Otx2 is required to respond to signals from anterior neural ridge for forebrain specification

Previous analysis employing chimeric and transgenic rescue experiments has suggested that Otx2 is required in the neuroectoderm for development of the forebrain region. In order to elucidate the precise role of Otx2 in forebrain development, we attempted to generate an allelic series of Otx2 mutations by Flp- and Cre-mediated recombination for the production of conditional knock-out mice. Unexpectedly, the neo-cassette insertion created a hypomorphic Otx2 allele; consequently, the phenotype of compound mutant embryos carrying both a hypomorphic and a null allele (Otx2(frt-neo/-)) was analyzed. Otx2(frt-neo/-) mutant mice died at birth, displaying rostral head malformations. Molecular marker analysis demonstrated that Otx2(frt-neo/-) mutant embryos appeared to undergo anterior-posterior axis generation and induction of anterior neuroectoderm normally; however, these mutants subsequently failed to correctly specify the forebrain region. As the rostral margin of the neural plate, termed the anterior neural ridge (ANR), plays crucial roles with respect to neural plate specification, we examined expression of molecular markers for the ANR and the neural plate; moreover, neural plate explant studies were performed. Analyses revealed that telencephalic gene expression did not occur in mutant embryos due to defects of the neural plate; however, the mutant ANR bore normal induction activity on gene expression. These results further suggest that Otx2 dosage may be crucial in the neural plate with respect to response to inductive signals primarily from the ANR for forebrain specification.     

Fate map and morphogenesis of presumptive neural crest and dorsal neural tube

In contrast to the classical assumption that neural crest cells are induced in chick as the neural folds elevate, recent data suggest that they are already specified during gastrulation. This prompted us to map the origin of the neural crest and dorsal neural tube in the early avian embryo. Using a combination of focal dye injections and time-lapse imaging, we find that neural crest and dorsal neural tube precursors are present in a broad, crescent-shaped region of the gastrula. Surprisingly, static fate maps together with dynamic confocal imaging reveal that the neural plate border is considerably broader and extends more caudally than expected. Interestingly, we find that the position of the presumptive neural crest broadly correlates with the BMP4 expression domain from gastrula to neurula stages. Some degree of rostrocaudal patterning, albeit incomplete, is already evident in the gastrula. Time-lapse imaging studies show that the neural crest and dorsal neural tube precursors undergo choreographed movements that follow a spatiotemporal progression and include convergence and extension, reorientation, cell intermixing, and motility deep within the embryo. Through these rearrangement and reorganization movements, the neural crest and dorsal neural tube precursors become regionally segregated, coming to occupy predictable rostrocaudal positions along the embryonic axis. This regionalization occurs progressively and appears to be complete in the neurula by stage 7 at levels rostral to Hensen's node.     

Grhl2 is required in nonneural tissues for neural progenitor survival and forebrain development

Grainyhead‐like genes are part of a highly conserved gene family that play a number of roles in ectoderm development and maintenance in mammals. Here we identify a novel allele of Grhl2, cleft‐face 3 (clft3), in a mouse line recovered from an ENU mutagenesis screen for organogenesis defects. Homozygous clft3 mutants have a number of phenotypes in common with other alleles of Grhl2. We note a significant effect of genetic background on the clft3 phenotype. One of these is a reduction in size of the telencephalon where we find abnormal patterns of neural progenitor mitosis and apoptosis in mutant brains. Interestingly, Grhl2 is not expressed in the developing forebrain, suggesting this is a survival factor for neural progenitors exerting a paracrine effect on the neural tissue from the overlying ectoderm where Grhl2 is highly expressed. genesis 53:573–582, 2015. © 2015 Wiley Periodicals, Inc.     

