Evolutionary conserved role of ptf1a in the specification of exocrine pancreatic fates

We have characterized and mapped the zebrafish ptf1a gene, analyzed its embryonic expression, and studied its role in pancreas development. In situ hybridization experiments show that from the 12-somite stage to 48 hpf, ptf1a is dynamically expressed in the spinal cord, hindbrain, cerebellum, retina, and pancreas of zebrafish embryos. Within the endoderm, ptf1a is initially expressed at 32 hpf in the ventral portion of the pdx1 expression domain; ptf1a is expressed in a subset of cells located on the left side of the embryo posteriorly to the liver primordium and anteriorly to the endocrine islet that arises from the posterodorsal pancreatic anlage. Then the ptf1a expression domain buds giving rise to the anteroventral pancreatic anlage that grows posteriorly to eventually engulf the endocrine islet. By 72 hpf, ptf1a continues to be expressed in the exocrine compartment derived from the anteroventral anlage. Morpholino-induced ptf1a loss of function suppresses the expression of the exocrine markers, while the endocrine markers in the islet are unaffected. In mind bomb (mib) mutants, in which delta-mediated notch signalling is defective [Dev. Cell 4 (2003) 67], ptf1a is normally expressed. In addition, the slow-muscle-omitted (smu) mutants that lack expression of endocrine markers because of a defective hedgehog signalling [Curr. Biol. 11(2001) 1358] exhibit normal levels of ptf1a. This indicates that hedgehog signaling plays a different genetic role in the specification of the anteroventral (mostly exocrine) and posterodorsal (endocrine) pancreatic anlagen.     

Expression of protocadherin 18 in the CNS and pharyngeal arches of zebrafish embryos

Here, we report the results of molecular cloning and expression analyses of a non-clustered protocadherin (pcdh), pcdh18 in zebrafish embryos. The predicted zebrafish pcdh18 protein shows 6566% identity and 7879% homology with its mammalian and Xenopus counterparts. It has a Disabled-1 binding motif in its cytoplasmic domain, which is characteristic of pcdh18. Zebrafish embryos expressed pcdh18 by the early gastrula stage, 6 h post-fertilization (hpf), in their animal cap but not in the germ ring or the shield. pcdh18 was expressed in the neural tube and the central nervous system (CNS) from 12 hpf. Some populations of cells in the lateral neural tube and spinal cord of 1218 hpf embryos expressed pcdh18, but expression in these cells disappeared by 24 hpf. The hindbrain of embryos at 2456 hpf expressed pcdh18 in cells closely adjacent to the rostral and caudal rhombomeric boundaries in a thread-like pattern running in the dorsoventral direction. The pcdh18-positive cells were localized in the ventral part of the hindbrain at 24 hpf and in the dorsal part from 36 hpf. pcdh18 was also expressed in the telencephalon, diencephalon, tectum, upper rhombic lip, retina and otic vesicle. Expression in the CNS decreased markedly before hatching. Pharyngeal arch primordia, arches, jaws and gills expressed pcdh18, and the molecule was also expressed in some endodermal cells in late embryos.     

Expression of the delta-protocadherin gene Pcdh19 in the developing mouse embryo

Protocadherins constitute a large family of transmembrane proteins primarily involved in weak homophilic adhesion in the brain and several other tissues. In a screen for potential regulators of kidney development, we have identified Pcdh19, a poorly characterized member of the delta-protocadherin subfamily. Here, we report the spatio-temporal expression pattern of Pcdh19 during mouse embryonic development. In midgestation embryos, Pcdh19 mRNA was detected in the mesonephros and in the neuroepithelium of the forebrain and midbrain. At later stages, Pcdh19 was expressed in other neural tissues such as the neural retina, nasal epithelium and spinal cord, as well as in the collecting duct and differentiating nephrons of the metanephros, in the glandular stomach, the exocrine pancreas and the hair follicles. Hence, the Pcdh19 gene is developmentally regulated during mouse organogenesis and shows a unique expression profile among protocadherins.     

