Biomedical subjects
Publications and source records attributed to D L Stemple.
Nodal signalling is essential for vertebrate germ-layer formation. How this single signal can generate such a diverse array of tissues remains a mystery and is an area of intense research. Three recent reports reveal unanticipated subtleties to the process and provide new mechanisms for generating distinct responses.
Zebrafish embryos homozygous for the masterblind (mbl) mutation exhibit a striking phenotype in which the eyes and telencephalon are reduced or absent and diencephalic fates expand to the front of the brain. Here we show that mbl(-/-) embryos carry an amino-acid change at a conserved site in the Wnt pathway scaffolding protein, Axin1. The amino-acid substitution present in the mbl allele abolishes the binding of Axin to Gsk3 and affects Tcf-dependent transcription. Therefore, Gsk3 activity may be decreased in mbl(-/-) embryos and in support of this possibility, overexpression of either wild-type Axin1 or Gsk3beta can restore eye and telencephalic fates to mbl(-/-) embryos. Our data reveal a crucial role for Axin1-dependent inhibition of the Wnt pathway in the early regional subdivision of the anterior neural plate into telencephalic, diencephalic, and eye-forming territories.
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The transcription factors Fast-1 and Mixer are important terminal components of Smad2-mediated TGF-beta signal transduction. Recent studies demonstrate that Fast-1 and Mixer play critical roles in the formation of endoderm and dorsal mesoderm in zebrafish.
Vertebrate gastrulation involves the specification and coordinated movement of large populations of cells that give rise to the ectodermal, mesodermal and endodermal germ layers. Although many of the genes involved in the specification of cell identity during this process have been identified, little is known of the genes that coordinate cell movement. Here we show that the zebrafish silberblick (slb) locus encodes Wnt11 and that Slb/Wnt11 activity is required for cells to undergo correct convergent extension movements during gastrulation. In the absence of Slb/Wnt11 function, abnormal extension of axial tissue results in cyclopia and other midline defects in the head. The requirement for Slb/Wnt11 is cell non-autonomous, and our results indicate that the correct extension of axial tissue is at least partly dependent on medio-lateral cell intercalation in paraxial tissue. We also show that the slb phenotype is rescued by a truncated form of Dishevelled that does not signal through the canonical Wnt pathway, suggesting that, as in flies, Wnt signalling might mediate morphogenetic events through a divergent signal transduction cascade. Our results provide genetic and experimental evidence that Wnt activity in lateral tissues has a crucial role in driving the convergent extension movements underlying vertebrate gastrulation.
In zebrafish, neuronal differentiation progresses across the retina in a pattern that is reminiscent of the neurogenic wave that sweeps across the developing eye in Drosophila. We show that expression of a zebrafish homolog of Drosophila atonal, ath5, sweeps across the eye predicting the wave of neuronal differentiation. By analyzing the regulation of ath5 expression, we have elucidated the mechanisms that regulate initiation and spread of neurogenesis in the retina. ath5 expression is lost in Nodal pathway mutant embryos lacking axial tissues that include the prechordal plate. A likely role for axial tissue is to induce optic stalk cells that subsequently regulate ath5 expression. Our results suggest that a series of inductive events, initiated from the prechordal plate and progressing from the optic stalks, regulates the spread of neuronal differentiation across the zebrafish retina.
We have investigated axis-inducing activities and cellular fates of the zebrafish organizer using a new method of transplantation that allows the transfer of both deep and superficial organizer tissues. Previous studies have demonstrated that the zebrafish embryonic shield possesses classically defined dorsal organizer activity. When we remove the morphologically defined embryonic shield, embryos recover and are completely normal by 24 hours post-fertilization. We find that removal of the morphological shield does not remove all goosecoid- and floating head-expressing cells, suggesting that the morphological shield does not comprise the entire organizer region. Complete removal of the embryonic shield and adjacent marginal tissue, however, leads to a loss of both prechordal plate and notochord. In addition, these embryos are cyclopean, show a significant loss of floor plate and primary motorneurons and display disrupted somite patterning. Motivated by apparent discrepancies in the literature we sought to test the axis-inducing activity of the embryonic shield. A previous study suggested that the shield is capable of only partial axis induction, specifically being unable to induce the most anterior neural tissues. Contrary to this study, we find shields can induce complete secondary axes when transplanted into host ventral germ-ring. In induced secondary axes donor tissue contributes to notochord, prechordal plate and floor plate. When explanted shields are divided into deep and superficial fragments and separately transplanted we find that deep tissue is able to induce the formation of ectopic axes with heads but lacking posterior tissues. We conclude that the deep tissue included in our transplants is important for proper head formation.
The change in vascular smooth muscle cells (SMC) from a differentiated to a dedifferentiated state is the critical phenotypic response that promotes occlusive arteriosclerotic disease. Despite its importance, research into molecular mechanisms regulating smooth muscle differentiation has been hindered by the lack of an in vitro cell differentiation system. We identified culture conditions that promote efficient differentiation of Monc-1 pluripotent neural crest cells into SMC. Exclusive Monc-1 to SMC differentiation was indicated by cellular morphology and time-dependent induction of the SMC markers smooth muscle alpha-actin, smooth muscle myosin heavy chain, calponin, SM22alpha, and APEG-1. The activity of the SM22alpha promoter was low in Monc-1 cells. Differentiation of these cells into SMC caused a 20-30-fold increase in the activity of the wild-type SM22alpha promoter and that of a hybrid promoter containing three copies of the CArG element. By gel mobility shift analysis, we identified new DNA-protein complexes in nuclear extracts prepared from differentiated Monc-1 cells. One of the new complexes contained serum response factor. This Monc-1 to SMC model should facilitate the identification of nodal regulators of smooth muscle development and differentiation.