Irreversible effects of retinoic acid pulse on Xenopus jaw morphogenesis: new insight into cranial neural crest specification

Jaws are formed by cephalic neural crest (CNCCs) and mesodermal cells migrating to the first pharyngeal arch (PA1). A complex signaling network involving different PA1 components then establishes the jaw morphogenetic program. To gather insight on this developmental process, in this study, we analyze the teratogenic effects of brief (1–15 min) pulses of low doses of retinoic acid (RA: 0.25–2 µM) or RA agonists administered to early Xenopus laevis (X.l.) embryos. We show that these brief pulses of RA cause permanent craniofacial defects specifically when treatments are performed during a 6‐hr window (developmental stages NF15–NF23) that covers the period of CNCCs maintenance, migration, and specification. Earlier or later treatments have no effect. Similar treatments performed at slightly different developmental stages within this temporal window give rise to different spectra of malformations. The RA‐dependent teratogenic effects observed in Xenopus can be partially rescued by folinic acid. We provide evidence suggesting that in Xenopus, as in the mouse, RA causes craniofacial malformations by perturbing signaling to CNCCs. Differently from the mouse, where RA affects CNCCs only at the end of their migration, in Xenopus, RA has an effect on CNCCs during all the period ranging from their exit from the neural tube until their arrival in the PA1. Our findings provide a conceptual framework to understand the origin of individual facial features and the evolution of different craniofacial morphotypes. Birth Defects Res (Part B) 89:493–503, 2010. © 2010 Wiley‐Liss, Inc.     

Temporally- and spatially regulated generation of distinct descendants by sonic hedgehog-expressing progenitors in the forebrain

The generation of distinct neural subtypes depends on the activities of cell-extrinsic and -intrinsic factors during the development of the vertebrate CNS. Previous studies have provided a molecular basis for how neural progenitors are patterned and generate distinct descendants that are spatially and temporally regulated by inductive signals secreted by polarized sources. However, it still remains unknown how the generation of neural descendants by progenitors located at polarized sources of inductive signals is controlled. Sonic hedgehog (Shh), which is expressed at the ventral midline in the forebrain, has been shown to play a critical role for the patterning and specification of distinct neural subtypes in the forebrain. Here, we analyzed the identities and distributions of Shh-descendants generated at discrete time points in the forebrain by using a ShhcreER(T2) mouse driver line in which a tamoxifen-inducible Cre cassette was inserted into the Shh locus together with a Z/EG mouse reporter line. Our results showed that Shh-expressing neural progenitors generated neuronal and glial descendants distributed throughout the telencephalon and diencephalon in a temporally distinct manner. Furthermore, our results showed that Shh-progenitors are located at two spatially distinct sub-domains that can be characterized by their temporally distinct patterns of Shh expression. These results suggest that temporally- and spatially controlled mechanisms that specify neural subtypes operate in the Shh-expressing progenitor domain, and raise the possibility that the distinct temporal gradient of Shh activity might be responsible for the generation of distinct neural subtypes in the telencephalon.     

Repulsive guidance molecule A (RGM A) and its receptor neogenin during neural and neural crest cell development of Xenopus laevis

Background information. RGM A (repulsive guidance molecule A) is a GPI (glycosylphosphatidylinositol)‐anchored glycoprotein which has repulsive properties on axons due to the interaction with its receptor neogenin. In addition, RGM A has been demonstrated to function as a BMP (bone morphogenetic protein) co‐receptor. Results. In the present study, we provide the first analysis of early RGM A and neogenin expression and function in Xenopus laevis neural development. Tissue‐specific RGM A expression starts at stage 12.5 in the anterior neural plate. Loss‐of‐function analyses suggest a function of RGM A and neogenin in regulating anterior neural marker genes, as well as eye development and neural crest cell migration. Furthermore, overexpression of RGM A leads to ectopic expression of neural crest cell marker genes. Conclusions. These data indicate that RGM A and neogenin have important functions during early neural development, in addition to their role during axonal guidance and synapse formation.     

Fate of the mammalian cranial neural crest during tooth and mandibular morphogenesis