The expression pattern of Follistatin-like 1 in mouse central nervous system development

Follistatin-like 1 (Fstl1), also named TSC-36 (TGF-β-stimulated clone 36), was first cloned from the mouse osteoblastic MC3T3-E1 cell line and can be up-regulated by TGF-β. To better study the function of Fstl1 during the development of the mouse central nervous system (CNS), we examined Fstl1 expression in the developing mouse CNS, in detail, by in situ hybridization. Our results show that Fstl1 is strongly expressed in the telencephalon, diencephalon, brainstem, limbic system and spinal cord. In the telencephalon, Fstl1 positive cells are mainly located in the ventricular zone (VZ) and the subventricular zone (SVZ); a relatively weak signal was observed in layers II and III of the neocortex at postnatal stages. Fstl1 expression is robust in the developing hippocampus and persists to P20. In the developing diencephalon and hindbrain, abundant Fstl1 signals were also detected in nuclei including the medial habenular nucleus, the medial dorsal nucleus, the cochlear nuclei and so on. In addition, a strong expression of Fstl1 was detected in the thalamencephalic signal center, as well as in the olfactory cortex from E14.5 to P0. Meanwhile, Fstl1 was expressed in the septal area and the cingulate gyrus of the limbic system after birth. A high level of expression was also observed in the ventral horn of the spinal cord. These results indicate that Fstl1 may play an important role during CNS development in the mouse.     

Cloning of zebrafish vsx1: Expression of a paired-like homeobox gene during CNS development

vsx1 is a homeobox gene encoding a paired-type homeodomain and a CVC domain that was originally cloned from an adult goldfish retinal library. We previously reported the spatiotemporal expression pattern of vsx1in the adult and developing retina of zebrafish and goldfish, and we suggested that vsx1 plays a role in determining the cell fate and maintenance of retinal interneurons. Other related genes encoding a CVC domain, such as vsx2 (alx) and chx10, are expressed both within and outside the retina during development. In this study, we report the cloning of zebrafish vsx1 and its developmental expression in both retinal and nonretinal regions of the CNS in zebrafish embryos. vsx1expression was detected in a subset of hindbrain and spinal cord neurons before it was expressed in the retina. At about the same time that retinal expression began, the level of vsx1 was decreased in the spinal cord. The expression of vsx1 was progressively restricted, and eventually it was detected only in the inner nuclear layer (INL) of the developing retina. The combined expression patterns of teleost vsx1 and vsx2 (alx) during early zebrafish development encompasses the expression pattern observed for murine Chx10, and indicates a partitioning of function for CVC genes in lower vertebrates. Dev. Genet. 23:128–141, 1998. © 1998 Wiley-Liss, Inc.     

Antisense attenuation of Wnt-1 and Wnt-3a expression in whole embryo culture reveals roles for these genes in craniofacial,spinal cord,and cardiac morphogenesis

Wnt-1 and Wnt-3a proto-on-cogenes have been implicated in the development of midbrain and hindbrain structures. Evidence for such a role has been derived from in situ hybridization studies showing Wnt-1 and -3a expression in developing cranial and spinal cord regions and from studies of mutant mice whose Wnt-1 genes have undergone targeted disruption by homologous recombination. Wnt-1 null mutants exhibit cranial defects but no spinal cord abnormalities, despite expression of the gene in these regions. The absence of spinal cord abnormalities is thought to be due to a functional compensation of the Wnt-1 deficiency by related genes, a problem that has complicated the analysis of null mutants of other developmental genes as well. Herein, we describe the attenuation of Wnt-1 expression using antisense oligonucleotide inhibition in mouse embryos grown in culture. We induce similar mid- and hindbrain abnormalities as those seen in the Wnt-1 null mutant mice. Attentuation of Wnt-1 expression was also associated with cardiomegaly resulting in hemostasis. These findings are consistent with the possibility that a subset of Wnt-1 expressing cells include neural crest cells known to contribute to septation of the truncus arteriosus and to formation of the visceral arches. Antisense knockout of Wnt-3a, a gene structurely related to Wnt-1, targeted the forebrain and midbrain region, which were hy-poplastic and failed to expand, and the spinal cord, which exhibited lateral outpocketings at the level of the forelimb buds. Dual antisense knockouts of Wnt-1 and Wnt-3a targeted all brain regions leading to incomplete closure of the cranial neural folds, and an increase in the number and severity of outpocketings along the spinal cord, suggesting that these genes complement one another to produce normal patterning of the spinal cord. The short time required to assess the mutant phenotype (2 days) and the need for limited sequence information of the target gene (20-25 nu-cleotides) make this antisense oligonucleotide/ whole embryo culture system ideal for testing the importance of specific genes and their interactions in murine embryonic development. © 1993 Wiley-Liss, Inc.     

