Ruth A. BreMiller

Regional expression of three homeobox transcripts in the inner ear of zebrafish embryos

The inner ear of all jawed vertebrates arises from the epithelium of the otic vesicle and contains three semicircular canals, otoliths, and sets of sensory neurons, all positioned precisely within the cranium to detect head orientation and movement. The msh-C gene and two new homebox genes, msh-D and a gene related to distal-less, dlx-3, are each expressed in distinct regions of the otic vesicle during its early development in zebrafish embryos. Cells in the ectoderm express dlx-3 before induction of the otic vesicle, suggesting that dlx-3 has an early function in this process. Later, cells aligned with the future axes of the semicircular canals specifically express either dlx-3 or msh-D. Even later, sensory hair cells express msh-C and msh-D, while other cells of the epithelium express dlx-3. The early expression of these genes could specify the orientation and morphogenesis of the inner ear, whereas their later expression could specify the fates of particular cell types.

Sex Reversal in Zebrafish fancl Mutants Is Caused by Tp53-Mediated Germ Cell Apoptosis

The molecular genetic mechanisms of sex determination are not known for most vertebrates, including zebrafish. We identified a mutation in the zebrafish fancl gene that causes homozygous mutants to develop as fertile males due to female-to-male sex reversal. Fancl is a member of the Fanconi Anemia/BRCA DNA repair pathway. Experiments showed that zebrafish fancl was expressed in developing germ cells in bipotential gonads at the critical time of sexual fate determination. Caspase-3 immunoassays revealed increased germ cell apoptosis in fancl mutants that compromised oocyte survival. In the absence of oocytes surviving through meiosis, somatic cells of mutant gonads did not maintain expression of the ovary gene cyp19a1a and did not down-regulate expression of the early testis gene amh; consequently, gonads masculinized and became testes. Remarkably, results showed that the introduction of a tp53 (p53) mutation into fancl mutants rescued the sex-reversal phenotype by reducing germ cell apoptosis and, thus, allowed fancl mutants to become fertile females. Our results show that Fancl function is not essential for spermatogonia and oogonia to become sperm or mature oocytes, but instead suggest that Fancl function is involved in the survival of developing oocytes through meiosis. This work reveals that Tp53-mediated germ cell apoptosis induces sex reversal after the mutation of a DNA–repair pathway gene by compromising the survival of oocytes and suggests the existence of an oocyte-derived signal that biases gonad fate towards the female developmental pathway and thereby controls zebrafish sex determination.

Larval and adult visual pigments of the zebrafish, Brachydanio rerio

Photoreceptors of adult and larval zebrafish were assayed by microspectrophotometry to characterize their visual pigments, and to determine when the visual pigments first appear during development. Short single cone outer segments contained a pigment with a lambdamax near 417 nm. Long single cones and long members of double cone outer segments contained a pigment with a lambdamax near 480 nm. Short members of double cone outer segments contained a pigment with a lambdamax near 556 nm. Rod outer segments contained a rhodopsin with a lambdamax near 501 nm. All four visual pigment types found in adult photoreceptors were present in the earliest measurable larval photoreceptors.

Further evidence for latent learning in hippocampal-lesioned rats

Rats with substantial dorsolateral lesions to the hippocampal formation showed significant latent learning of a complex spatial maze. Their overall maze performance, however, was characteristically impaired. Behavior during the pre-exposure phase of the experiment was useful in predicting test maze behavior. Three measures in particular had predictive value: entrances into the maze alley leading to the final choice point, behavior at that choice point, and goal box entries.

Hippocampal lesions, superior cervical ganglia removal, and behavior in rats.

Abstract Rats were subjected to either bilateral removal of the superior cervical ganglia (SCG) or to a sham operation of the neck. Subsequently, these rats received either bilateral neopallial lesions, bilateral dorsal hippocampal lesions or no further operation. The SCG removal prevented the anomalous noradrenergic innervation of remaining hippocampal tissue. This anomalous innervation, as reported by Loy and Moore [19], originates from neurons in the SCG. All animals were then evaluated on behaviors previously shown to be sensitive to hippocampal destruction: open field activity, spontaneous alternation and spatial maze learning, in order to determine if any behavioral consequences could be related to the anomalous hippocampal innervation. No statistically significant differences appeared which could be unambiguously related to the anomalous innervation.

Fetal brain implants improve maze performance in hippocampal-lesioned rats

Hippocampal tissue from day 16 fetal rats was implanted into the damaged hippocampal formation of adults. Significant recovery of the lesion-induced deficit in spatial maze performance was observed, particularly in those animals which showed integration of the implanted fetal tissue with the host hippocampal formation. A two-stage procedure in which 2 weeks or more elapsed between the initial lesion and the implantation resulted in more successful tissue integration and better behavioral recovery.

Expression profiling of zebrafish sox9 mutants reveals that Sox9 is required for retinal differentiation

The transcription factor gene Sox9 plays various roles in development, including differentiation of the skeleton, gonads, glia, and heart. Other functions of Sox9 remain enigmatic. Because Sox9 protein regulates expression of target genes, the identification of Sox9 targets should facilitate an understanding of the mechanisms of Sox9 action. To help identify Sox9 targets, we used microarray expression profiling to compare wild-type embryos to mutant embryos lacking activity for both sox9a and sox9b, the zebrafish co-orthologs of Sox9. Candidate genes were further evaluated by whole-mount in situ hybridization in wild-type and sox9 single and double mutant embryos. Results identified genes expressed in cartilage (col2a1a and col11a2), retina (calb2a, calb2b, crx, neurod, rs1, sox4a and vsx1) and pectoral fin bud (klf2b and EST AI722369) as candidate targets for Sox9. Cartilage is a well-characterized Sox9 target, which validates this strategy, whereas retina represents a novel Sox9 function. Analysis of mutant phenotypes confirmed that Sox9 helps regulate the number of Müller glia and photoreceptor cells and helps organize the neural retina. These roles in eye development were previously unrecognized and reinforce the multiple functions that Sox9 plays in vertebrate development.

