RESUMO
The telencephalon is one of the most-elaborated tissues. A broad variety of cell types is produced by spatiotemporally regulated mechanisms and is involved, in different combinations, in subregional formation. The dorsal half of the telencephalon, the pallium or cerebral cortex, is subdivided along the dorsal-ventral (D-V) axis into the medial, dorsal, lateral, and ventral pallium (MP, DP, LP and VP, respectively). An in vitro differentiation system has been achieved using mouse embryonic stem cells, and major telencephalic neurons can be obtained in this way; however, in using the in vitro differentiation system, many telencephalic neuron subtypes remain undifferentiated, although some of them are related to neuronal diseases. In the current study, we found that inhibiting the TGFbeta signal was efficient for neural induction. A continuous arrangement of Emx1+/Pax6-, Emx1+/Pax6+, and Emx1-/Pax6+ cells was achieved in Foxg1+ neuroepithelia, corresponding approximately to cortical progenitors derived from MP, DP/LP, and VP, respectively. A small portion of Dbx1+ cells resided in the VP fraction. These findings suggested that the D-V axis of the pallium was recapitulated in the in vitro-derived pallium.
Assuntos
Córtex Cerebral/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Neurônios/metabolismo , Telencéfalo/metabolismo , Animais , Diferenciação Celular , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/metabolismo , Proteínas de Homeodomínio/farmacocinética , Camundongos , Fator de Transcrição PAX6/metabolismo , Fatores de Transcrição/metabolismoRESUMO
The development of the mammalian cerebral cortex involves a variety of temporally organized events such as successive waves of neuronal production and the transition of progenitor competence for each neuronal subtype generated. The number of neurons generated in a certain time period, that is, the rate of neuron production, varies across the regions of the brain and the specific developmental stage; however, the underlying mechanism of this process is poorly understood. We have recently found that nascent neurons communicate with undifferentiated progenitors and thereby regulate neurogenesis, through a transiently retained apical endfoot that signals via the Notch pathway. Here, we report that the retention time length of the neuronal apical endfoot correlates with the rate of neuronal production in the developing mouse cerebral cortex. We further demonstrate that a forced reduction or extension of the retention period through the disruption or stabilization of adherens junction, respectively, resulted in the acceleration or deceleration of neurogenesis, respectively. Our results suggest that the apical endfeet of differentiating cells serve as a pace controller for neurogenesis, thereby assuring the well-proportioned laminar organization of the neocortex.
Assuntos
Córtex Cerebral/embriologia , Neurogênese , Neurônios/fisiologia , Animais , Proliferação de Células , Camundongos Endogâmicos ICRRESUMO
The piriform cortex (paleocortex) is the olfactory cortex or the primary cortex for the sense of smell. It receives the olfactory input from the mitral and tufted cells of the olfactory bulb and is involved in the processing of information pertaining to odors. The piriform cortex and the adjoining neocortex have different cytoarchitectures; while the former has a three-layered structure, the latter has a six-layered structure. The regulatory mechanisms underlying the building of the six-layered neocortex are well established; in contrast, less is known about of the regulatory mechanisms responsible for structure formation of the piriform cortex. The differences as well as similarities in the regulatory mechanisms between the neocortex and the piriform cortex remain unclear. Here, the expression of neocortical layer-specific genes in the piriform cortex was examined. Two sublayers were found to be distinguished in layer II of the piriform cortex using Ctip2/Bcl11b and Brn1/Pou3f3. The sequential expression pattern of Ctip2 and Brn1 in the piriform cortex was similar to that detected in the neocortex, although the laminar arrangement in the piriform cortex exhibited an outside-in arrangement, unlike that observed in the neocortex.
