Charline Walker

Cardiac troponin T is essential in sarcomere assembly and cardiac contractility.

Mutations of the gene (TNNT2) encoding the thin-filament contractile protein cardiac troponin T are responsible for 15% of all cases of familial hypertrophic cardiomyopathy, the leading cause of sudden death in young athletes. Mutant proteins are thought to act through a dominant-negative mode that impairs function of heart muscle. TNNT2 mutations can also lead to dilated cardiomyopathy, a leading cause of heart failure. Despite the importance of cardiac troponin T in human disease, its loss-of-function phenotype has not been described. We show that the zebrafish silent heart (sih) mutation affects the gene tnnt2. We characterize two mutated alleles of sih that severely reduce tnnt2 expression: one affects mRNA splicing, and the other affects gene transcription. Tnnt2, together with α-tropomyosin (Tpma) and cardiac troponins C and I (Tnni3), forms a calcium-sensitive regulatory complex within sarcomeres. Unexpectedly, in addition to loss of Tnnt2 expression in sih mutant hearts, we observed a significant reduction in Tpma and Tnni3, and consequently, severe sarcomere defects. This interdependence of thin-filament protein expression led us to postulate that some mutations in tnnt2 may trigger misregulation of thin-filament protein expression, resulting in sarcomere loss and myocyte disarray, the life-threatening hallmarks of TNNT2 mutations in mice and humans.

The Zebrafish Glypican Knypek Controls Cell Polarity during Gastrulation Movements of Convergent Extension

Mutations in the zebrafish knypek locus impair gastrulation movements of convergent extension that narrow embryonic body and elongate it from head to tail. We demonstrate that knypek regulates cellular movements but not cell fate specification. Convergent extension movement defects in knypek are associated with abnormal cell polarity, as mutant cells fail to elongate and align medio-laterally. Positional cloning reveals that knypek encodes a member of the glypican family of heparan sulfate proteoglycans. Double mutant and overexpression analyses show that Knypek potentiates Wnt11 signaling, mediating convergent extension. These studies provide experimental and genetic evidence that glypican Knypek acts during vertebrate gastrulation as a positive modulator of noncanonical Wnt signaling to establish polarized cell behaviors underlying convergent extension movements.

A neural degeneration mutation that spares primary neurons in the zebrafish

We describe an embryonic lethal mutation in the zebrafish Brachydanio rerio that specifically affects the viability of most cells in the embryonic central nervous system (CNS). The mutation ned-1 (b39rl) was induced with gamma-irradiation and segregates as a single recessive allele closely linked to its centromere. It produces massive cell death in the CNS but a small set of specific neurons, including Rohon-Beard sensory neurons, large hindbrain interneurons, and primary motoneurons, survive embryogenesis and are functional. Synaptic connections between embryonic motoneurons and muscle cells appear physiologically normal, and the normally observed spontaneous flexions are present. Correlated with the presence of sensory neurons and interneurons, mutant embryos display reflexive movements in response to mechanical stimulation. Together, the surviving neurons, called primary neurons, form a class of cells that are prominent in size and arise early during development. Thus, this mutation may define a function that is differentially required by developmentally distinguishable sets of cells in the embryonic CNS.

Pathfinding and synapse formation in a zebrafish mutant lacking functional acetylcholine receptors

We induced and characterized a recessive lethal mutation, nic-1, in zebrafish that blocks the function of muscle acetylcholine (ACh) receptors. Homozygous nic-1 embryos are nonmotile and fail to respond to exogenous application of cholinergic agonists, although their muscles contract in response to direct electrical stimulation. Moreover, we do not detect cell surface labeling by alpha-bungarotoxin or monoclonal antibodies that recognize the other three subunits of ACh receptors. Motoneurons, however, establish morphologically normal patterns of innervation and normal neuromuscular junctions. We suggest that neither transmitter-mediated nerve signaling nor any other aspect of ACh receptor function is required for the formation of appropriate nerve connections in this system.

HAPLOID SCREENS AND GAMMA-RAY MUTAGENESIS

Publisher Summary A number of largescale mutagenesis screens have been conducted to identify developmental mutations in zebrafish. In addition, smaller screens are ongoing in many other laboratories. Regardless of which type of mutagen is used, it is important to screen as efficiently as possible in terms of time, space, and the number of fish used. This chapter describes the use of haploids to screen for early developmental mutations in mutagenized zebrafish, with an emphasis on the use of gamma-rays as a mutagen. Screening haploids allow rapid identification of mutation-bearing females in a parental P or an F1 screen, avoiding the necessity of raising several generations of fish stocks prior to screening. Gynogenetic haploid embryos are produced when eggs are fertilized by UV-irradiated sperm, the resulting haploid embryos develop solely from maternal genetic information. Although the UV-irradiated sperm provides no male genetic contribution to the embryo, they are necessary to activate embryonic development.

Clonal origins of cells in the pigmented retina of the zebrafish eye.

Mosaic analysis has been used to study the clonal basis of the development of the pigmented retina of the zebrafish, Brachydanio rerio. Zebrafish embryos heterozygous for a recessive mutation at the gol-1 locus were exposed to gamma-irradiation at various developmental stages to create mosaic individuals consisting of wild-type pigmented cells and a clone of pigmentless (golden) cells in the eye. The contribution of individual embryonic cells to the pigmented retina was measured and the total number of cells in the embryo that contributed descendants to this tissue was determined. Until the 32-cell stage, almost every blastomere has some descendants that participate in the formation of the pigmented retina of the zebrafish. During subsequent cell divisions, up to the several thousand-cell stage, the number of ancestral cells is constant: approximately 40 cells are present that will give rise to progeny in the pigmented retina. Analysis of the size of clones in the pigmented retina indicates that the cells of this tissue do not arise through a rigid series of cell divisions originating in the early embryo. The findings that each cleavage stage cell contributes to the pigmented retina and yet the contribution of such cells is highly variable are consistent with the interpretation that clonal descendants of different blastomeres normally intermix extensively prior to formation of the pigmented retina.

