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Derek L Stemple

Publications and source records attributed to Derek L Stemple.

17 recordsLinked to original sources
PubMed2006

Genetic screens for mutations affecting development of Xenopus tropicalis.

We present here the results of forward and reverse genetic screens for chemically-induced mutations in Xenopus tropicalis. In our forward genetic screen, we have uncovered 77 candidate phenotypes in diverse organogenesis and differentiation processes. Using a gynogenetic screen design, which minimizes time and husbandry space expenditures, we find that if a phenotype is detected in the gynogenetic F2 of a given F1 female twice, it is highly likely to be a heritable abnormality (29/29 cases). We have also demonstrated the feasibility of reverse genetic approaches for obtaining carriers of mutations in specific genes, and have directly determined an induced mutation rate by sequencing specific exons from a mutagenized population. The Xenopus system, with its well-understood embryology, fate map, and gain-of-function approaches, can now be coupled with efficient loss-of-function genetic strategies for vertebrate functional genomics and developmental genetics.

Animal Diseases↗
PubMed2006

Genetic and genomic prospects for Xenopus tropicalis research.

Research using Xenopus laevis has made enormous contributions to our understanding of vertebrate development, control of the eukaryotic cell cycle and the cytoskeleton. One limitation, however, has been the lack of systematic genetic studies in Xenopus to complement molecular and cell biological investigations. Work with the closely related diploid frog Xenopus tropicalis is beginning to address this limitation. Here, we review the resources that will make genetic studies using X. tropicalis a reality.

Animals↗
PubMed2006

Essential and overlapping roles for laminin alpha chains in notochord and blood vessel formation.

Laminins are major constituents of basement membranes and have wide ranging functions during development and in the adult. They are a family of heterotrimeric molecules created through association of an alpha, beta and gamma chain. We previously reported that two zebrafish loci, grumpy (gup) and sleepy (sly), encode laminin beta1 and gamma1, which are important both for notochord differentiation and for proper intersegmental blood vessel (ISV) formation. In this study we show that bashful (bal) encodes laminin alpha1 (lama1). Although the strongest allele, bal(m190), is fully penetrant, when compared to gup or sly mutant embryos, bal mutants are not as severely affected, as only anterior notochord fails to differentiate and ISVs are unaffected. This suggests that other alpha chains, and hence other isoforms, act redundantly to laminin 1 in posterior notochord and ISV development. We identified cDNA sequences for lama2, lama4 and lama5 and disrupted the expression of each alone or in mutant embryos also lacking laminin alpha1. When expression of laminin alpha4 and laminin alpha1 are simultaneously disrupted, notochord differentiation and ISVs are as severely affected as sly or gup mutants. Moreover, live imaging of transgenic embryos expressing enhanced green fluorescent protein in forming ISVs reveals that the vascular defects in these embryos are due to an inability of ISV sprouts to migrate correctly along the intersegmental, normally laminin-rich regions.

Animals↗
PubMed2006

DNA detection using recombination proteins.

DNA amplification is essential to most nucleic acid testing strategies, but established techniques require sophisticated equipment or complex experimental procedures, and their uptake outside specialised laboratories has been limited. Our novel approach, recombinase polymerase amplification (RPA), couples isothermal recombinase-driven primer targeting of template material with strand-displacement DNA synthesis. It achieves exponential amplification with no need for pretreatment of sample DNA. Reactions are sensitive, specific, and rapid and operate at constant low temperature. We have also developed a probe-based detection system. Key aspects of the combined RPA amplification/detection process are illustrated by a test for the pathogen methicillin-resistant Staphylococcus aureus. The technology proves to be sensitive to fewer than ten copies of genomic DNA. Furthermore, products can be detected in a simple sandwich assay, thereby establishing an instrument-free DNA testing system. This unique combination of properties is a significant advance in the development of portable and widely accessible nucleic acid-based tests.

Bacillus subtilis↗
PubMed2005

Multiple mutations in mouse Chd7 provide models for CHARGE syndrome.

Mouse ENU mutagenesis programmes have yielded a series of independent mutations on proximal chromosome 4 leading to dominant head-bobbing and circling behaviour due to truncations of the lateral semicircular canal of the inner ear. Here, we report the identification of mutations in the Chd7 gene in nine of these mutant alleles including six nonsense and three splice site mutations. The human CHD7 gene is known to be involved in CHARGE syndrome, which also shows inner ear malformations and a variety of other features with varying penetrance and appears to be due to frequent de novo mutation. We found widespread expression of Chd7 in early development of the mouse in organs affected in CHARGE syndrome including eye, olfactory epithelium, inner ear and vascular system. Closer inspection of heterozygous mutant mice revealed a range of defects with reduced penetrance, such as cleft palate, choanal atresia, septal defects of the heart, haemorrhages, prenatal death, vulva and clitoral defects and keratoconjunctivitis sicca. Many of these defects mimic the features of CHARGE syndrome. There were no obvious features of the gene that might make it more mutable than other genes. We conclude that the large number of mouse mutants and human de novo mutations may be due to the combination of the Chd7 gene being a large target and the fact that many heterozygous carriers of the mutations are viable individuals with a readily detectable phenotype.