Roles of brca2 (fancd1) in Oocyte Nuclear Architecture, Gametogenesis, Gonad Tumors, and Genome Stability in Zebrafish

Mild mutations in BRCA2 (FANCD1) cause Fanconi anemia (FA) when homozygous, while severe mutations cause common cancers including breast, ovarian, and prostate cancers when heterozygous. Here we report a zebrafish brca2 insertional mutant that shares phenotypes with human patients and identifies a novel brca2 function in oogenesis. Experiments showed that mutant embryos and mutant cells in culture experienced genome instability, as do cells in FA patients. In wild-type zebrafish, meiotic cells expressed brca2; and, unexpectedly, transcripts in oocytes localized asymmetrically to the animal pole. In juvenile brca2 mutants, oocytes failed to progress through meiosis, leading to female-to-male sex reversal. Adult mutants became sterile males due to the meiotic arrest of spermatocytes, which then died by apoptosis, followed by neoplastic proliferation of gonad somatic cells that was similar to neoplasia observed in ageing dead end (dnd)-knockdown males, which lack germ cells. The construction of animals doubly mutant for brca2 and the apoptotic gene tp53 (p53) rescued brca2-dependent sex reversal. Double mutants developed oocytes and became sterile females that produced only aberrant embryos and showed elevated risk for invasive ovarian tumors. Oocytes in double-mutant females showed normal localization of brca2 and pou5f1 transcripts to the animal pole and vasa transcripts to the vegetal pole, but had a polarized rather than symmetrical nucleus with the distribution of nucleoli and chromosomes to opposite nuclear poles; this result revealed a novel role for Brca2 in establishing or maintaining oocyte nuclear architecture. Mutating tp53 did not rescue the infertility phenotype in brca2 mutant males, suggesting that brca2 plays an essential role in zebrafish spermatogenesis. Overall, this work verified zebrafish as a model for the role of Brca2 in human disease and uncovered a novel function of Brca2 in vertebrate oocyte nuclear architecture.

Retinoic acid metabolic genes, meiosis, and gonadal sex differentiation in zebrafish.

To help understand the elusive mechanisms of zebrafish sex determination, we studied the genetic machinery regulating production and breakdown of retinoic acid (RA) during the onset of meiosis in gonadogenesis. Results uncovered unexpected mechanistic differences between zebrafish and mammals. Conserved synteny and expression analyses revealed that cyp26a1 in zebrafish and its paralog Cyp26b1 in tetrapods independently became the primary genes encoding enzymes available for gonadal RA-degradation, showing lineage-specific subfunctionalization of vertebrate genome duplication (VGD) paralogs. Experiments showed that zebrafish express aldh1a2, which encodes an RA-synthesizing enzyme, in the gonad rather than in the mesonephros as in mouse. Germ cells in bipotential gonads of all zebrafish analyzed were labeled by the early meiotic marker sycp3, suggesting that in zebrafish, the onset of meiosis is not sexually dimorphic as it is in mouse and is independent of Stra8, which is required in mouse but was lost in teleosts. Analysis of dead-end knockdown zebrafish depleted of germ cells revealed the germ cell-independent onset and maintenance of gonadal aldh1a2 and cyp26a1 expression. After meiosis initiated, somatic cell expression of cyp26a1 became sexually dimorphic: up-regulated in testes but not ovaries. Meiotic germ cells expressing the synaptonemal complex gene sycp3 occupied islands of somatic cells that lacked cyp26a1 expression, as predicted by the hypothesis that Cyp26a1 acts as a meiosis-inhibiting factor. Consistent with this hypothesis, females up-regulated cyp26a1 in oocytes that entered prophase-I meiotic arrest, and down-regulated cyp26a1 in oocytes resuming meiosis. Co-expression of cyp26a1 and the pluripotent germ cell stem cell marker pou5f1(oct4) in meiotically arrested oocytes was consistent with roles in mouse to promote germ cell survival and to prevent apoptosis, mechanisms that are central for tipping the sexual fate of gonads towards the female pathway in zebrafish.

Hippocampal lesions slow extinction of a conditioned taste aversion in rats

Abstract Rats were subjected to either a bilateral removal of the superior cervical ganglia or to a sham operation of the neck. In subsequent operations, these animals underwent either bilateral dorsal hippocampal lesions, bilateral neopallial lesions or no further operation. Removal of the superior cervical ganglia prevented the anomalous innervation of the remaining hippocampal tissue. All animals then learned a conditioned taste aversion to a sweetened milk solution (CS) following LiCl-induced gastrointestinal illness (UCS). The rats with hippocampal lesions were significantly slower to extinguish the aversion conditioning across the 14 day recovery period than were controls. There was no effect of any manipulation on the acquisition of the conditioned taste aversion, and there were no behavioral consequences of the presence or absence of the anomalous innervation. The slower extinction of the taste aversion shown by the hippocampal lesioned rats was discussed in terms of a lessened impact of the pre-illness exposure to the CS in these animals.