Assuntos
Neocórtex/anatomia & histologia , Córtex Piriforme/anatomia & histologia , Animais , Camundongos , Neocórtex/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Fatores do Domínio POU/metabolismo , Córtex Piriforme/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Tempo , Proteínas Supressoras de Tumor/metabolismoRESUMO
The development of the vertebrate brain requires an exquisite balance between proliferation and differentiation of neural progenitors. Notch signaling plays a pivotal role in regulating this balance, yet the interaction between signaling and receiving cells remains poorly understood. We have found that numerous nascent neurons and/or intermediate neurogenic progenitors expressing the ligand of Notch retain apical endfeet transiently at the ventricular lumen that form adherens junctions (AJs) with the endfeet of progenitors. Forced detachment of the apical endfeet of those differentiating cells by disrupting AJs resulted in precocious neurogenesis that was preceded by the downregulation of Notch signaling. Both Notch1 and its ligand Dll1 are distributed around AJs in the apical endfeet, and these proteins physically interact with ZO-1, a constituent of the AJ. Furthermore, live imaging of a fluorescently tagged Notch1 demonstrated its trafficking from the apical endfoot to the nucleus upon cleavage. Our results identified the apical endfoot as the central site of active Notch signaling to securely prohibit inappropriate differentiation of neural progenitors.
Assuntos
Caderinas/metabolismo , Neurogênese , Neurônios/citologia , Neurônios/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais , Vertebrados/metabolismo , Junções Aderentes/metabolismo , Junções Aderentes/ultraestrutura , Animais , Encéfalo/citologia , Encéfalo/embriologia , Encéfalo/ultraestrutura , Proteínas de Ligação ao Cálcio , Adesão Celular , Galinhas , Imageamento Tridimensional , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Camundongos , Modelos Biológicos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Frações Subcelulares/metabolismoRESUMO
Thalamocortical axons (TCAs) pass through the prethalamus in the first step of their neural circuit formation. Although it has been supposed that the prethalamus is an intermediate target for thalamocortical projection formation, much less is known about the molecular mechanisms of this targeting. Here, we demonstrated the functional implications of the prethalamus in the formation of this neural circuit. We show that Olig2 transcription factor, which is expressed in the ventricular zone (VZ) of prosomere 3, regulates prethalamus formation, and loss of Olig2 results in reduced prethalamus size in early development, which is accompanied by expansion of the thalamic eminence (TE). Extension of TCAs is disorganized in the Olig2-KO dorsal thalamus, and initial elongation of TCAs is retarded in the Olig2-KO forebrain. Microarray analysis demonstrated upregulation of several axon guidance molecules, including Epha3 and Epha5, in the Olig2-KO basal forebrain. In situ hybridization showed that the prethalamus in the wild type excluded the expression of Epha3 and Epha5, whereas loss of Olig2 resulted in reduction of this Ephas-negative area and the corresponding expansion of the Ephas-positive TE. Dissociated cultures of thalamic progenitor cells demonstrated that substrate-bound EphA3 suppresses neurite extension from dorsal thalamic neurons. These results indicate that Olig2 is involved in correct formation of the prethalamus, which leads to exclusion of the EphA3-expressing region and is crucial for proper TCA formation. Our observation is the first report showing the molecular mechanisms underlying how the prethalamus acts on initial thalamocortical projection formation.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Rede Nervosa/embriologia , Proteínas do Tecido Nervoso/fisiologia , Vias Neurais/embriologia , Tálamo/embriologia , Animais , Axônios/fisiologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Células Cultivadas , Embrião de Galinha , Embrião de Mamíferos , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Camundongos Endogâmicos ICR , Camundongos Knockout , Rede Nervosa/metabolismo , Proteínas do Tecido Nervoso/genética , Vias Neurais/metabolismo , Fator de Transcrição 2 de Oligodendrócitos , Fatores de Transcrição/fisiologiaRESUMO
The cerebral cortex in mammals, the neocortex specifically, is highly diverse among species with respect to its size and morphology, likely reflecting the immense adaptiveness of this lineage. In particular, the pattern and number of convoluted ridges and fissures, called gyri and sulci, respectively, on the surface of the cortex are variable among species and even individuals. However, little is known about the mechanism of cortical folding, although there have been several hypotheses proposed. Recent studies on embryonic neurogenesis revealed the differences in cortical progenitors as a critical factor of the process of gyrification. Here, we investigated the gyrification processes using developing guinea pig brains that form a simple but fundamental pattern of gyri. In addition, we established an electroporation-mediated gene transfer method for guinea pig embryos. We introduce the guinea pig brain as a useful model system to understand the mechanisms and basic principle of cortical folding.