Animals↗
PubMed2005

Structure and function of the notochord: an essential organ for chordate development.

The notochord is the defining structure of the chordates, and has essential roles in vertebrate development. It serves as a source of midline signals that pattern surrounding tissues and as a major skeletal element of the developing embryo. Genetic and embryological studies over the past decade have informed us about the development and function of the notochord. In this review, I discuss the embryonic origin, signalling roles and ultimate fate of the notochord, with an emphasis on structural aspects of notochord biology.

Animals↗
PubMed2004

The notochord.

Explore the source record for details and available documents.

Animals↗
PubMed2004

Repression of nodal expression by maternal B1-type SOXs regulates germ layer formation in Xenopus and zebrafish.

B1-type SOXs (SOXs 1, 2, and 3) are the most evolutionarily conserved subgroup of the SOX transcription factor family. To study their maternal functions, we used the affinity-purified antibody antiSOX3c, which inhibits the binding of Xenopus SOX3 to target DNA sequences [Development. 130(2003)5609]. The antibody also cross-reacts with zebrafish embryos. When injected into fertilized Xenopus or zebrafish eggs, antiSOX3c caused a profound gastrulation defect; this defect could be rescued by the injection of RNA encoding SOX3DeltaC-EnR, a SOX3-engrailed repression domain chimera. In antiSOX3c-injected Xenopus embryos, normal animal-vegetal patterning of mesodermal and endodermal markers was disrupted, expression domains were shifted toward the animal pole, and the levels of the endodermal markers SOX17 and endodermin increased. In Xenopus, SOX3 acts as a negative regulator of Xnr5, which encodes a nodal-related TGFbeta-family protein. Two nodal-related proteins are expressed in the early zebrafish embryo, squint and cyclops; antiSOX3c-injection leads to an increase in the level of cyclops expression. In both Xenopus and zebrafish, the antiSOX3c phenotype was rescued by the injection of RNA encoding the nodal inhibitor Cerberus-short (CerS). In Xenopus, antiSOX3c's effects on endodermin expression were suppressed by injection of RNA encoding a dominant negative version of Mixer or a morpholino against SOX17alpha2, both of which act downstream of nodal signaling in the endoderm specification pathway. Based on these data, it appears that maternal B1-type SOX functions together with the VegT/beta-catenin system to regulate nodal expression and to establish the normal pattern of germ layer formation in Xenopus. A mechanistically conserved system appears to act in a similar manner in the zebrafish.

Amino Acid Sequence↗
PubMed2004

Differential requirements for COPI transport during vertebrate early development.

The coatomer vesicular coat complex is essential for normal Golgi and secretory activities in eukaryotic cells. Through positional cloning of genes controlling zebrafish notochord development, we found that the sneezy, happy, and dopey loci encode the alpha, beta, and beta' subunits of the coatomer complex. Export from mutant endoplasmic reticulum is blocked, Golgi structure is disrupted, and mutant embryos eventually degenerate due to widespread apoptosis. The early embryonic phenotype, however, demonstrates that despite its "housekeeping" functions, coatomer activity is specifically and cell autonomously required for normal chordamesoderm differentiation, perinotochordal basement membrane formation, and melanophore pigmentation. Hence, differential requirements for coatomer activity among embryonic tissues lead to tissue-specific developmental defects. Moreover, we note that the mRNA encoding alpha coatomer is strikingly upregulated in notochord progenitors, and we present data suggesting that alpha coatomer transcription is tuned to activity- and cell type-specific secretory loads.

Animals↗
PubMed2003

Nodal signaling and vertebrate germ layer formation.

The understanding of germ layer formation in vertebrates began with classical experimental embryology. Early in the 20th century, Spemann and Mangold (1924) identified a region of the early embryo capable of inducing an entire embryonic axis. Termed the dorsal organizer, the tissue and the activity have been shown to exist in all vertebrates examined. In mice, for example, the activity resides in a region of the gastrula embryo known as the node. Experiments by the Dutch embryologist Nieuwkoop (1967a, 1967b, 1973, 1977) showed that a signal derived from the vegetal half of the amphibian embryo is responsible for the formation of mesoderm. Nieuwkoop's results allowed the development of in vitro assays that led, in the late 1980s and early 1990s, to the identification of growth factors essential for germ layer formation. Through more recent genetic investigations in mice and zebrafish, we now know that one class of secreted growth factor, called Nodal because of its localized expression in the mouse node, is essential for formation of mesoderm and endoderm and for the morphological rearrangements that occur during gastrulation.

