins. In this way, Neurog3 establishes the posterior boundary of the serotonergic system by actively suppressing serotonergic specification in the spinal cord. These results explain how equivalent p3 progenitors within the hindbrain and the spinal cord produce functionally distinct neuron cell types.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Diferenciação Celular , Proteínas do Tecido Nervoso/fisiologia , Rombencéfalo/citologia , Neurônios Serotoninérgicos/citologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/biossíntese , Embrião de Galinha , Regulação da Expressão Gênica no Desenvolvimento , Interneurônios/fisiologia , Camundongos , Proteínas do Tecido Nervoso/biossíntese , Proteínas Repressoras/biossíntese , Proteínas Repressoras/fisiologia , Rombencéfalo/metabolismo , Medula Espinal/metabolismo , Medula Espinal/fisiologia , Células-Tronco/metabolismo , Células-Tronco/fisiologiaRESUMO
Mammary gland morphogenesis and differentiation are mediated through the combined activities of systemic hormones and locally synthesized growth factors. Activin, a member of the transforming growth factor (TGF)-beta superfamily, is known to regulate the growth and differentiation of several cell types. In the present study, we investigated the role of activin in rat mammary gland on different stages of development. We found that activin A in vitro inhibits the proliferation of isolated acini, and this effect increases with the development of the gland. This factor also produces in vitro an inhibition of the final differentiation of acini obtained from 19th day pregnant rats. We also report the expression of activin receptors IIA and IIB mRNA in whole rat mammary gland and acini, with decreased levels of expression of type IIA (in both compartments) and IIB (in acini) during pregnancy and lactogenesis. In addition, we show that activin betaB-subunit mRNA decreases throughout pregnancy, and that the mRNA levels of follistatin (Fst) (its ligand protein) are high in cycling rats and at the beginning of pregnancy and diminish thereafter, having the acini higher levels of expression. Our data show that activin betaB-subunit, follistatin and ActRIIA and IIB transcripts are expressed in rat mammary gland at appropriate times and locations during development, allowing an interplay that might regulate activin action on growth and differentiation of the gland.
Assuntos
Ativinas/fisiologia , Folistatina/biossíntese , Subunidades beta de Inibinas/fisiologia , Glândulas Mamárias Animais/crescimento & desenvolvimento , Receptores de Ativinas/biossíntese , Receptores de Ativinas/genética , Ativinas/genética , Ativinas/farmacologia , Animais , Caseínas/biossíntese , Caseínas/genética , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Feminino , Folistatina/genética , Folistatina/farmacologia , Regulação da Expressão Gênica , Subunidades beta de Inibinas/biossíntese , Subunidades beta de Inibinas/genética , Subunidades beta de Inibinas/farmacologia , Inibinas/biossíntese , Inibinas/genética , Lactação/fisiologia , Glândulas Mamárias Animais/efeitos dos fármacos , Glândulas Mamárias Animais/fisiologia , Gravidez , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Fator de Crescimento Transformador beta/farmacologiaRESUMO
We studied the expression of TGF-beta/T(beta)R system and its biological role in tumor development, in M3 and MM3 murine mammary adenocarcinomas with different metastasizing capability and in LM3 and LMM3 derived cell lines. All the studied cells secreted TGF-beta(s) and expressed T(beta)Rs. While the proliferation of the poorly metastatic M3 cells was significantly inhibited by 4 ng/ml TGF-beta(s), the highly metastatic MM3 cells were only slightly inhibited in response to the highest dose used. LM3 and LMM3 cells, highly invasive and metastatic, were totally refractory to TGF-beta antiproliferative effect. The role of TGF-beta in modulating key proteolytic cascades in tumor progression was also studied. TGF-beta(s) enhanced metalloproteinases production in all the studied cells while induced a stimulatory net effect on plasmin system activity only in the more metastatic cells. Our results in this murine mammary tumor lineage support the concept that dissociation of TGF-beta regulated growth control versus proteolytic enzyme pathways promotes tumor dissemination.