Assuntos
Encéfalo/embriologia , Córtex Cerebral/embriologia , Cobaias , Modelos Animais , Animais , EletroporaçãoRESUMO
The mammalian neocortex is composed of various types of neurons that reflect its laminar and area structures. It has been suggested that not only intrinsic but also afferent-derived extrinsic factors are involved in neuronal differentiation during development. However, the role and molecular mechanism of such extrinsic factors are almost unknown. Here, we attempted to identify molecules that are expressed in the thalamus and affect cortical cell development. First, thalamus-specific molecules were sought by comparing gene expression profiles of the developing rat thalamus and cortex using microarrays, and by constructing a thalamus-enriched subtraction cDNA library. A systematic screening by in situ hybridization showed that several genes encoding extracellular molecules were strongly expressed in sensory thalamic nuclei. Exogenous and endogenous protein localization further demonstrated that two extracellular molecules, Neuritin-1 (NRN1) and VGF, were transported to thalamic axon terminals. Application of NRN1 and VGF to dissociated cell culture promoted the dendritic growth. An organotypic slice culture experiment further showed that the number of primary dendrites in multipolar stellate neurons increased in response to NRN1 and VGF, whereas dendritic growth of pyramidal neurons was not promoted. These molecules also increased neuronal survival of multipolar neurons. Taken together, these results suggest that the thalamus-specific molecules NRN1 and VGF play an important role in the dendritic growth and survival of cortical neurons in a cell type-specific manner.
Assuntos
Sobrevivência Celular/efeitos dos fármacos , Córtex Cerebral/citologia , Dendritos/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Tálamo/química , Tálamo/fisiologia , Animais , Anticorpos Bloqueadores/farmacologia , Células Cultivadas , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/crescimento & desenvolvimento , Primers do DNA , DNA Complementar/biossíntese , DNA Complementar/genética , Eletroporação , Feminino , Proteínas Ligadas por GPI/antagonistas & inibidores , Proteínas Ligadas por GPI/química , Proteínas Ligadas por GPI/farmacologia , Vetores Genéticos , Imuno-Histoquímica , Hibridização In Situ , Masculino , Análise em Microsséries , Neuropeptídeos/antagonistas & inibidores , Neuropeptídeos/química , Neuropeptídeos/farmacologia , Plasmídeos/genética , Gravidez , Terminações Pré-Sinápticas/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , TransfecçãoRESUMO
Tsukushi (TSK) proteoglycan dysfunction leads to hydrocephalus, a condition defined by excessive fluid collection in the ventricles and lateral ventricular enlargement. TSK injections into the LV at birth are effective at rescuing the lateral ventricle (LV). TSK regulates the activation of the Wnt signaling to facilitate the proper expansion of the LV and maintain the fate of the neural stem cell lineage. However, the molecular mechanism by which TSK acts on neural stem/progenitor cells (NSCs) during LV development is unknown. We demonstrated that TSK is crucial for the splicing and development-associated gene regulation of GFAP-expressing subventricular zone (SVZ) NSCs. We isolated GFAP-expressing NSCs from the SVZ of wild-type (GFAPGFP/+/TSK+/+) and TSK knock-out (GFAPGFP/+/TSK-/-) mice on postnatal day 3 and compared their transcriptome and splicing profiles. TSK deficiency in NSCs resulted in genome-wide missplicing (alteration in exon usage) and transcriptional dysregulation affecting the post-transcriptional regulatory processes (including splicing, cell cycle, and circadian rhythm) and developmental signaling networks specific to the cell (including Wnt, Sonic Hedgehog, and mTOR signaling). Furthermore, TSK deficiency prominently affected the splicing of genes encoding RNA and DNA binding proteins in the nervous SVZ and non-nervous muscle tissues. These results suggested that TSK is involved in the maintenance of correct splicing and gene regulation in GFAP-expressing NSCs, thereby protecting cell fate and LV development. Hence, our study provides a critical insight on hydrocephalus development.