Activins↗
PubMed2002

Lefty antagonism of Squint is essential for normal gastrulation.

Activities of a variety of signaling proteins that regulate embryogenesis are limited by endogenous antagonists. The zebrafish Nodal-related ligands, Squint and Cyclops, and their antagonists, Lefty1 and Lefty2, belong to the TGFbeta-related protein superfamily, whose members have widespread biological activities. Among other activities, Nodals direct the formation of most mesendoderm. By inducing their own transcription and that of the Lefties, Nodal signals establish positive and negative autoregulatory loops. To investigate how these autoregulatory pathways regulate development, we depleted zebrafish embryos of Lefty1 and/or Lefty2 by using antisense morpholino oligonucleotides. Loss of Lefty1 causes aberrations during somitogenesis stages, including left-right patterning defects, whereas Lefty2 depletion has no obvious consequences. Depletion of both Lefty1 and Lefty2, by contrast, causes unchecked Nodal signaling, expansion of mesendoderm, and loss of ectoderm. The expansion of mesendoderm correlates with an extended period of rapid cellular internalization and a failure of deep-cell epiboly. The gastrulation defects of embryos depleted of Lefty1 and Lefty2 result from the deregulation of Squint signaling. In contrast, deregulation of Cyclops does not affect morphology or the transcription of Nodal target genes during gastrulation. Furthermore, we find that Cyclops is specifically required for the maintenance of lefty1 and lefty2 transcription.

Animals↗
PubMed2002

Pioneering work in vertebrate neural development.

Control of directional axon growth is fundamental to correct wiring in the nervous system, and glia are thought to play an important role. New work in the zebrafish lateral line shows that glia are not required for axonal pathfinding but are required for normal mature nerve organization.

Animals↗
PubMed2002

Zebrafish mutants identify an essential role for laminins in notochord formation.

Basement membranes are thought to be essential for organ formation, providing the scaffold on which individual cells organize to form complex tissues. Laminins are integral components of basement membranes. To understand the development of a simple vertebrate organ, we have used positional cloning to characterize grumpy and sleepy, two zebrafish loci known to control notochord formation, and find that they encode laminin beta1 and laminin gamma1, respectively. Removal of either chain results in the dramatic loss of laminin 1 staining throughout the embryo and prevents formation of the basement membrane surrounding the notochord. Notochord cells fail to differentiate and many die by apoptosis. By transplantation, we demonstrate that, for both grumpy and sleepy, notochord differentiation can be rescued by exogenous sources of the missing laminin chain, although notochordal sources are also sufficient for rescue. These results demonstrate a clear in vivo requirement for laminin beta1 and laminin gamma1 in the formation of a specific vertebrate organ and show that laminin or the laminin-dependent basement membrane is essential for the differentiation of chordamesoderm to notochord.

Animals↗
PubMed2002

Removal of dystroglycan causes severe muscular dystrophy in zebrafish embryos.

Muscular dystrophy is frequently caused by disruption of the dystrophin-glycoprotein complex (DGC), which links muscle cells to the extracellular matrix. Dystroglycan, a central component of the DGC, serves as a laminin receptor via its extracellular alpha subunit, and interacts with dystrophin (and thus the actin cytoskeleton) through its integral membrane beta subunit. We have removed the function of dystroglycan in zebrafish embryos. In contrast to mouse, where dystroglycan mutations lead to peri-implantation lethality, dystroglycan is dispensable for basement membrane formation during early zebrafish development. At later stages, however, loss of dystroglycan leads to a disruption of the DGC, concurrent with loss of muscle integrity and necrosis. In addition, we find that loss of the DGC leads to loss of sarcomere and sarcoplasmic reticulum organisation. The DGC is required for long-term survival of muscle cells in zebrafish, but is dispensable for muscle formation. Dystroglycan or the DGC is also required for normal sarcomere and sarcoplasmic reticulum organisation. Because zebrafish embryos lacking dystroglycan share several characteristics with human muscular dystrophy, they should serve as a useful model for the disease. In addition, knowing the dystroglycan null phenotype in zebrafish will facilitate the isolation of other molecules involved in muscular dystrophy pathogenesis.

Animals↗