Expression of the zebrafish CD133/prominin1 genes in cellular proliferation zones in the embryonic central nervous system and sensory organs

The CD133/prominin1 gene encodes a pentamembrane glycoprotein cell surface marker that is expressed in stem cells from neuroepithelial, hematopoietic, and various organ tissues. Here we report the analysis of two zebrafish CD133/prominin1 orthologues, prominin1a and prominin1b. The expression patterns of the zebrafish prominin1a and b genes were analyzed during embryogenesis using whole mount in situ hybridization. prominin1a and b show novel complementary and overlapping patterns of expression in proliferating zones in the developing sensory organs and central nervous system. The expression patterns suggest functional conservation of the zebrafish prominin1 genes. Initial analyses of prominin1a and b in neoplastic tissue show increased expression of both genes in a subpopulation of cells in malignant peripheral nerve sheath tumors in tp53 mutants. Based on these analyses, the zebrafish prominin1 genes will be useful markers for examining proliferating cell populations in adult organs, tissues, and tumors. Developmental Dynamics 239:1849–1857, 2010. © 2010 Wiley‐Liss, Inc.     

Expression of strawberry notch family genes during zebrafish embryogenesis

Our previous study suggested a possible role for Sbno1, a mouse homologue of strawberry notch gene during brain development. In this report, we cloned the zebrafish homologues of sbno, and examined their expression pattern during embryogenesis by whole‐mount in situ hybridization. Zebrafish have three sbno genes: one Sbno1 homologue and two Sbno2 homologues, sbno2a and sbno2b. We observed that the expression of sbno1 and sbno2a was initially ubiquitous and gradually became predominant in the central nervous system as development progressed. The expression of sbno2b was observed in non‐neural tissues in contrast to the other two genes. sbno1 and sbno2a exhibited higher expression in distinct regions within the nervous system of pharyngula‐stage embryos, suggesting possible differing roles for sbno1 and sbno2a during later stages of embryogenesis. Together, the observed gene expression patterns suggest an important role of sbno‐family genes during development of the vertebrate central nervous system. Developmental Dynamics 239:1789–1796, 2010. © 2010 Wiley‐Liss, Inc.     

Comparison of Iroquois gene expression in limbs/fins of vertebrate embryos

In Drosophila, Iroquois (Irx) genes have various functions including the specification of the identity of wing veins. Vertebrate Iroquois (Irx) genes have been reported to be expressed in the developing digits of mouse limbs. Here we carry out a phylogenetic analysis of vertebrate Irx genes and compare expression in developing limbs of mouse, chick and human embryos and in zebrafish pectoral fin buds. We confirm that the six Irx gene families in vertebrates are well defined and that Clusters A and B are duplicates; in contrast, Irx1 and 3, Irx2 and 5, and Irx4 and 6 are paralogs. All Irx genes in mouse and chick are expressed in developing limbs. Detailed comparison of the expression patterns in mouse and chick shows that expression patterns of genes in the same cluster are generally similar but paralogous genes have different expression patterns. Mouse and chick Irx1 are expressed in digit condensations, whereas mouse and chick Irx6 are expressed interdigitally. The timing of Irx1 expression in individual digits in mouse and chick is different. Irx1 is also expressed in digit condensations in developing human limbs, thus showing conservation of expression of this gene in higher vertebrates. In zebrafish, Irx genes of all but six of the families are expressed in early stage pectoral fin buds but not at later stages, suggesting that these genes are not involved in patterning distal structures in zebrafish fins.     

Zebrafish scarb2a insertional mutant reveals a novel function for the Scarb2/Limp2 receptor in notochord development

