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Background: R‐spondins (Rspos) are secreted proteins that modulate Wnt/β‐catenin signaling. At the early stages of spinal cord development, Wnts (Wnt1, Wnt3a) and Rspos (Rspo1, Rspo3) are co‐expressed in the roof plate, suggesting that Rspos are involved in development of dorsal spinal cord and neural crest cells in cooperation with Wnt ligands. Results: Here, we found that Rspo1 and Rspo3, as well as Wnt1 and Wnt3a, maintained roof‐plate‐specific expression until late embryonic stages. Rspo1‐ and Rspo3‐double‐knock‐out (dKO) embryos partially exhibited the phenotype of Wnt1 and Wnt3a dKO embryos. While the number of Ngn2‐positive sensory lineage neural crest cells is reduced in Rspo‐dKO embryos, development of dorsal spinal cord, including its size and dorso‐ventral patterning in early development, elongation of the roof plate, and proliferation of ependymal cells, proceeded normally. Consistent with these slight defects, Wnt/β‐catenin signaling was not obviously changed in developing spinal cord of dKO embryos. Conclusions: Our results show that Rspo1 and Rspo3 are dispensable for most developmental processes involving roof plate‐derived Wnt ligands, except for specification of a subtype of neural crest cells. Thus, Rspos may modulate Wnt/β‐catenin signaling in a context‐dependent manner. Key Findings: Rspo1‐ and Rspo3‐double‐knock‐out (dKO) embryos partially exhibited the phenotype of Wnt1 and Wnt3a dKO embryos.The number of Ngn2‐positive sensory lineage neural crest cells is reduced in Rspo‐dKO embryos.Development of dorsal spinal cord proceeded normally in Rspo‐dKO embryos.Wnt/β‐catenin signaling was not obviously changed in developing spinal cord of dKO embryos. [ABSTRACT FROM AUTHOR]

Topographic neuronal maps arise as a consequence of axon trajectory choice correlated with the localisation of neuronal soma, but the identity of the pathways coordinating these processes is unknown. We addressed this question in the context of the myotopic map formed by limb muscles innervated by spinal lateral motor column (LMC) motor axons where the Eph receptor signals specifying growth cone trajectory are restricted by Foxp1 and Lhx1 transcription factors. We show that the localisation of LMC neuron cell bodies can be dissociated from axon trajectory choice by either the loss or gain of function of the Reelin signalling pathway. The response of LMC motor neurons to Reelin is gated by Foxp1- and Lhx1-mediated regulation of expression of the critical Reelin signalling intermediate Dab1. Together, these observations point to identical transcription factors that control motor axon guidance and soma migration and reveal the molecular hierarchy of myotopic organisation.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2010.03.008 Byline: Yoko Ito (a), Hideomi Tanaka (a)(b), Hitoshi Okamoto (a)(b), Toshio Ohshima (a) Keywords: Adult neurogenesis; Neural stem cells; Optic tectum; Radial glia; Zebrafish Abstract: In the adult teleost brain, proliferating cells are observed in a broad area, while these cells have a restricted distribution in adult mammalian brains. In the adult teleost optic tectum, most of the proliferating cells are distributed in the caudal margin of the periventricular gray zone (PGZ). We found that the PGZ is largely divided into 3 regions: 1 mitotic region and 2 post-mitotic regions -- the superficial and deep layers. These regions are distinguished by the differential expression of several marker genes: pcna, sox2, msi1, elavl3, gfap, fabp7a, and s100[beta]. Using transgenic zebrafish Tg (gfap:GFP), we found that the deep layer cells specifically express gfap:GFP and have a radial glial morphology. We noted that bromodeoxyuridine (BrdU)-positive cells in the mitotic region did not exhibit glial properties, but maintained neuroepithelial characteristics. Pulse chase experiments with BrdU-positive cells revealed the presence of self-renewing stem cells within the mitotic region. BrdU-positive cells differentiate into glutamatergic or GABAergic neurons and oligodendrocytes in the superficial layer and into radial glial cells in the deep layer. These results demonstrate that the proliferating cells in the PGZ contribute to neuronal and glial lineages to maintain the structure of the optic tectum in adult zebrafish. Author Affiliation: (a) Department of Life Science and Medical Bio-Science, School of Advanced Science and Engineering, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo, 162-8480, Japan (b) Laboratory for Developmental Gene Regulation, Brain Science Institute, The Institute of Physical and Chemical Research (RIKEN), 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan Article History: Received 26 September 2009; Revised 11 March 2010; Accepted 15 March 2010