Neural crest cells are multipotential stem cells that contribute extensively to vertebrate development and give rise to various cell and tissue types. Determination of the fate of mammalian neural crest has been inhibited by the lack of appropriate markers. Here, we make use of a two-component genetic system for indelibly marking the progeny of the cranial neural crest during tooth and mandible development. In the first mouse line, Cre recombinase is expressed under the control of the Wnt1 promoter as a transgene. Significantly, Wnt1 transgene expression is limited to the migrating neural crest cells that are derived from the dorsal CNS. The second mouse line, the ROSA26 conditional reporter (R26R), serves as a substrate for the Cre-mediated recombination. Using this two-component genetic system, we have systematically followed the migration and differentiation of the cranial neural crest (CNC) cells from E9.5 to 6 weeks after birth. Our results demonstrate, for the first time, that CNC cells contribute to the formation of condensed dental mesenchyme, dental papilla, odontoblasts, dentine matrix, pulp, cementum, periodontal ligaments, chondrocytes in Meckel's cartilage, mandible, the articulating disc of temporomandibular joint and branchial arch nerve ganglia. More importantly, there is a dynamic distribution of CNC- and non-CNC-derived cells during tooth and mandibular morphogenesis. These results are a first step towards a comprehensive understanding of neural crest cell migration and differentiation during mammalian craniofacial development. Furthermore, this transgenic model also provides a new tool for cell lineage analysis and genetic manipulation of neural-crest-derived components in normal and abnormal embryogenesis.     

Fate mapping study of the endoderm in the posterior part of the 1.5-day-old chick embryo

Various endodermal sites posterior to the caudal-most somite were marked in ovo with the vital dye Dil, and the fate of marked endoderm was analyzed after 2 or 3 days' reincubation. The endoderm in this area became gut epithelium posterior to the caudal jejunum and yolk sac. The area occupied by the cells that were to contribute to the dorsal part of the digestive tube lay centrally around the area overlaid by axial and paraxial mesoderm, with the preventral digestive area lying outside with considerable overlapping, which was surrounded by the preyolk sac area. During the formation of the posterior digestive tube, the endoderm was elongated anteroposteriorly to a considerable degree. Cells that contributed to the cloaca and those that produced descendants in the large intestine occupied similar areas posterior to the center of the sinus rhomboidalis, which were included in the pre-ileal area extending more anteriorly. Prejejunal cells generally localized in a more anterior position than pre-ileal cells. Pre-allantoic cells were located in a rather small area around the posterior primitive streak.     

神经发育中的细胞周期时程

哺乳动物的神经发育经历一系列神经前体细胞的形态结构和机能改变,其细胞周期时程也呈现动态变化,从神经发生早期至后期,神经前体细胞的细胞周期时程逐渐延长,并与细胞发育命运转归有关,其调节因素包括周期蛋白复合体、Notch信号通路、原神经基因靶向蛋白、微管与分子马达蛋白等。细胞周期长度假说认为,细胞周期的长度影响到命运决定子的积累,因而决定细胞的命运。文章综述了相关的研究进展。     

Social experience organizes parallel networks in sensory and limbic forebrain

Successful social behavior can directly influence an individual's reproductive success. Therefore, many organisms readily modify social behavior based on past experience. The neural changes induced by social experience, however, remain to be fully elucidated. We hypothesize that social modulation of neural systems not only occurs at the level of individual nuclei, but also of functional networks, and their relationships with behavior. We used the green anole lizard (Anolis carolinensis), which displays stereotyped, visually triggered social behaviors particularly suitable for comparisons of multiple functional networks in a social context, to test whether repeated aggressive interactions modify behavior and metabolic activity in limbic-hypothalamic and sensory forebrain regions, assessed by quantitative cytochrome oxidase (a slowly accumulating endogenous metabolic marker) histochemistry. We found that aggressive interactions potentiate aggressive behavior, induce changes in activities of individual nuclei, and organize context-specific functional neural networks. Surprisingly, this experiential effect is not only present in a limbic-hypothalamic network, but also extends to a sensory forebrain network directly relevant to the behavioral expression. Our results suggest that social experience modulates organisms' social behavior via modifying sensory and limbic neural systems in parallel both at the levels of individual regions and networks, potentially biasing perceptual as well as limbic processing.     

