RESUMEN
The wiring of the nervous system arises from extensive directional migration of neuronal cell bodies and growth of processes that, somehow, end up forming functional circuits. Thus far, this feat of biological engineering appears to rely on sequences of pathfinding decisions upon local cues, each with little relationship to the anatomical and physiological outcome. Here, we uncover a straightforward cellular mechanism for circuit building whereby a neuronal type directs the development of its future partners. We show that visceral afferents of the head (that innervate taste buds) provide a scaffold for the establishment of visceral efferents (that innervate salivatory glands and blood vessels). In embryological terms, sensory neurons derived from an epibranchial placode--that we show to develop largely independently from the neural crest--guide the directional outgrowth of hindbrain visceral motoneurons and control the formation of neural crest-derived parasympathetic ganglia.
Asunto(s)
Región Branquial/embriología , Ganglios/embriología , Cresta Neural/embriología , Animales , Región Branquial/metabolismo , Femenino , Ganglios/metabolismo , Ganglios Parasimpáticos/embriología , Ganglios Parasimpáticos/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Ratones , Ratones Transgénicos , Neuronas Motoras/citología , Neuronas Motoras/metabolismo , Cresta Neural/metabolismo , Neuronas Aferentes/citología , Neuronas Aferentes/metabolismo , Embarazo , Células Receptoras Sensoriales/citología , Células Receptoras Sensoriales/metabolismo , Factores de Transcripción/deficiencia , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The retrotrapezoid nucleus (RTN) is a group of neurons in the rostral medulla, defined here as Phox2b-, Vglut2-, neurokinin1 receptor-, and Atoh1-expressing cells in the parafacial region, which have been proposed to function both as generators of respiratory rhythm and as central respiratory chemoreceptors. The present study was undertaken to assess these two putative functions using genetic tools. We generated two conditional Phox2b mutations, which target different subsets of Phox2b-expressing cells, but have in common a massive depletion of RTN neurons. In both conditional mutants as well as in the previously described Phox2b(27Ala) mutants, in which the RTN is also compromised, the respiratory-like rhythmic activity normally seen in the parafacial region of fetal brainstem preparations was completely abrogated. Rhythmic motor bursts were recorded from the phrenic nerve roots in the mutants, but their frequency was markedly reduced. Both the rhythmic activity in the RTN region and the phrenic nerve discharges responded to a low pH challenge in control, but not in the mutant embryos. Together, our results provide genetic evidence for the essential role of the Phox2b-expressing RTN neurons both in establishing a normal respiratory rhythm before birth and in providing chemosensory drive.
Asunto(s)
Células Quimiorreceptoras/metabolismo , Proteínas de Homeodominio/genética , Respiración , Centro Respiratorio/metabolismo , Rombencéfalo/metabolismo , Factores de Transcripción/genética , Potenciales de Acción/fisiología , Animales , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación/genética , Red Nerviosa/embriología , Red Nerviosa/metabolismo , Red Nerviosa/fisiopatología , Técnicas de Cultivo de Órganos , Nervio Frénico/fisiología , Centro Respiratorio/embriología , Centro Respiratorio/fisiopatología , Rombencéfalo/embriología , Rombencéfalo/fisiopatologíaRESUMEN
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.
Asunto(s)
Corteza Cerebral/crecimiento & desarrollo , Proteínas de Unión al ADN/fisiología , Proteínas de Choque Térmico/fisiología , Fosfotransferasas/genética , Factores de Transcripción/fisiología , Animales , Sitios de Unión/genética , Movimiento Celular , Corteza Cerebral/embriología , Corteza Cerebral/metabolismo , ADN/genética , ADN/metabolismo , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Femenino , Regulación del Desarrollo de la Expresión Génica , Factores de Transcripción del Choque Térmico , Proteínas de Choque Térmico/deficiencia , Proteínas de Choque Térmico/genética , Técnicas In Vitro , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuroglía/citología , Neuroglía/fisiología , Neuronas/citología , Neuronas/fisiología , Embarazo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteína Reelina , Transducción de Señal , Factores de Transcripción/deficiencia , Factores de Transcripción/genéticaRESUMEN
Hedgehog signaling is required for multiple aspects of brain development, including growth, the establishment of both dorsal and ventral midline patterning and the generation of specific cell types such as oligodendrocytes and interneurons. To identify more precisely when during development hedgehog signaling mediates these events, we directed the removal of hedgehog signaling within the brain by embryonic day 9 of development, using a FoxG1(Cre) driver line to mediate the removal of a conditional smoothened null allele. We observed a loss of ventral telencephalic patterning that appears to result from an initial lack of specification of these structures rather than by changes in proliferation or cell death. A further consequence of the removal of smoothened in these mice is the near absence of both oligodendrocytes and interneurons. Surprisingly, the dorsal midline appears to be patterned normally in these mutants. Together with previous analyses, the present results demonstrate that hedgehog signaling in the period between E9.0 and E12 is essential for the patterning of ventral regions and the generation of cell types that are thought to largely arise from them.