RESUMO
Neuronal abundance and thickness of each cortical layer are specific to each area, but how this fundamental feature arises during development remains poorly understood. While some of area-specific features are controlled by intrinsic cues such as morphogens and transcription factors, the exact influence and mechanisms of action by cues extrinsic to the cortex, in particular the thalamic axons, have not been fully established. Here, we identify a thalamus-derived factor, VGF, which is indispensable for thalamocortical axons to maintain the proper amount of layer 4 neurons in the mouse sensory cortices. This process is prerequisite for further maturation of the primary somatosensory area, such as barrel field formation instructed by a neuronal activity-dependent mechanism. Our results provide an actual case in which highly site-specific axon projection confers further regional complexity upon the target field through locally secreting signaling molecules from axon terminals.
Assuntos
Neocórtex , Animais , Axônios/fisiologia , Camundongos , Neocórtex/fisiologia , Neurônios/fisiologia , Terminações Pré-Sinápticas , Córtex Somatossensorial/fisiologia , Tálamo/fisiologiaRESUMO
Microtubule (MT) dynamics are modulated through the coordinated action of various MT-associated proteins (MAPs). However, the regulatory mechanisms underlying MT dynamics remain unclear. We show that the MAP7 family protein Map7D2 stabilizes MTs to control cell motility and neurite outgrowth. Map7D2 directly bound to MTs through its N-terminal half and stabilized MTs in vitro. Map7D2 localized prominently to the centrosome and partially on MTs in mouse N1-E115 neuronal cells, which expresses two of the four MAP7 family members, Map7D2 and Map7D1. Map7D2 loss decreased the resistance to the MT-destabilizing agent nocodazole without affecting acetylated/detyrosinated stable MTs, suggesting that Map7D2 stabilizes MTs via direct binding. In addition, Map7D2 loss increased the rate of random cell migration and neurite outgrowth, presumably by disturbing the balance between MT stabilization and destabilization. Map7D1 exhibited similar subcellular localization and gene knockdown phenotypes to Map7D2. However, in contrast to Map7D2, Map7D1 was required for the maintenance of acetylated stable MTs. Taken together, our data suggest that Map7D2 and Map7D1 facilitate MT stabilization through distinct mechanisms in cell motility and neurite outgrowth.
Assuntos
Microtúbulos , Neurônios , Animais , Movimento Celular/genética , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Neurônios/metabolismo , Nocodazol/metabolismo , Nocodazol/farmacologiaRESUMO
To define the roles of α-catenin in cell-cell adhesion, the E-cadherin, α-catenin, ß-catenin, and/or plakoglobin genes were inactivated in F9 teratocarcinoma cells. An E-cadherin-α-catenin fusion protein (Eα) restored full cell-adhesion function and organized the actin-based cytoskeleton and ZO-1, an actin filament binding protein, in F9 cells lacking all endogenous cadherin-catenin complex components. There were two types of cadherin-based cell-adhesion junctions in parental F9 cells, those with ZO-1 and those without ZO-1, and only junctions with ZO-1 were associated with thick actin bundles. Additionally, ZO-1 localized to most Eα-based cell-adhesion junctions. These data demonstrated that Eα supported cadherin-based cell adhesion and recruited actin bundles and ZO-1 to cell-cell contact sites in the absence of cytoplasmic α-catenin. Moreover, the C-terminal half of α-catenin was involved in the formation of cell-adhesion junctions with ZO-1.
