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J H Postlethwait

Publications and source records attributed to J H Postlethwait.

At least 55 records · Page 3Linked to original sources
PubMed1995

Expression of snail2, a second member of the zebrafish snail family, in cephalic mesendoderm and presumptive neural crest of wild-type and spadetail mutant embryos.

Transcripts of a newly discovered gene called snail2, encoding a zinc finger protein of the Snail family, first appear in rows of cephalic mesendodermal cells in gastrulating zebrafish embryos. At the end of gastrulation, snail2 RNA accumulates in a domain of ectodermal cells that mark the border between the epidermal epithelium and the neural plate and includes precursors of the neural crest. During somitogenesis, snail2 expression becomes restricted to neural crest. snail2 is thus one of the earliest genes yet known to be specifically expressed in neural crest in zebrafish embryos. Since snail2 is expressed in mesendoderm, a tissue layer whose convergence in the trunk is known to be altered in embryos homozygous for the spadetail mutation, we examined snail2 expression in spadetail embryos. In these mutants, the number of cephalic mesendodermal cells expressing snail2 is strongly reduced and the distribution of cells containing snail2 and no tail transcripts in the axial mesoderm is much broader than normal Moreover, the embryos are shorter than normal at the end of gastrulation. This shows that, in addition to the failure of paraxial mesoderm to converge normally in the trunk during gastrulation, spadetail also affects the elongation of the embryo and the convergence of axial and lateral mesendoderm in both trunk and head.

Amino Acid Sequence↗
PubMed1995

Half-tetrad analysis in zebrafish: mapping the ros mutation and the centromere of linkage group I.

Analysis of meiotic tetrads is routinely used to determine genetic linkage in various fungi. Here we apply tetrad analysis to the study of genetic linkage in a vertebrate. The half-tetrad genotypes of gynogenetic diploid zebrafish produced by early-pressure (EP) treatment were used to investigate the linkage relationships of two recessive pigment pattern mutations, leopard (leo) and rose (ros). The results showed that ros is tightly linked to its centromere and leo maps 31 cM from its centromere. Analysis of half-tetrads segregating for ros and leo in repulsion revealed no homozygous ros individuals among 32 homozygous leo half-tetrads--i.e., a parental ditype (PD) to nonparental ditype (NPD) ratio of 32:0. This result shows that ros is linked to leo, a mutation previously mapped to Linkage Group I. Investigation of PCR-based DNA polymorphisms on Linkage Group I confirmed the location of ros near the centromere of this linkage group. We propose an efficient, generally useful method to assign new mutations to a linkage group in zebrafish by determining which of 25 polymerase chain reaction (PCR)-based centromere markers shows a significant excess of PD to NPD in half-tetrad fish.

Animals↗
PubMed1995

Cell-autonomous shift from axial to paraxial mesodermal development in zebrafish floating head mutants.

Zebrafish floating head mutant embryos lack notochord and develop somitic muscle in its place. This may result from incorrect specification of the notochord domain at gastrulation, or from respecification of notochord progenitors to form muscle. In genetic mosaics, floating head acts cell autonomously. Transplanted wild-type cells differentiate into notochord in mutant hosts; however, cells from floating head mutant donors produce muscle rather than notochord in wild-type hosts. Consistent with respecification, markers of axial mesoderm are initially expressed in floating head mutant gastrulas, but expression does not persist. Axial cells also inappropriately express markers of paraxial mesoderm. Thus, single cells in the mutant midline transiently co-express genes that are normally specific to either axial or paraxial mesoderm. Since floating head mutants produce some floor plate in the ventral neural tube, midline mesoderm may also retain early signaling capabilities. Our results suggest that wild-type floating head provides an essential step in maintaining, rather than initiating, development of notochord-forming axial mesoderm.

Animals↗
PubMed1994

A genetic linkage map for the zebrafish.