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2006.08.018 Byline: Motoko Aoki (a)(c), Michihiro Mieda (a), Toshio Ikeda (b), Yoshio Hamada (d), Harukazu Nakamura (c), Hitoshi Okamoto (a)(e) Keywords: R-spondin; Labyrinth; Placental development; Glial cells missing gene Abstract: Mouse R-spondin3 (Rspo3) is a member of the R-spondin protein family, which is characterized by furin-like cysteine-rich domains and a thrombospondin type 1 repeat. Rspo3 has been proposed to function as a secretory molecule that promotes the Wnt/[beta]-catenin signaling pathway. We generated mice bearing a mutant Rspo3 allele in which a lacZ-coding region replaced the coding region of the first exon. The homozygous mutant mice died at about embryonic day 10, due to impaired formation of the labyrinthine layer of the placenta. Rspo3 was expressed in the allantoic component of the labyrinth. In the homozygous mutant placentas, fetal blood vessels did not penetrate into the chorion, and expression of Gcm1, encoding the transcription factor glial cells missing-1 (Gcm1), was dramatically reduced in the chorionic trophoblast cells. These findings suggest a critical role for Rspo3 in the interaction between chorion and allantois in labyrinthine development. Author Affiliation: (a) Laboratory for Developmental Gene Regulation, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan (b) Laboratory for Behavioral Genetics, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan (c) Department of Molecular Neurobiology, Graduate School of Life Sciences, Tohoku University, Seiryo-machi 4-1, Aoba-ku, Sendai 980-8575, Japan (d) National Institute for Basic Biology, Okazaki, Aichi 444-8585, Japan (e) Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation (JST), 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan Article History: Received 12 April 2006; Revised 2 August 2006; Accepted 7 August 2006

The telencephalon shows the greatest degree of size variation in the vertebrate brain. Understanding the genetic cascade that regulates telencephalon growth is crucial to our understanding of how evolution of the normal human brain has supported such a variation in size. Here, we present a simple and quick approach to analyze this cascade that combines caged-mRNA technology and the use of antisense morpholino oligonucleotides in zebrafish embryos. Lhx2, a LIM-homeodomain protein, and Six3s (Six3b and Six3a), another homeodomain proteins, show very similar expression patterns early in forebrain development, and these are known to be involved in the growth of this part of the brain. The telencephalon of six3b and six3a double morphant (six3 morphant) embryos is markedly reduced in size due to impaired cellular proliferation. Head-specific overexpression of Lhx2 by photoactivation of a caged-lhx2 mRNA completely rescued this size reduction, whereas similar head-specific activation of Six3b could not rescue the knockdown effect of lhx2. In the forebrain of medaka embryos, Six3 facilitates cellular proliferation by sequestration of Geminin from Cdtl, a key component in the assembly of the prereplication complex. Our results suggest that Lhx2 may mediate an alternative or parallel pathway for control of cellular proliferation in the developing forebrain via Six3. Keywords: mRNA caging; lhx2; six3; Antisense morpholinos; Forebrain; Cell proliferation; Zebrafish

Islet-1 (Isl1) is a member of the Isl1 family of LIM-homeodomain transcription factors (LIM-HD) that is expressed in a defined subset of motor and sensory neurons during vertebrate embryogenesis. To investigate how this specific expression of isl1 is regulated, we searched for enhancers of the isl1 gene that are conserved in vertebrate evolution. Initially, two enhancer elements, CREST1 and CREST2, were identified downstream of the isl1 locus in the genomes of fugu, chick, mouse, and human by BLAST searching for highly similar elements to those originally identified as motor and sensory neuron-specific enhancers in the zebrafish genome. The combined action of these elements is sufficient for completely recapitulating the subtype-specific expression of the isl1 gene in motor neurons of the mouse spinal cord. Furthermore, by direct comparison of the upstream flanking regions of the zebrafish and human isl1 genes, we identified another highly conserved noncoding element, CREST3, and subsequently C3R, a similar element to CREST3 with two CDP CR1 recognition motifs, in the upstream regions of all other isl1 family members. In mouse and human, CRESTs are located as far as more than 300 kb away from the isl1 locus, while they are much closer to the isl1 locus in zebrafish. Although all of zebrafish CREST2, CREST3, and C3R activate gene expression in the sensory neurons of zebrafish, CREST2 of mouse and human does not have the sequence necessary for sensory neuronspecific expression. Our results revealed both a remarkable conservation of the regulatory elements regulating subtype-specific gene expression in motor and sensory neurons and the dynamic process of reorganization of these elements whereby each element increases the level of cell-type specificity by losing redundant functions with the other elements during vertebrate evolution. Keywords: Enhancer; Islet-1; Islet-2; Islet-3; Motor neuron; Sensory neuron; Subtype specification; Comparative functional genomics; Zebrafish- Mouse