Time course of the development of motor behaviors in the zebrafish embryo

The development and properties of locomotor behaviors in zebrafish embryos raised at 28.5°C were examined. When freed from the chorion, embryonic zebrafish showed three sequential stereotyped behaviors: a transient period of alternating, coiling contractions followed by touch-evoked rapid coils, then finally, organized swimming. The three different behaviors were characterized by video microscopy. Spontaneous, alternating contractions of the trunk appeared suddenly at 17 h postfertilization (hpf), with a frequency of 0.57 Hz, peaked at 19 hpf at 0.96 Hz, and gradually decreased to <0.1 Hz by 27 hpf. Starting at 21 hpf, touching either the head or the tail of the embryos resulted in vigorous coils. The coils accelerated with development, reaching a maximum speed of contraction before 48 hpf, which is near the time of hatching. After 27 hpf, touching the embryos, particularly on the tail, could induce partial coils (instead of full coils). At this time, embryos started to swim in response to a touch, preferentially to the tail. The swim cycle frequency gradually increased with age from 7 Hz at 27 hpf to 28 Hz at 36 hpf. Lesions of the central nervous system rostral to the hindbrain had no effect on the three behaviors. Lesioning the hindbrain eliminated swimming and touch responses, but not the spontaneous contractions. Our observations suggest that the spontaneous contractions result from activation of a primitive spinal circuit, while touch and swimming require additional hindbrain inputs to elicit mature locomotor behaviors. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 622–632, 1998     

Expression of<Emphasis Type="Italic"> neurochondrin</Emphasis> in the developing and adult mouse brain

Here we describe a detailed analysis of the expression of neurochondrin (ncdn) in the developing and adult mouse brain. Ncdn is first expressed in the hindbrain and spinal cord at embryonic day 10.5 (E10.5) followed by expression in the midbrain at E11.5. By E18 ncdn is also expressed in the diencephalon and telencephalon. However, strongest expression is still observed in the hindbrain. In adults, the expression in the forebrain is as strong as in the hindbrain. Ncdn is highly expressed in the hippocampus, piriform cortex, septum, amygdaloid complex, medial geniculate nucleus, inferior colliculus, cerebellar nuclei and the nuclei of the Vth, VIIth, and XIIth cranial nerves.Edited by B. HerrmannR. Istvánffy and D.M. Vogt Weisenhorn contributed equally to this paper     

Identification of initially appearing glycine‐immunoreactive neurons in the embryonic zebrafish brain

Glycine is a major inhibitory neurotransmitter in the central nervous system of vertebrates. Here, we report the initial development of glycine‐immunoreactive (Gly‐ir) neurons and fibers in zebrafish. The earliest Gly‐ir cells were found in the hindbrain and rostral spinal cord by 20 h post‐fertilization (hpf). Gly‐ir cells in rhombomeres 5 and 6 that also expressed glycine transporter 2 (glyt2) mRNA were highly stereotyped; they were bilaterally located and their axons ran across the midline and gradually turned caudally, joining the medial longitudinal fascicles in the spinal cord by 24 hpf. Gly‐ir neurons in rhombomere 5 were uniquely identified, since there was one per hemisegment, whereas the number of Gly‐ir neurons in rhombomere 6 were variable from one to three per hemisegment. Labeling of these neurons by single‐cell electroporation and tracing them until the larval stage revealed that they became MiD2cm and MiD3cm, respectively. The retrograde labeling of reticulo‐spinal neurons in Tg(glyt2:gfp) larva, which express GFP in Gly‐ir cells, and a genetic mosaic analysis with glyt2:gfp DNA construct also supported this notion. Gly‐ir cells were also distributed widely in the anterior brain by 27 hpf, whereas glyt2 was hardly expressed. Double staining with anti‐glycine and anti‐GABA antibodies demonstrated distinct distributions of Gly‐ir and GABA‐ir cells, as well as the presence of doubly immunoreactive cells in the brain and placodes. These results provide evidence of identifiable glycinergic (Gly‐ir/glyt2‐positive) neurons in vertebrate embryos, and they can be used in further studies of the neurons' development and function at the single‐cell level. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 74: 616–632, 2014     