To facilitate molecular genetic analysis of vertebrate development, haploid genetics was used to construct a recombination map for the zebrafish Danio (Brachydanio) rerio. The map consists of 401 random amplified polymorphic DNAs (RAPDs) and 13 simple sequence repeats spaced at an average interval of 5.8 centimorgans. Strategies that exploit the advantages of haploid genetics and RAPD markers were developed that quickly mapped lethal and visible mutations and that placed cloned genes on the map. This map is useful for the position-based cloning of mutant genes, the characterization of chromosome rearrangements, and the investigation of evolution in vertebrate genomes.

Animals↗
PubMed1994

Identification of RAPD primers that reveal extensive polymorphisms between laboratory strains of zebrafish.

The zebrafish has recently emerged as a useful system for understanding vertebrate developmental genetics, despite the lack of a linkage map. To identify DNA-based genetic polymorphisms for constructing a genetic map, we have screened a collection of RAPD primers for their utility in identifying genetic polymorphisms between two laboratory strains of zebrafish. Here, we report 116 primers that identify 721 strain-specific genetic markers and show how they can be used in haploid genetics of zebrafish.

Animals↗
PubMed1994

Goosecoid expression in neurectoderm and mesendoderm is disrupted in zebrafish cyclops gastrulas.

RNA from goosecoid, a homeobox-containing gene expressed during gastrulation in the anterior mesoderm of vertebrate embryos, can generate organizer activity when injected into ventral mesoderm, resulting in a secondary body axis; it is not yet understood, however, how goosecoid performs its organizer function. We report here that in the zebrafish gastrula, a domain of goosecoid expression arises in presumptive anterior neurectoderm which lies directly above goosecoid-expressing mesendodermal cells. From this position, goosecoid expression then spreads gradually across the ectodermal layer. In cyclops mutant embryos, which lack a ventral anterior brain, expression of goosecoid is abnormal in the mesendoderm and completely absent in the overlying neurectoderm. These results indicate that cyclops is required for correct specification of the mesendoderm and suggest that goosecoid expression in the ectoderm may result from vertical induction from the mesoderm. We propose that in the gastrula head, goosecoid may be important in organizing the ventral neurectoderm.

Animals↗
PubMed1993

Structure of the zebrafish snail1 gene and its expression in wild-type, spadetail and no tail mutant embryos.

Mesoderm formation is critical for the establishment of the animal body plan and in Drosophila requires the snail gene. This report concerns the cloning and expression pattern of the structurally similar gene snail1 from zebrafish. In situ hybridization shows that the quantity of snail1 RNA increases at the margin of the blastoderm in cells that involute during gastrulation. As gastrulation begins, snail1 RNA disappears from the dorsal axial mesoderm and becomes restricted to the paraxial mesoderm and the tail bud. snail1 RNA increases in cells that define the posterior border of each somite and then disappears when somitic cells differentiate. Later in development, expression appears in cephalic neural crest derivatives. Many snail1-expressing cells were missing from mutant spadetail embryos and the quantity of snail1 RNA was greatly reduced in mutant no tail embryos. The work presented here suggests that snail1 is involved in morphogenetic events during gastrulation, somitogenesis and development of the cephalic neural crest, and that no tail may act as a positive regulator of snail1.

Amino Acid Sequence↗
PubMed1990

Vitellogenesis in Drosophila: sequestration of a yolk polypeptide/invertase fusion protein into developing oocytes.