Assuntos
Caderinas/metabolismo , Citoesqueleto/ultraestrutura , alfa Catenina/metabolismo , Actinas/ultraestrutura , Animais , Caderinas/genética , Adesão Celular , Linhagem Celular Tumoral , Deleção de Genes , Proteínas de Membrana/análise , Camundongos , Fosfoproteínas/análise , Proteína da Zônula de Oclusão-1 , alfa Catenina/genéticaRESUMO
Proper brain development requires precisely controlled phases of stem cell proliferation, lineage specification, differentiation, and migration. Lineage specification depends partly on concentration gradients of chemical cues called morphogens. However, the rostral brain (telencephalon) expands prominently during embryonic development, dynamically altering local morphogen concentrations, and telencephalic subregional properties develop with a time lag. Here, we investigated how progenitor specification occurs under these spatiotemporally changing conditions using a three-dimensional in vitro differentiation model. We verified the critical contributions of three signaling factors for the lineage specification of subregional tissues in the telencephalon, ventralizing sonic hedgehog (Shh) and dorsalizing bone morphogenetic proteins (BMPs) and WNT proteins (WNTs). We observed that a short-lasting signal is sufficient to induce subregional progenitors and that the timing of signal exposure for efficient induction is specific to each lineage. Furthermore, early and late progenitors possess different Shh signal response capacities. This study reveals a novel developmental mechanism for telencephalon patterning that relies on the interplay of dose- and time-dependent signaling, including a time lag for specification and a temporal shift in cellular Shh sensitivity. This delayed fate choice through two-phase specification allows tissues with marked size expansion, such as the telencephalon, to compensate for the changing dynamics of morphogen signals.
RESUMO
Roles of interstitial tissue in morphogenesis of testicular structures remain less well understood. To analyze the roles of CD34+ cells in the reconstruction of interstitial tissue containing Leydig cells (LCs), and testicular structures, we used 3D-reaggregate culture of dissociated testicular cells from prepubertal mouse. After a week of culture, adult Leydig cells (ALCs) were preferentially incorporated within CD34+ cell-aggregates, but fetal LCs (FLCs) were not. Immunofluorescence studies showed that integrins α4, α9 and ß1, and VCAM1, one of the ligands for integrins α4ß1 and α9ß1, are expressed mainly in CD34+ cells and ALCs, but not in FLCs. Addition of function-blocking antibodies against each integrin and VCAM1 to the culture disturbed the reconstruction of testicular structures. Antibodies against α4 and ß1 integrins and VCAM1 robustly inhibited cell-to-cell adhesion between testicular cells and between CD34+ cells. Cell-adhesion assays indicated that CD34+ cells adhere to VCAM1 through the interaction with α4ß1 integrin. Live cell imaging showed that CD34+ cells adhered around ALC-aggregates. CD34+ cells on the dish moved toward the aggregates, extending filopodia, and entered into them, which was disturbed by VCAM1 antibody. These results indicate that VCAM1-α4ß1 integrin interaction plays pivotal roles in formation of testicular interstitial tissues in vitro and also in vivo.