Cloning and characterization of zebrafish protocadherin–17

Protocadherins constitute the largest subgroup within the cadherin superfamily of cell surface molecules. In this study, we report the molecular cloning and expression analysis of the non-clustered protocadherin-17 (pcdh17) in the embryonic zebrafish nervous system. The zebrafish Pcdh17 protein is highly conserved, exhibiting 73% sequence homology with the human protein. The zebrafish pcdh17 gene consists of four exons spread over 150 kb, and this organization is highly conserved throughout vertebrates. Pcdh17 message is first detectable by 6 h postfertilization in the developing embryo, and the expression is maintained throughout development. Zebrafish embryos express pcdh17 in all of the major subdivisions of the central nervous system, including the telencephalon, diencephalon, mesencephalon, and rhombencephalon. Analysis of the genomic sequence upstream of pcdh17 in several species reveals a pattern of paired CpG islands. While the CpG islands in zebrafish are further upstream than in other teleosts, alignment of the identified sequences reveals a high degree of conservation, suggesting that the sequences may be important for the regulation of pcdh17 expression.     

Flounder (Paralichthys olivaceus) FoxD1 and its regulation on the expression of myogenic regulatory factor, MyoD

It has been reported that FoxD1 plays important roles in formation of several different tissues, such as retina and kidney in vertebrates. The function of FoxD1 in muscle development is, however, unclear although it is expressed in muscle cells in zebrafish. Muscles are the major tissue in fish, which serves as a rich protein source in our diet. To further understand the function of FoxD1 in fish muscle development, here we isolated and characterized the FoxD1 gene from flounder (Paralichthys olivaceus), a valuable sea food and an important fish species in aquaculture in Asia. We analyzed its expression pattern and function in regulating myogenic regulatory factor, MyoD, one of the earliest marker of myogenic commitment. In situ hybridization revealed that FoxD1 was expressed in the tailbud, adaxial cells, posterior intestine, forebrain, midbrain and half of the retina in flounder embryos. Functional studies demonstrated that when flounder FoxD1 was over-expressed in zebrafish by microinjection, MyoD expression was decreased, suggesting that FoxD1 may be involved in myogenesis by regulating the expression of MyoD.     

GFAP transgenic zebrafish

We have generated transgenic zebrafish that express green fluorescent protein (GFP) in glial cells driven by the zebrafish glial fibrillary acidic protein (GFAP) regulatory elements. Transgenic lines Tg(gfap:GFP) were generated from three founders; the results presented here are from the mi2001 line. GFP expression was first visible in the living embryo at the tail bud-stage, then in the developing brain by the 5-somite-stage ( approximately 12 h post-fertilization, hpf) and then spreading posteriorly along the developing spinal cord by the 12-somite stage (approximately 15 hpf). At 24 hpf GFP-expressing cells were in the retina and lens. By 72 hpf GFP expression levels were strong and localized to the glia of the brain, neural retina, spinal cord, and ventral spinal nerves, with moderate expression in the enteric nervous system and weaker levels in the olfactory sensory placode and otic capsule. GFP expression in glia co-localized with anti-GFAP antibodies, but did not co-localize with the neuronal antibodies HuC/D or calretinin in mature neurons.     

Characterization and Developmental Expression of Pax9, a Paired-Box-Containing Gene Related to Pax1

Pax9, a recently identified mouse paired-box-containing gene, is highly homologous to Pax1 and belongs to the same subfamily as Pax1, Hup48, PAX9, and pox meso. Two overlapping cDNA clones spanning the entire coding region of Pax9 were isolated and sequenced. A comparison of the Pax1 and -9 protein sequences reveals a high degree of similarity even outside the paired box, while the carboxy-terminus of the two proteins diverges completely. We demonstrate that Pax9 can bind to the e5 sequence from the Drosophila even skipped promoter, which is also recognized by Pax1. We analyzed the expression of Pax9 during embryo-genesis of wildtype, Undulated short-tail (Un^s), and Danforth's short tail (Sd) mice. In wildtype embryos Pax9 is expressed in the pharyngeal pouches and their derivatives, the developing vertebral column, the tail, the head, and the limbs. Expression of Pax9 is unaffected in Un^s embryos, in which the Pax1 gene is deleted, arguing that expression of Pax9 is not dependent on Pax1. The expression of Pax9 is lost in the caudal part of Sd homozygous embryos, suggesting that expression of Pax9 in the vertebral column independent on the notochord. These results indicate that both Pax9 and -1 may act in parallel during morphogenesis of the vertebral column.