The mechanism of yolk deposition into developing oocytes of Drosophila was investigated by following the fate of a reporter protein fused to a vitellogenin, or yolk polypeptide (YP). Embryos were transformed with a hybrid gene consisting of the promotor and amino terminal 430 codons of the Yp2 gene fused to the cytoplasmic form of the invertase gene from the yeast Saccharomyces cerevisiae. RNA hybridization experiments with established lines of transformed flies showed that the hybrid gene was expressed in female fat bodies and ovaries but not in any male cells. Immunoblotting and endoglycosidase digestion showed that the hybrid protein was secreted from fat body cells via the secretory pathway, transported in hemolymph, and sequestered into developing oocytes. Transfusion experiments with hemolymph and pure invertase showed that sequestration of invertase depended on its attachment to YP. Immunocytochemistry demonstrated that the hybrid protein became localized in yolk granules as oocytes developed. Females homozygous for the fusion gene are generally sterile; their eggs containing the hybrid protein often collapse and their embryos fail to develop, suggesting that the structure of the yolk polypeptides is important for embryonic development. These experiments show that YP2 carries structural information sufficient to direct a reporter protein from fat body cells, through the hemolymph, and into the yolk granules of developing oocytes. This work provides a means of identifying the features of yolk polypeptides that are responsible for their deposition into yolk during oogenesis.

Amino Acid Sequence↗
PubMed1987

Sequence homologies among the three yolk polypeptide (Yp) genes in Drosophila melanogaster.

To identify candidates for cis-acting sequences that regulate the sex-, stage-, and cell-specific expression of three coordinately regulated yolk polypeptide genes (Yp) in Drosophila melanogaster, we have mapped the Yp3 transcript, sequenced a 4278 bp DNA fragment containing the Yp3 gene, compared Yp3 region sequences to corresponding parts of Yp1 and Yp2, and compared the predicted amino acid sequence of YP3 to YP1 and YP2. The results showed that the Yps are largely homologous in translated regions, especially in the 3' half of the genes. Untranscribed flanking regions had little homology. A conserved inverted repeat (the H-box) has homology both to vertebrate steroid hormone receptor binding sites and to the ecdysone control region of Drosophila's hsp23. These results identify sequences to mutate in order to define elements that regulate Yp gene expression and govern YP polypeptide function.

Amino Acid Sequence↗
PubMed1986

The humoral antibacterial response of Drosophila adults.

Hemolymph from a normal adult Drosophila melanogaster lacks factors that block the growth of Escherichia coli, but hemolymph from a fly previously inoculated with Enterobacter cloacae inhibits bacterial growth. Antibacterial activity appears within two hours after inoculation, and is still detectable sixty days later. Activity is potent, and can be detected in as little as a quarter of the hemolymph from a single inoculated male fly. After inoculation, at least eight new polypeptides not of bacterial origin appear in hemolymph with a time course similar to the appearance of antibacterial activity; these are called Antibacterial Response Polypeptides, or ARs. The most prominent polypeptides are AR24, AR22, and AR19 with molecular weights of about 24, 22, and 19 kilodaltons (kd). Other bands with as much as 75 kd and as little as 5 kd were also found. Electrophoresis of active hemolymph under non-denaturing conditions, and isoelectric focusing separate several protein species that block bacterial growth (Antibacterial Proteins, or ABs); one AB is neutral (AB7.1) and three are basic (AB8.7, AB9.0 and AB9.2). Two dimensional gels show that AR24, AR22 and AR19 have pIs identical to the basic antibacterial proteins. Radiolabelling experiments proved that the ARs were synthesized de novo after bacterial inoculation. ARs in six species of Drosophila showed fundamentally similar electrophoretic patterns.

Animals↗
PubMed1985

Development of gap junctions in normal and mutant ovaries of Drosophila melanogaster.

An ovarian follicle of Drosophila consists of an oocyte, 15 nurse cells, and hundreds of follicular epithelial cells. A freeze-fracture analysis of the surfaces between glutaraldehyde-fixed ovarian cells showed that all three cell types were interconnected by gap junctions. This is the first report of gap junctions between adjacent nurse cells, between nurse cells and oocytes, and between follicle cells and oocytes in Drosophila. Since we did not observe intramembranous particle clumping into crystalline patterns and since structurally different gap junctions occurred at different times in development and at different cell-cell interfaces, it is unlikely that fixation artifacts influenced particle distribution in our experiments. A computer-assisted morphometric analysis showed that the extent, size, and morphology of gap junctions varied with development and that these junctions can cover up to 9% of the cell surfaces. To test the role of gap junctions in follicular maturation, we studied ovaries from flies homozygous for the female sterile mutation fs(2)A17, in which follicles develop normally until yolk deposition commences. During the development of mutant follicles, gap junctions became abnormal before any other morphological aspect of the follicle. These studies show that gap junctions are available to play an important role in coordinating intercellular activities between all three cell types in ovarian follicles of Drosophila.