Asunto(s)
Tipificación del Cuerpo/fisiología , Transducción de Señal/fisiología , Telencéfalo/embriología , Transactivadores/metabolismo , Animales , Corteza Cerebral/anomalías , Corteza Cerebral/citología , Corteza Cerebral/embriología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción Forkhead , Proteínas Hedgehog , Interneuronas/metabolismo , Ratones , Mutación , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Oligodendroglía/citología , Oligodendroglía/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptor Smoothened , Telencéfalo/metabolismo , Factores de Tiempo , Transactivadores/genéticaRESUMEN
Considerable data suggest that sonic hedgehog (Shh) is both necessary and sufficient for the specification of ventral pattern throughout the nervous system, including the telencephalon. We show that the regional markers induced by Shh in the E9.0 telencephalon are dependent on the dorsoventral and anteroposterior position of ectopic Shh expression. This suggests that by this point in development regional character in the telencephalon is established. To determine whether this prepattern is dependent on earlier Shh signaling, we examined the telencephalon in mice carrying either Shh- or Gli3-null mutant alleles. This analysis revealed that the expression of a subset of ventral telencephalic markers, including Dlx2 and Gsh2, although greatly diminished, persist in Shh(-/-) mutants, and that these same markers were expanded in Gli3(-/-) mutants. To understand further the genetic interaction between Shh and Gli3, we examined Shh/Gli3 and Smoothened/Gli3 double homozygous mutants. Notably, in animals carrying either of these genetic backgrounds, genes such as Gsh2 and Dlx2, which are expressed pan-ventrally, as well as Nkx2.1, which demarcates the ventral most aspect of the telencephalon, appear to be largely restored to their wild-type patterns of expression. These results suggest that normal patterning in the telencephalon depends on the ventral repression of Gli3 function by Shh and, conversely, on the dorsal repression of Shh signaling by Gli3. In addition these results support the idea that, in addition to hedgehog signaling, a Shh-independent pathways must act during development to pattern the telencephalon.
Asunto(s)
Tipificación del Cuerpo/fisiología , Proteínas de Unión al ADN/deficiencia , Proteínas del Tejido Nervioso , Receptores Acoplados a Proteínas G , Telencéfalo/embriología , Transactivadores/deficiencia , Factores de Transcripción/deficiencia , Animales , Tipificación del Cuerpo/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Regulación del Desarrollo de la Expresión Génica , Edad Gestacional , Proteínas Hedgehog , Factores de Transcripción de Tipo Kruppel , Ratones , Ratones Noqueados , Modelos Neurológicos , Fenotipo , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/fisiología , Transducción de Señal , Receptor Smoothened , Transactivadores/genética , Transactivadores/fisiología , Factores de Transcripción/genética , Factores de Transcripción/fisiología , Proteína Gli3 con Dedos de ZincRESUMEN
Heat shock transcription factors (HSFs) are characterized by their ability, upon activation, to bind to heat shock response elements (HSE) present in the promoter of their target genes. HSE are composed of inverted repeats of the pentamer nGAAm. In this study, we compare the embryonic HSF2 protein, purified from F9 embryonal carcinoma cells tumor, and the in vitro synthesized HSF2. We show that the context of HSF2 synthesis influences its thermosensitivity and DNA-binding properties. Therefore, we determined the consensus binding sequence for the purified embryonic HSF2 by the technique of systematic evolution of ligands by exponential enrichment (SELEX). We show that embryonic HSF2 prefers sites containing three or four nGAAm inverted pentamers and that its optimal binding sequence contains the 8-mer palindromic core 5'-TTCTAGAA-3'. The consensus binding sequence for the embryonic HSF2 will be very helpful to identify new targets for this factor, during developmental and differentiation processes.
Asunto(s)
Proteínas de Choque Térmico/metabolismo , Factores de Transcripción/metabolismo , Animales , Sitios de Unión , Clonación Molecular , Secuencia de Consenso , ADN/metabolismo , Biblioteca de Genes , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/aislamiento & purificación , Calor , Ratones , Unión Proteica , Factores de Transcripción/genética , Factores de Transcripción/aislamiento & purificación , Células Tumorales CultivadasRESUMEN
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.