Assuntos
Integrina alfa4beta1/metabolismo , Testículo/citologia , Testículo/metabolismo , Molécula 1 de Adesão de Célula Vascular/metabolismo , Animais , Antígenos CD34/metabolismo , Biomarcadores , Adesão Celular/efeitos dos fármacos , Adesão Celular/genética , Células Cultivadas , Expressão Gênica , Integrina alfa4beta1/genética , Células Intersticiais do Testículo/metabolismo , Masculino , Camundongos , Organogênese/genética , Ligação Proteica , Isoformas de Proteínas , Maturidade Sexual , Esferoides Celulares , Molécula 1 de Adesão de Célula Vascular/genética , Molécula 1 de Adesão de Célula Vascular/farmacologiaRESUMO
The lateral ventricle (LV) is flanked by the subventricular zone (SVZ), a neural stem cell (NSC) niche rich in extrinsic growth factors regulating NSC maintenance, proliferation, and neuronal differentiation. Dysregulation of the SVZ niche causes LV expansion, a condition known as hydrocephalus; however, the underlying pathological mechanisms are unclear. We show that deficiency of the proteoglycan Tsukushi (TSK) in ependymal cells at the LV surface and in the cerebrospinal fluid results in hydrocephalus with neurodevelopmental disorder-like symptoms in mice. These symptoms are accompanied by altered differentiation and survival of the NSC lineage, disrupted ependymal structure, and dysregulated Wnt signaling. Multiple TSK variants found in patients with hydrocephalus exhibit reduced physiological activity in mice in vivo and in vitro. Administration of wild-type TSK protein or Wnt antagonists, but not of hydrocephalus-related TSK variants, in the LV of TSK knockout mice prevented hydrocephalus and preserved SVZ neurogenesis. These observations suggest that TSK plays a crucial role as a niche molecule modulating the fate of SVZ NSCs and point to TSK as a candidate for the diagnosis and therapy of hydrocephalus.
Assuntos
Hidrocefalia , Células-Tronco Neurais , Neurogênese , Proteoglicanas , Animais , Proliferação de Células , Humanos , Camundongos , Camundongos Knockout , Nicho de Células-TroncoRESUMO
The morphogenesis of mammalian embryonic external genitalia (eExG) shows dynamic differences between males and females. In genotypic males, eExG are masculinized in response to androgen signaling. Disruption of this process can give rise to multiple male reproductive organ defects. Currently, mechanisms of androgen-driven sexually dimorphic organogenesis are still unclear. We show here that mesenchymal-derived actomyosin contractility, by MYH10, is essential for the masculinization of mouse eExG. MYH10 is expressed prominently in the bilateral mesenchyme of male eExG. Androgen induces MYH10 protein expression and actomyosin contractility in the bilateral mesenchyme. Inhibition of actomyosin contractility through blebbistatin treatment and mesenchymal genetic deletion induced defective urethral masculinization with reduced mesenchymal condensation. We also suggest that actomyosin contractility regulates androgen-dependent mesenchymal directional cell migration to form the condensation in the bilateral mesenchyme leading to changes in urethral plate shape to accomplish urethral masculinization. Thus, mesenchymal-derived actomyosin contractility is indispensable for androgen-driven urethral masculinization.
Assuntos
Actomiosina/metabolismo , Androgênios/metabolismo , Uretra/fisiologia , Animais , Biomarcadores , Imunofluorescência , Imuno-Histoquímica , Masculino , Camundongos , Modelos Biológicos , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Miosina não Muscular Tipo IIB/genética , Miosina não Muscular Tipo IIB/metabolismoRESUMO
In vitro whole-embryo culture of chick embryos, originally invented by New, has been widely used for studies of early embryogenesis. Here, a method for electroporation using the New culture and its derivatives is described, to achieve misexpression of exogenous gene in a temporally and spatially controlled manner in gastrulating chick embryos. Detailed information for the devices and procedures, and some experimental examples are presented.
Assuntos
Embrião de Galinha , Biologia do Desenvolvimento/métodos , Eletroporação/métodos , Técnicas de Cultura Embrionária/métodos , Técnicas de Transferência de Genes , Animais , Proteínas de Fluorescência Verde/química , Plasmídeos/metabolismo , Fatores de Tempo , TransfecçãoRESUMO
A method for predicting pharmacokinetics in pediatric patients for intravenous beta-lactam antibiotics is proposed. We focused on the allometric relationships of pharmacokinetic parameters with individual body weights (BW) in human including healthy adults and pediatric patients. Drug concentration data for 15 intravenous beta-lactam antibiotics were collected retrospectively from the published articles and the individual pharmacokinetic parameters were re-calculated. A mixed effect modeling (MEM) was applied for the allometric relationship for those beta-lactam antibiotics, and mean and variances of inter-drug variability for the allometric parameters and also variance for intra-drug (residual) variability were estimated. Then drug-specific allometric parameters were estimated by an empirical Bayesian method using the pharmacokinetic parameters for a drug only in healthy adults as observations, and finally the individual pharmacokinetic parameters in pediatric patients were predicted. The predictability of the method was evaluated by the leave-one-out method. We also demonstrated a method for simulating plasma concentration-time profiles in pediatric patients, and the predicted time-course curves generally coincided well with the actual plasma concentration data for the tested drugs.