Expanding EMC foldopathies: Topogenesis deficits alter the neural crest

The endoplasmic reticulum (ER) membrane protein complex (EMC) is essential for the insertion of a wide variety of transmembrane proteins into the plasma membrane across cell types. Each EMC is composed of Emc1-7, Emc10, and either Emc8 or Emc9. Recent human genetics studies have implicated variants in EMC genes as the basis for a group of human congenital diseases. The patient phenotypes are varied but appear to affect a subset of tissues more prominently than others. Namely, craniofacial development seems to be commonly affected. We previously developed an array of assays in Xenopus tropicalis to assess the effects of emc1 depletion on the neural crest, craniofacial cartilage, and neuromuscular function. We sought to extend this approach to additional EMC components identified in patients with congenital malformations. Through this approach, we determine that EMC9 and EMC10 are important for neural crest development and the development of craniofacial structures. The phenotypes observed in patients and our Xenopus model phenotypes similar to EMC1 loss of function likely due to a similar mechanism of dysfunction in transmembrane protein topogenesis.     

Murine forebrain and midbrain crest cells generate different characteristic derivatives in vitro

Neural crest (NC) is a transient structure that gives rise to various types of tissues. Many NC cells are pluripotent in the sense that their progeny can generate more than one derivative. However, the potentiality to differentiate into certain derivatives, such as cartilage and bone, seems to be specified with respect to the neuraxial levels at which the NC generates. In order to compare the differentiation potentiality of different regions of head NC, the derivatives of forebrain and midbrain mouse NC have been investigated in vitro using explant cultures of neuroepithelial fragments. From morphology and expression of specific markers, the midbrain crest cultures obviously generated earlier and were greater in number of neuronal cells than were the forebrain ones. Moreover, collagen type II positive cells were detected in the midbrain but not in the forebrain crest cultures. Finally, pigment cells were only observed in the forebrain cultures. The results suggest that the forebrain and midbrain crest cells have a different potentiality to differentiate.     

Elimination of heparan sulfate by heparitinases induces abnormal mesodermal and neural formation in Xenopus embryos

The expression of heparan sulfate glycosaminoglycan (HS-GAG) was examined in Xenopus embryos during the developmental stages. Chemical analysis showed the existence of HS-GAG in the ³⁵S-labeled embryos. By western blot analysis using a specific anti-HS monoclonal antibody, HS-GAG related epitope was found after the neurulation on two protein bands, whose molecular weights were approximately 90 kDa and 100 kDa, respectively. Immunohistochemistry revealed that HS-GAG occurred exclusively in the animal hemisphere in early gastrulae, and then appeared predominantly on the sheath of the neural tube, the notochord and epithelium. To address whether HS-GAG chains contribute to Xenopus embryonic development, we eliminated the embryonic HS-GAG by injecting purified Flavobacterium heparitinases (HSase) into their blastocoels. Most of the injected embryos were aberrant in mesodermal and neural formation, and became acephalic. Histological examination showed that these embryos were completely devoid of the central nervous system and the mesodermal tissues. Neither heat-inactivated heparitinase nor chondroitinase showed such abnormality. The HS-GAG-eliminated embryos showed decreased expression of both muscular and neural-specific markers. These results suggest that HS-GAG plays an indispensable role in establishing the fundamental body plan during early Xenopus development.     

Dual roles of the chemorepellent axon guidance molecule RGMa in establishing pioneering axon tracts and neural fate decisions in embryonic vertebrate forebrain

Repulsive guidance molecule A (RGMa) is a glycosylphosphatidylinositol‐anchored plasma membrane protein that was originally identified based on its chemorepulsive activity during axon navigation in the developing nervous system. Knock down of RGMa has previously shown to perturb axon navigation in the developing Xenopus forebrain (Wilson and Key, 2006). In order to further understand the in vivo role of RGMa in axon guidance, we have adopted an in vivo gain‐of‐function approach. RGMa was mosaically overexpressed in the developing Xenopus embryo by the injection of mRNA into single blastomeres. Ectopic expression of RGMa affected the morphology and the topography of developing axon tracts in vivo. Pioneer axons misrouted or aberrantly projected in response to ectopic RGMa in the developing Xenopus forebrain, confirming the in vivo chemorepulsive activity of this ligand. In addition, we show here for the first time that overexpression of RGMa acts cell‐autonomously to generate ectopic neurons in the developing embryonic brain. Taken together, the current study reveals a pleiotropic role of RGMa in early vertebrate embryonic brain in the spatial organization of axon tracts, pioneer axon guidance, and neural cell differentiation. © 2011 Wiley Periodicals, Inc. Develop Neurobiol, 2012