Zebrafish Mef2ca and Mef2cb are essential for both first and second heart field cardiomyocyte differentiation

Mef2 transcription factors have been strongly linked with early heart development. D-mef2 is required for heart formation in Drosophila, but whether Mef2 is essential for vertebrate cardiomyocyte (CM) differentiation is unclear. In mice, although Mef2c is expressed in all CMs, targeted deletion of Mef2c causes lethal loss of second heart field (SHF) derivatives and failure of cardiac looping, but first heart field CMs can differentiate. Here we examine Mef2 function in early heart development in zebrafish. Two Mef2c genes exist in zebrafish, mef2ca and mef2cb. Both are expressed similarly in the bilateral heart fields but mef2cb is strongly expressed in the heart poles at the primitive heart tube stage. By using fish mutants for mef2ca and mef2cb and antisense morpholinos to knock down either or both Mef2cs, we show that Mef2ca and Mef2cb have essential but redundant roles in myocardial differentiation. Loss of both Mef2ca and Mef2cb function does not interfere with early cardiogenic markers such as nkx2.5, gata4 and hand2 but results in a dramatic loss of expression of sarcomeric genes and myocardial markers such as bmp4, nppa, smyd1b and late nkx2.5 mRNA. Rare residual CMs observed in mef2ca;mef2cb double mutants are ablated by a morpholino capable of knocking down other Mef2s. Mef2cb over-expression activates bmp4 within the cardiogenic region, but no ectopic CMs are formed. Surprisingly, anterior mesoderm and other tissues become skeletal muscle. Mef2ca single mutants have delayed heart development, but form an apparently normal heart. Mef2cb single mutants have a functional heart and are viable adults. Our results show that the key role of Mef2c in myocardial differentiation is conserved throughout the vertebrate heart.

Studies on the metabolic role of peptidyl-tRNA hydrolase

SummaryA mutant strain of Eschrichia coli that is temperature-sensitive for growth stopped protein biosynthesis at 43° C after a brief lag (Fig. 1). Cell-free extracts from the strain showed no specific defect in aminoacyl-tRNA synthetases, binding initiator tRNA to ribosomes (Table 1), protein chain elongation (Tables 2, 5) or protein chain termination (Tables 3, 4) at high temperature.The partially purified enzyme peptidyl-tRNA hydrolase, however, was temperature-sensitive (Table 6); the mutant hydrolase was inactivated rapidly at 43° C (Table 7). Mixing experiments ruled out the presence, in the mutant enzyme preparation, of an inhibitor and also demonstrated, on the mutant enzyme, a protective effect by wild type enzyme that was not shown by general coli proteins (Tables 8, 9).Interrupted mating allowed the temperature-sensitive growth phenotype to be mapped near to and before trp (Figs. 4, 5). Co-transsduction, mediated by bacteriophage P1, with trp+ (frequency 7.5%) located the marker at 24 min on the coli map. All transductants for temperature-sensitive growth also had temperature-sensitive peptidyl-tRNA hydrolase activity in crude sonicates (Table 10). We provisionally conclude that the temperature-sensitive protein synthesis and growth are caused by a single genetic change in the structural gene (pth) for peptidyl-tRNA hydrolase.After shift to 43° C the polysomes of the mutant cells broke down into 70S particles (Figs. 2, 3). A defect in protein biosynthesis thus appeared to be located after termination and before reformation of new polysomes.The metabolic role of peptidyl-tRNA hydrolase is discussed in the light of these experiments.

Production of haploid and diploid androgenetic zebrafish (including methodology for delayed in vitro fertilization).

Publisher Summary This chapter describes how to produce androgenotes, how they have been used, and their future potential. It describes two procedures that are required for androgenesis, but which can also be used for other applications: one is the technique of delayed in vitro fertilization, and the other is the technique of interfering with the first mitotic division to double the chromosome complement. Androgenetic haploid and diploid zebrafish larva can be efficiently produced. The production of both andro- and gynogenetic fish indicates that the irreversible inactivation of genes essential for development by parent-of-origin-genome imprinting does not occur in zebrafish, in contrast to some mammals. The success rate in producing haploid androgenotes with nearly normal morphology should make them suitable for some types of genetic screens. Being able to delay the fertilization of zebrafish eggs for periods up to a few hours after they are extruded from the female can facilitate experiments that involve in vitro fertilization. It allows manipulations prior to fertilization; it aids studying fertilization itself; and it can also provide working time for postfertilization manipulations.

[31] An assay for protein chain termination using peptidyl-tRNA

Publisher Summary This assay for protein chain termination has the virtues of being performed at low magnesium ion concentrations in the absence of solvent perturbants (e.g., ethanol) with unpurified transfer RNA and a random copolymer message. It may therefore be of use in exploring the process of protein chain termination in eukaryote systems where inefficient isolation methods preclude the extensive purification of components. Because the nucleic acid moiety of the substrate for the assay is a typical transfer RNA, use of this technique gives answers complementary to those involving the unique methionine tRNA F . The ability to perform the measurements in the absence of, e.g., ethanol or acetone may allow one to perceive the control of protein chain termination unimpeded by the distorting effects of structure perturbants.