Assuntos
Antibacterianos/farmacocinética , beta-Lactamas/farmacocinética , Adolescente , Adulto , Antibacterianos/sangue , Criança , Pré-Escolar , Humanos , Lactente , Injeções Intravenosas , beta-Lactamas/sangueRESUMO
Tissue reconstruction in vitro can provide, if successful, a refined and simple system to analyze the underlying mechanisms that drive the morphogenesis and maintain the ordered structure. We have recently succeeded in reconstruction of seminiferous cord-like and tubule-like structures using 3-D re-aggregate culture of dissociated testicular cells. In testis formation, endothelial cells that migrated from mesonephroi to embryonic gonads have been shown to be critical for development of testis cords, but how endothelial cells contribute to testis cord formation remains unknown. To decipher the roles of endothelial and peritubular cells in the reconstruction of cord-like and tubule-like structures, we investigated the behavior of CD34+ endothelial and p75+ cells, and peritubular myoid cells (PTMCs) in 3-D re-aggregate cultures of testicular cells. The results showed that these 3 types of cells had the capacity of re-aggregation on their own and with each other, and of segregation into 3 layers in a re-aggregate, which were very similar to interstitial and peritubular tissues in vivo. Observation of behaviors of fluorescent Sertoli cells and other non-fluorescent types of cells using testes from Sox9-EGFP transgenic mice showed dynamic cell movement and segregation in re-aggregate cultures. Cultures of testicular cells deprived of interstitial and peritubular cells resulted in dysmorphic structures, but re-addition of them restored tubule-like structures. Purified CD34+ cells in culture differentiated into p75+ cells and PTMCs. These results indicate that CD34+ cells differentiate into p75+ cells, which then differentiate into PTMCs. TGFß signaling inhibitors, SB431542 and ALK5i, disturbed the reconstruction of cord-like and tubule-like structures, and the latter compromised re-construction of interstitial-like and peritubular-like structures, as well as the proliferation of CD34+, p75+, PTMCs, and Sertoli cells, and their movement and differentiation. These results indicate that CD34+ cells and signaling through ALK5 play pivotal roles in the morphogenesis of interstitial-like, peritubular-like and cord-like structures.
Assuntos
Antígenos CD34/imunologia , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Túbulos Seminíferos/anatomia & histologia , Transdução de Sinais , Animais , Animais Recém-Nascidos , Proliferação de Células , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptor do Fator de Crescimento Transformador beta Tipo I , Túbulos Seminíferos/citologiaRESUMO
Notch signaling in neural progenitor cell is triggered by ligands expressed in adjacent cells. To identify the sources of active Notch ligands in the mouse retina, we negatively regulated Notch ligand activity in various neighbors of retinal progenitor cells (RPCs) by eliminating mindbomb E3 ubiquitin protein ligase 1 (Mib1). Mib1-deficient retinal cells failed to induce Notch activation in intra-lineage RPCs, which prematurely differentiated into neurons; however, Mib1 in post-mitotic retinal ganglion cells was not important. Interestingly, Mib1 in the retinal pigment epithelium (RPE) also contributed to Notch activation in adjacent RPCs by supporting the localization of active Notch ligands at RPE-RPC contacts. Combining this RPE-driven Notch signaling and intra-retinal Notch signaling, we propose a model in which one RPC daughter receives extra Notch signals from the RPE to become an RPC, whereas its sister cell receives only a subthreshold level of intra-retinal Notch signal and differentiates into a neuron.