Retinoic acid (RA) plays a critical role in neural patterning and organogenesis in the vertebrate embryo. Here we characterize a mutant of the zebrafish named giraffe (gir) in which the gene for the RA-degrading enzyme Cyp26a1 is mutated. The gir mutant displayed patterning defects in multiple organs including the common cardinal vein, pectoral fin, tail, hindbrain, and spinal cord. Analyses of molecular markers suggested that the lateral plate mesoderm is posteriorized in the gir mutant, which is likely to cause the defects of the common cardinal vein and pectoral fin. The cyp26a1 expression in the rostral spinal cord was strongly upregulated in the gir mutant suggesting a strong feedback control of its expression by RA signaling. We also found that the rostral spinal cord territory was expanded at the expense of the hindbrain territory in the gir mutant. Such a phenotype is the opposite of that of the mutant for Raldh2, an enzyme that synthesizes RA. We propose a model in which Cyp26a1 attenuates RA signaling in the prospective rostral spinal cord to limit the expression of hox genes and to determine the hindbrain spinal cord boundary. Keywords: giraffe; neckless; cyp26a1; rahdh2; hox; Retinoic acid; Common cardinal vein; Hindbrain; Spinal cord; Zebrafish

In vertebrate embryos, spinal motor neurons project through segmentally reiterated nerves into the somites. Here, we report that zebrafish secondary motor neurons, which are similar to motor neurons in birds and mammals, depend on myotomal cues to navigate into the periphery. We show that the absence of myotomal adaxial cells in you-too/gli2 embryos severely impairs secondary motor axonal pathfinding, including their ability to project into the somites. Moreover, in diwanka mutant embryos, in which adaxial cells are present but fail to produce cues essential for primary motor growth cones to pioneer into the somites, secondary motor axons display similar pathfinding defects. The similarities between the axonal defects in you-too/gli2 and diwanka mutant embryos strongly suggest that pathfinding of secondary motor axons depends on myotome-derived cues, and that the diwanka gene is a likely candidate to produce or encode such a cue. Our experiments also demonstrate that diwanka plays a central role in the migration of primary and secondary motor neurons, suggesting that both neural populations share mechanisms underlying axonal pathfinding. In summary, we provide compelling evidence that myotomal cells produce multiple signals to initiate and control the migration of spinal nerve axons into the somites. Key Words: spinal cord; motor axon; migration; neural development; adaxial cells; axon guidance; zebrafish.

Newborn neurons migrate extensively in the radial and tangential directions to organize the developing vertebrate nervous system. We show here that mutations in zebrafish trilobite (tri) that affect gastrulation-associated cell movements also eliminate tangential migration of motor neurons in the hindbrain. In the wild-type hindbrain, facial (nVII) and glossopharyngeal (nIX) motor neurons are induced in rhombomeres 4 and 6, respectively, and migrate tangentially into r6 and r7 (nVII) and r7 (nIX). In all three tri alleles examined, although normal numbers of motor neurons are induced, nVII motor neurons are found exclusively in r4, and nIX-like motor neurons are found exclusively in r6. The migration of other neuronal and nonneuronal cell types is unaffected in tri mutants. Rhombomere formation and the development of other hindbrain neurons are also unaffected in tri mutants. Furthermore, tangential neuronal migration occurs normally in the gastrulation mutant knypek, indicating that the trilobite neuron phenotype does not arise nonspecifically from aberrant gastrulation-associated movements. We conclude that trilobite function is specifically required for two types of cell migration that occur at different stages of zebrafish development. Key Words: zebrafish; hindbrain; motor neuron; tangential; radial; neuronal migration; gastrulation; rhombomere; green fluorescent protein; time-lapse microscopy.