Background: Scarb2 or Limp2 belong to a subfamily of Scavenger receptors described as lysosomal transmembrane glycosylated receptors, that are mutated in the human syndrome AMRF (action myoclonus‐renal failure). The zebrafish insertional mutant scarb2a^hi1463Tg has notochord defects, the notochord is a defining feature of chordates running along the center of the longitudinal axis and it is essential for forming the spinal column in all vertebrates. Results: There are three paralogous scarb2 genes in zebrafish; scarb2a, scarb2b, and scarb2c. Both Scarb2a and Scarb2b proteins lack the classical di‐leucine motif. We found that scarb2a^hi1463Tg homozygous zebrafish embryos have a null mutation impairing vacuole formation in the notochord and simultaneously disrupting proper formation of the basement membrane resulting in its thickening at the ventral side of the notochord, which may be the cause for the anomalous upward bending observed in the trunk. Through whole‐mount in situ hybridization, we detected scarb2a mRNA expression in the notochord and in the brain early in development. However, it is puzzling that scarb2a notochord mRNA expression is short‐lived in the presumptive notochord and precedes the complete differentiation of the notochord. Conclusions: This work describes a novel function for the Scarb2 receptor as an essential glycoprotein for notochord development. Developmental Dynamics 245:508–519, 2016. © 2015 Wiley Periodicals, Inc.     

Identification and expression of voltage‐gated calcium channel β subunits in Zebrafish

Voltage‐gated calcium channels (VGCC) play important roles in electrically excitable cells and embryonic development. The VGCC β subunits are essential for membrane localization of the channel and exert modulatory effects on channel functions. In mammals, the VGCC β subunit gene family contains four members. In zebrafish, there appear to be seven VGCC β subunits including the previously identified β1 subunit. cDNAs for six additional VGCC β subunit homologs were identified in zebrafish, their chromosomal locations determined and their expression patterns characterized during embryonic development. These six genes are primarily expressed in the nervous system with cacnb4a also expressed in the developing heart. Sequence homology, genomic synteny and expression patterns suggest that there are three pairs of duplicate genes for β2, β3, and β4 in zebrafish with distinct expression patterns during embryonic development. Developmental Dynamics 237:3842–3852, 2008. © 2008 Wiley‐Liss, Inc.     

Expression and retinoic acid regulation of the zebrafish nr2f orphan nuclear receptor genes

Background : The vertebrate nuclear receptor subfamily 2, group f (nr2f) genes encode orphan receptors that have the capacity to act as negative regulators of retinoic acid (RA) signaling. Results : We describe embryonic and larval expression of four of the six zebrafish nr2f genes, nr2f1a, nr2f1b, nr2f2, and nr2f5. These genes show highly regulated patterns of expression within the central nervous system, including in the developing hindbrain, as well as in the mesoderm and endoderm. We also investigated the role of RA and fibroblast growth factor (Fgf) signaling in regulating early nr2f gene expression. RA is not required for nr2f expression in the hindbrain; however, exogenous RA can repress this expression. Conversely, we find that RA positively regulates nr2f1a expression in trunk endoderm and mesoderm. Fgf signaling is not required for nr2f expression onset in the hindbrain; however, it may play a role in maintaining rhombomere‐specific expression. Conclusions : We report detailed expression analysis of four nr2f genes in all three germ layers. The onset of nr2f expression in the hindbrain does not require RA or Fgf signals. Our finding that RA positively regulates nr2f1a expression in the trunk supports the possibility that Nr2fs function in a negative feedback loop to modulate RA signaling in this region. Developmental Dynamics 241:1603–1615, 2012. © 2012 Wiley Periodicals, Inc.     

Sequence and expression of the zebrafish alpha‐actinin gene family reveals conservation and diversification among vertebrates

alpha‐actinins are actin microfilament crosslinking proteins. Vertebrate actinins fall into two classes: the broadly‐expressed actinins 1 and 4 (actn1 and actn4) and muscle‐specific actinins, actn2 and actn3. Members of this family have numerous roles, including regulation of cell adhesion, cell differentiation, directed cell motility, intracellular signaling, and stabilization of f‐actin at the sarcomeric Z‐line in muscle. Here we identify five zebrafish actinin genes including two paralogs of ACTN3. We describe the temporal and spatial expression patterns of these genes through embryonic development. All zebrafish actinin genes have unique expression profiles, indicating specialization of each gene. In particular, the muscle actinins display preferential expression in different domains of axial, pharyngeal, and cranial musculature. There is no identified avian actn3 and approximately 16% of humans are null for ACTN3. Duplication of actn3 in the zebrafish indicates that variation in actn3 expression may promote physiological diversity in muscle function among vertebrates. Developmental Dynamics 238:2936–2947, 2009. © 2009 Wiley‐Liss, Inc.     