Assuntos
Adenocarcinoma/imunologia , Adenocarcinoma/fisiopatologia , Regulação Neoplásica da Expressão Gênica , Neoplasias Mamárias Animais/imunologia , Neoplasias Mamárias Animais/fisiopatologia , Metástase Neoplásica/fisiopatologia , Fator de Crescimento Transformador beta/farmacologia , Animais , Diferenciação Celular , Divisão Celular , Progressão da Doença , Metaloproteases/farmacologia , Camundongos , Células Tumorais CultivadasRESUMO
Tumour necrosis factor-alpha (TNF-alpha) has been proposed as an intraovarian modulator of granulosa cell function. The effect of TNF-alpha on DNA synthesis in cultured rat granulosa cells was examined. Tumour necrosis factor-alpha stimulated thymidine incorporation when added in the presence of transforming growth factor-beta (TGF-beta). In contrast, the co-mitogenic effect of follicle-stimulating hormone (FSH) and TGF-beta was inhibited in a dose-dependent manner by TNF-alpha. Inhibition of FSH-dependent DNA synthesis by TNF-alpha was also found when cultures were co-stimulated with activin A. The inhibitory action of TNF-alpha on FSH-treated cultures was not associated with changes in cell viability. The inhibitory effects of TNF-alpha could not be solely explained by a decrease in cAMP levels, since TNF-alpha was also able to inhibit the stimulation by dibutyryl-cAMP and TGF-beta on granulosa cell DNA synthesis. These results suggest that TNF-alpha regulation of granulosa cell growth is elicited either independently or downstream from gonadotrophin-induced cAMP production. The actions of TNF-alpha could be only partially mimicked by a cell-permeable analogue of ceramide, thus indicating that actions of this cytokine can not be fully ascribed to an activation of sphingomyelinase. Data presented here indicate that, in addition to its previously demonstrated inhibitory effects on gonadotrophin-induced cell differentiation, TNF-alpha may also exert a marked inhibition on hormonally stimulated immature granulosa cell proliferation. In contrast to this inhibitory action, this cytokine could amplify the mitogenic action of putative intraovarian growth regulators such as TGF-beta. These observations add further support to the notion that TNF-alpha plays a physiological role as a paracrine modulator of follicle development and may be also relevant to the alteration of ovarian function during physiopathological processes.
Assuntos
DNA/biossíntese , Células da Granulosa/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Ativinas/farmacologia , Animais , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Ceramidas/farmacologia , AMP Cíclico/metabolismo , Interações Medicamentosas , Feminino , Hormônio Foliculoestimulante/farmacologia , Células da Granulosa/citologia , Células da Granulosa/efeitos dos fármacos , Subunidades beta de Inibinas/farmacologia , Ratos , Ratos Sprague-DawleyRESUMO
We evaluated the effects of transforming growth factor beta1 (TGFbeta1), alone or in combination with FSH and estradiol, on DNA synthesis in primary cultures of immature rat granulosa cells. 3H-Thymidine incorporation was significantly stimulated by TGFbeta1 (5.6-fold). This effect was enhanced by FSH (20 ng/ml, 27.7-fold) or estradiol (100 ng/ml, 13.4-fold) or by a combination of both hormones (59.2-fold). Measurement of TGFbeta bioactivity showed the presence of significant amounts of TGFbeta in conditioned medium from granulosa cell cultures, and most of the activity was present in the latent form. FSH alone or in combination with estradiol produced a marked suppression of the production of latent and active TGFbeta. Activated conditioned medium from control cultures of granulosa cell elicited a 1.4-fold increase in thymidine incorporation. This effect was markedly amplified by FSH (3-fold) and estradiol (4.3-fold) and by a combination of both (8.7-fold). The peptide containing the cell-binding domain of fibronectin (RGDSPC) partially inhibited thymidine incorporation stimulated by TGFbeta1. Fibronectin did not synergize with FSH, and the interaction between TGFbeta1 and FSH was even observed in the presence of this protein. The conclusions reached were as follows: 1) TGFbeta1 is an autocrine stimulator of rat granulosa cell DNA synthesis, 2) FSH and estradiol produce a suppression of latent and active TGFbeta production but markedly amplify TGFbeta action, presumably at a postreceptor level, and 3) the stimulatory effects of TGFbeta1 may be only partly mediated by the increased fibronectin secretion.