Animals↗
PubMed1985

Naturally occurring quantitative variants of acid phosphatase-1 in Drosophila melanogaster.

We have examined 111 wild Drosophila melanogaster lines for cis-acting quantitative variants of the Acph-1 gene, which codes for acid phosphatase-1 (ACPH). Three variants with obvious, reproducible phenotypes were isolated. All variants acted equally on all tissues and developmental stages examined. No recombinants were detected between one quantitative variant and the site determining the electrophoretic mobility of Acph-1 among 3885 flies examined. Several enzymatic properties of the variant enzymes were tested, including the Km values for two substrates, inhibition by three different inhibitors, and thermal stability; the variant enzymes behaved identically to the wild-type enzyme in all cases. Immunological titration experiments showed that the variant enzymes had the same enzyme activity per molecule of ACPH as the wild-type enzyme. These results suggest that the quantitative variants we have identified are altered in the regulatory portion of Acph-1 so as to produce altered numbers of normal ACPH molecules.

Acid Phosphatase↗
PubMed1985

Processing and secretion of a mutant yolk polypeptide in Drosophila.

Flies homozygous for the female sterile mutation fs(1)1163 produce eggs deficient in YP1, one of the three major yolk polypeptides. Genetic studies showed that fs(1)1163 is cis acting on YP1 quantity, so that mutation does not control a diffusible substance regulating YP1 production. The sterility and YP1 quantity phenotypes were not genetically separated from each other or from the structural gene for YP1, indicating that the mutation is located in or near Yp1. The amount of translatable YP1 message in mutant and wild-type cells was approximately equal, but the primary translation products were different in size and, hence, different in structure. The signal peptide was cleaved normally from the mutant polypeptide, and phosphorylation and glycosylation of the mutant YP1 also occur. However, YP1 processing intermediates that are transient in wild-type cells become major species in fs(1)1163 cells. We conclude that fs(1)1163 alters the primary structure of YP1 in a way that does not block signal-peptide cleavage but does alter later processing steps and hence its rate of secretion, leading to the YP1 deficiency found in the hemolymph and eggs.

Animals↗
PubMed1983

20-Hydroxyecdysone stimulates the accumulation of translatable yolk polypeptide gene transcript in adult male Drosophila melanogaster.

Yolk polypeptide (YP) synthesis is hormonally stimulated during maturation of adult female Drosophila melanogaster. Synthesis of the three YPs is sex specific and occurs in fat body cells and follicle cells of adult females. However, males have been shown to produce YPs when treated with the steroid hormone 20-hydroxyecdysone (20-HE). By using a cell-free translation system as an assay for YP mRNA, we found that 20-HE also causes the accumulation of translatable YP message in males. In addition, hybridization of cloned copies of genes for both YP1 and YP3 to total RNA from males showed that 20-HE caused the appearance of YP gene transcripts in males. Eight hours after treatment of males with 20-HE, YP gene transcript levels had increased at least 25-fold to approximately 2.7 x 10(6) copies of YP1 gene transcript per adult male fly. In normal adult females, there were 42 x 10(6) copies per fly by 24 hr. There was neither detectable YP synthesis nor translatable YP gene transcript in either normal 1- to 3-day-old males or 24-hr-old males treated with a juvenile hormone analogue. This evidence shows that 20-HE acts to regulate the levels of translatable YP mRNA in male Drosophila.

Animals↗