Dynamic miRNA expression patterns during retinal regeneration in zebrafish: Reduced dicer or miRNA expression suppresses proliferation of Müller Glia‐derived neuronal progenitor cells

Background: Adult zebrafish spontaneously regenerate their retinas after damage. Although a number of genes and signaling pathways involved in regeneration have been identified, the exact mechanisms regulating various aspects of regeneration are unclear. microRNAs (miRNAs) were examined for their potential roles in regulating zebrafish retinal regeneration. Results: To investigate the requirement of miRNAs during zebrafish retinal regeneration, we knocked down the expression of Dicer in retinas prior to light‐induced damage. Reduced Dicer expression significantly decreased the number of proliferating Müller glia‐derived neuronal progenitor cells during regeneration. To identify individual miRNAs with roles in neuronal progenitor cell proliferation, we collected retinas at different stages of light damage and performed small RNA high‐throughput sequencing. We identified subsets of miRNAs that were differentially expressed during active regeneration but returned to basal levels once regeneration was completed. We then knocked down five different miRNAs that increased in expression and assessed the effects on retinal regeneration. Reduction of miR‐142b and miR‐146a expression significantly reduced INL proliferation at 51h of light treatment, while knockdown of miR‐7a, miR‐27c, and miR‐31 expression significantly reduced INL proliferation at 72h of constant light. Conclusions: miRNAs exhibit dynamic expression profiles during retinal regeneration and are necessary for neuronal progenitor cell proliferation. Developmental Dynamics 243:1591–1605, 2014. © 2014 The Authors. Developmental Dynamics published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists     

Characterization of the Zebrafish atxn1/axh Gene Family

In mammals, ataxin-1 (ATXN1) is a member of a family of proteins in which each member contains an AXH domain. Expansion of the polyglutamine tract in ATXN1 causes the neurodegenerative disease, spinocerebellar ataxia type 1 (SCA1) with prominent cerebellar pathology. Toward a further characterization of the genetic diversification of the ATXN1/AXH gene family, we identified and characterized members of this gene family in zebrafish, a lower vertebrate with a cerebellum. The zebrafish genome encodes two ATXN1 homologs, atxn1a and atxn1b, and one ATXN1L homolog, atxn1l. Key biochemical features of the human ATXN1 protein not seen in the invertebrate homologs (a nuclear localization sequence and a site of phosphorylation at serine 776) are conserved in the zebrafish homologs, and all three zebrafish Atxn1/Axh proteins behave similarly to their human counterparts in tissue-culture cells. Importantly, each of the three homologs is expressed in the zebrafish cerebellum, which in humans, is a prominent site of SCA1 pathogenesis. In addition, atxn1a and atxn1b are expressed in the developing zebrafish cerebellum. These data show that in zebrafish, a lower vertebrate, the complexity of the atxn1/axh gene family is more similar to higher vertebrates than invertebrates with a simple central nervous system and suggests a relationship between the diversification of the ATXN1/AXH gene family and the development of a complex central nervous system, including a cerebellum.     

Differential expression of neuroligin genes in the nervous system of zebrafish

The establishment and maturation of appropriate synaptic connections is crucial in the development of neuronal circuits. Cellular adhesion is believed to play a central role in this process. Neuroligins are neuronal cell adhesion molecules that are hypothesized to act in the initial formation and maturation of synaptic connections. In order to establish the zebrafish as a model to investigate the in vivo role of Neuroligin proteins in nervous system development, we identified the zebrafish orthologs of neuroligin family members and characterized their expression. Zebrafish possess seven neuroligin genes. Synteny analysis and sequence comparisons show that NLGN2, NLGN3, and NLGN4X are duplicated in zebrafish, but NLGN1 has a single zebrafish ortholog. All seven zebrafish neuroligins are expressed in complex patterns in the developing nervous system and in the adult brain. The spatial and temporal expression patterns of these genes suggest that they occupy a role in nervous system development and maintenance. Developmental Dynamics 239:703–714, 2010. © 2010 Wiley‐Liss, Inc.