Zebrafish connexin 79.8 (Gja8a): A lens connexin used as an electrical synapse in some neurons.

In the mammalian central nervous system, a remarkably small number of connexins is used in electrical synapses, with the majority formed from Cx36. A larger number has been detected in teleosts, with some seeming to serve restricted roles. Here, we report the discovery of a new connexin expressed in the zebrafish lens and a limited set of neurons. Zebrafish cx79.8 (gja8a), previously annotated incorrectly as cx50.5 based on a partial cDNA sequence, is a homologue of mammalian Cx50 (Gja8). We examined its expression through transgenic promoter-reporter constructs, in situ hybridization, and immunolabeling, and examined regulation of coupling in transfected HeLa cells. cx79.8 was expressed most strongly in the lens, but expression was also found in several groups of neurons in the cerebellum and related areas at the midbrain-hindbrain boundary, in cone photoreceptors, and in neurons in the retinal inner nuclear and ganglion cell layers. Labeling in the retina with antibodies against two C-terminal epitopes revealed numerous small punctate spots in the inner plexiform layer and along the somata of cones. Abundant gap junctions were labeled in the outer 1/3 of the lens, but were absent from the center, suggesting that the epitopes or the entire protein was absent from the center. Cx79.8 tracer coupling was strongly regulated by phosphorylation, and was extremely low in control conditions in HeLa cells due to protein phosphatase 2A activity. These properties allow coupling to be strongly restricted in situ, a frequently observed property for electrical synapses. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 548-561, 2017.

G2R Cre reporter transgenic zebrafish.

The Cre/loxP site-specific recombination system has been widely used to manipulate DNA in vivo and to study gene function in the mouse by inducible transgenic and conditional gene targeting. To fully use this powerful genetic tool in a relatively new animal model, zebrafish, we generated reporter transgenic lines for easy detection of Cre recombinase activity in vivo. The transgenic fish lines, designated G2R, express two fluorescent protein genes, GFP and RFP, under the control of the ubiquitous promoter of the Xenopus EF1 alpha gene. The G2R animals change their color from green to red (G2R) after Cre-mediated recombination and are useful for development of cell type specific Cre transgenic lines and for cell lineage and fate mapping studies in zebrafish.

Spontaneous and inheritable R555Q mutation in the TGFBI/BIGH3 gene in two unrelated families exhibiting Bowman's layer corneal dystrophy.

PURPOSE: Bowman's layer corneal dystrophies (CDBs) include 2 distinct types: CDB1, or Reis-Bücklers (RBCD), and CDB2, or Thiel-Behnke (TBCD). We studied the genetic basis of 2 cases of apparent spontaneous CDB mutations and attempted to determine if these are sporadic and inheritable mutations. DESIGN: Retrospective molecular genetic study and case report. PARTICIPANTS: Twelve patients were recruited from 2 unrelated families for this study, including 2 affected individuals from one family (family A) and 1 affected individual from another (family B). METHODS: Slit-lamp examination was performed for each patient to determine the disease phenotype. Histological analysis of affected cornea specimens was used for identification of pathogenic corneal opacities in 2 affected patients from family A. MAIN OUTCOME MEASURES: Genomic DNA was isolated from the blood samples and used for mutation screening of the TGFBI/BIGH3 gene. Sixteen polymorphic DNA markers from 9 different chromosomes were used to establish the maternity and paternity of the 2 probands. RESULTS: The 2 families were confirmed to be unrelated. The age onset of ocular symptoms was <2 years for all 3 affected patients. Clinical diagnoses of CDB1 (RBCD) and CDB2 (TBCD) were made for probands A and B, respectively. The affected corneas showed epithelial haze with diffuse, irregular, patchy opacities in a honeycomb and geographic pattern. Subepithelial plaques, increased trichome staining of anterior stroma, and irregular Bowman's layer were observed. An R555Q mutation was found in TGFBI/BIGH3 in the 2 probands but not in their parents. The son of proband A was also affected and apparently inherited his disease allele from his father. CONCLUSION: The R555Q mutation occurred spontaneously and independently in the 2 unrelated CDB families and was confirmed to be transmitted to the next generation in 1 of the 2 families. These findings strongly support the notion that a genetic diagnosis should be determined for CDB and other dystrophies associated with mutations in TGFBI/BIGH3. The discovery of a spontaneous mutation should alert clinicians to be aware of the existence of genetic alterations for their patients without apparent family history of the disease.

Transgenic analysis of the anterior eye-specific enhancers of the zebrafish gelsolin-like 1 (gsnl1) gene.

The anterior segment of the eye includes such structures as the cornea, lens, iris, and ciliary body and is essential for many visual and physiological functions of the eye. The zebrafish gelsolin-like 1 (gsnl1) gene encodes an actin regulatory protein and is expressed in the anterior segment of the eye. We report the transgenic analyses of the gsnl1 promoter and enhancer that are required for expression in the anterior segment of the eye. A 6.4-kb genomic fragment upstream from the translation initiation site (ATG) was capable of driving green fluorescent protein (GFP) expression in transient transgenic embryos and stable transgenic adult fish, which mimics the endogenous gsnl1 expression. The GFP expression was localized in the corneal epithelium (CE) and the annular ligament (AL) at the iridocorneal angle. A unique enhancer for each of these two tissues was identified at 3.7-kb upstream from the ATG. The 60-bp AL and 25-bp CE enhancers were separated by 100-bp and functioned independently from each other. Deletion analysis indicated that the proximal promoter was located 1.6-kb upstream from the ATG. Stable GFP transgenic lines were established for future studies of genetic regulation in the anterior segment of the fish eye.

Distinct developmental programs require different levels of Bmp signaling during mouse retinal development.

The Bmp family of secreted signaling molecules is implicated in multiple aspects of embryonic development. However, the cell-type-specific requirements for this signaling pathway are often obscure in the context of complex embryonic tissue interactions. To define the cell-autonomous requirements for Bmp signaling, we have used a Cre-loxP strategy to delete Bmp receptor function specifically within the developing mouse retina. Disruption of a Bmp type I receptor gene, Bmpr1a, leads to no detectable eye abnormality. Further reduction of Bmp receptor activity by removing one functional copy of another Bmp type I receptor gene, Bmpr1b, in the retina-specific Bmpr1a mutant background, results in abnormal retinal dorsoventral patterning. Double mutants completely lacking both of these genes exhibit severe eye defects characterized by reduced growth of embryonic retina and failure of retinal neurogenesis. These studies provide direct genetic evidence that Bmpr1a and Bmpr1b play redundant roles during retinal development, and that different threshold levels of Bmp signaling regulate distinct developmental programs such as patterning, growth and differentiation of the retina.

BMP signaling through ACVRI is required for left-right patterning in the early mouse embryo.

Vertebrate organisms are characterized by dorsal-ventral and left-right asymmetry. The process that establishes left-right asymmetry during vertebrate development involves bone morphogenetic protein (BMP)-dependent signaling, but the molecular details of this signaling pathway remain poorly defined. This study tests the role of the BMP type I receptor ACVRI in establishing left-right asymmetry in chimeric mouse embryos. Mouse embryonic stem (ES) cells with a homozygous deletion at Acvr1 were used to generate chimeric embryos. Chimeric embryos were rescued from the gastrulation defect of Acvr1 null embryos but exhibited abnormal heart looping and embryonic turning. High mutant contribution chimeras expressed left-side markers such as nodal bilaterally in the lateral plate mesoderm (LPM), indicating that loss of ACVRI signaling leads to left isomerism. Expression of lefty1 was absent in the midline of chimeric embryos, but shh, a midline marker, was expressed normally, suggesting that, despite formation of midline, its barrier function was abolished. High-contribution chimeras also lacked asymmetric expression of nodal in the node. These data suggest that ACVRI signaling negatively regulates left-side determinants such as nodal and positively regulates lefty1. These functions maintain the midline, restrict expression of left-side markers, and are required for left-right pattern formation during embryogenesis in the mouse.

Centrifugal bisection of starfish oocytes.

Immature oocytes or mature eggs of starfish were centrifuged in a sucrose density gradient. They were then separated into two fractions of fragments, nucleate light fragments and anucleate heavy fragments. Vital-staining experiments showed that the oocytes were elongated along the animal-vegetal (AV) axis during the centrifugation in a contrast to centrifuged eggs whose centrifugal axis was not related to the AV axis. The light and heavy oocyte fragments were comprised of animal and vegetal halves of oocytes, respectively. When matured and fertilized, most of the light oocyte fragment-derived embryos failed gastrulation and developed into Dauerblastulae. Two-dimensional gel electrophoretic analysis of fragments revealed that three basic proteins were predominantly enriched in the heavy oocyte fragments but scarcely detected in the light oocyte fragments. One of these proteins, App20, was identified as a homologue of cyclophilin (peptidyl-prolyl cis-trans isomerase). The present study provides a simple means of separating a population of starfish oocytes into animal and vegetal halves, thereby enabling us to analyze any difference of components between animal and vegetal cytoplasm of the oocytes.

A novel antigen Apsi is expressed in a half population of endoderm cells in normal and LiCI-treated starfish embryos.

A novel antigen, Apsi, revealed a tissue specific expression in the starfish embryo. Apsi was detected in the stomach and intestine of the bipinnaria larva by immunofluorescence microscopy, but was not detected in the esophagus or ectoderm. The expression of Apsi was zygotic and first detected at day 3 after fertilization. Using this antigen as a molecular marker, the effect of LiCI treatment on development was examined by counting the cell number of each germ layer and endoderm tissues on serial paraffin sections. At day 5 larva stage, the ratio of the cell number of ectoderm, esophagus, Apsi-expressing tissue (stomach and intestine) and mesoderm was 75:10:10:5. The corresponding ratio in LiCI-treated embryo was 68:14:14:4. LiCI treatment increased the cell number of endoderm by 40%, at the expense of a 10% decrease in the cell number of ectoderm. In intact embryos, approximately half the endoderm cells expressed Apsi antigen, while the other half did not. LiCI treatment did not change this ratio of Apsi expression in endoderm tissues. These observations indicate that LiCI treatment of early blastulae affects the commitment of ectoderm/endoderm but does not affect the differentiation of the esophagus/stomach and intestine.