The zebrafish pronephric kidney provides a simplified model of nephron development and epithelial cell differentiation which is amenable to genetic analysis. The pronephros consists of two nephrons with fused glomeruli and paired pronephric tubules and ducts. Nephron formation occurs after the differentiation of the pronephric duct with both the glomeruli and tubules being derived from a nephron primordium. Fluorescent dextran injection experiments demonstrate that vascularization of the zebrafish pronephros and the onset of glomerular filtration occurs between 40 and 48 hpf. We isolated fifteen recessive mutations that affect development of the pronephros. All have visible cysts in place of the pronephric tubule at 2-2.5 days of development. Mutants were grouped in three classes: (1) a group of twelve mutants with defects in body axis curvature and manifesting the most rapid and severe cyst formation involving the glomerulus, tubule and duct, (2) the fleer mutation with distended glomerular capillary loops and cystic tubules, and (3) the mutation pao pao tang with a normal glomerulus and cysts limited to the pronephric tubules. double bubble was analyzed as a representative of mutations that perturb the entire length of the pronephros and body axis curvature. Cyst formation begins in the glomerulus at 40 hpf at the time when glomerular filtration is established suggesting a defect associated with the onset of pronephric function. Basolateral membrane protein targeting in the pronephric duct epithelial cells is also severely affected, suggesting a failure in terminal epithelial cell differentiation and alterations in electrolyte transport. These studies reveal the similarity of normal pronephric development to kidney organogenesis in all vertebrates and allow for a genetic dissection of genes needed to establish the earliest renal function.
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Two recent large-scale genetic screens in zebrafish have identified many mutations that affect differentiation in a variety of organ systems, particularly the notochord, the neural crest and the blood. The combination of these newly identified mutations and well established embryological methods makes zebrafish uniquely suited among vertebrate experimental systems to simultaneously address the roles of specific genes and specific cell-cell interactions during differentiation.
The notochord is critical for the normal development of vertebrate embryos. It serves both as the major skeletal element of the embryo and as a signaling source for the establishment of pattern within the neurectoderm, the paraxial mesoderm and other tissues. In a large-scale systematic screen of mutations affecting embryogenesis in zebrafish we identified 65 mutations that fall into 29 complementation groups, each leading to a defect in the formation and/or maintenance of the notochord. These mutations produce phenotypic abnormalities at numerous stages of notochord development, thereby establishing a phenotypic pathway, which in turn suggests a genetic pathway for the development of the notochord. Perturbations within adjacent tissues in mutant embryos further indicate the importance of notochord-derived signals for patterning within the embryo and suggest that these mutations will yield additional insight into the cues that regulate these patterning processes.
In a large scale mutagenesis screen for embryonic mutants in zebrafish, we have identified 63 mutations in 24 loci affecting the morphogenesis of the zebrafish brain. The expression of marker genes and the integrity of the axonal scaffold have been studied to investigate abnormalities in regionalization, neurogenesis and axonogenesis in the brain. Mutants can be broadly classified into two groups, one affecting regionalization along the anterior-posterior or dorsal-ventral axis, and the other affecting general features of brain morphology. The first group includes one locus that is required to generate the anlage of the midbrain-hindbrain boundary region at the beginning of somitogenesis. Four loci were identified that affect dorsal-ventral patterning of the brain, including the previously described cyclops locus. Mutant embryos of this class show a reduction of ventral neuroectodermal structures and variable fusion of the eyes. The second group includes a large class of mutations affecting the formation of brain ventricles. Analysis of this class reveals the requirement of a functional cardiovascular system for ventricle enlargement during embryogenesis. Mutations in one locus lead to the formation of supernumerary primary neurons, a phenotype reminiscent of neurogenic mutants in Drosophila. Other mutant phenotypes described here range from abnormalities in the fasciculation and outgrowth of axons to defects in the diameter of the neural tube. The identified loci establish the genetic foundation for a further analysis of the development of the zebrafish embryonic brain.
Programmed cell death is a prominent feature of normal animal development. During neurogenesis, naturally occurring cell death is a mechanism to eliminate neurons that fail to make appropriate connections. To prevent accidental cell death, mechanisms that trigger programmed cell death, as well as the genetic components of the cell death program, are tightly controlled. In a large-scale mutagenesis screen for embryonic lethal mutations in zebrafish Danio rerio we have found 481 mutations with a neural degeneration phenotype. Here, we present 50 mutations that fall into two classes (termed spacehead and fala-like) that are characterized by two main features: first, they appear to affect cell survival primarily within the neuroectodermal lineages during somitogenesis, and second, they show an altered brain morphology at or before 28 hours of development. Evidence for the specificity of cell death within the central nervous system comes from visual inspection of dying cells and analysis of DNA fragmentation, a process associated with apoptotic cell death. In mutants, the level of dying cells is significantly increased in brain and spinal cord. Furthermore, at the end of somitogenesis, the cell count of radial glia and trigeminal neurons is reduced in some mutants of the spacehead class. A variety of neurodegenerative disorders in mouse and humans have been associated with abnormal levels of programmed cell death within the central nervous system. The mutations presented here might provide a genetic framework to aid in the understanding of the etiology of degenerative and physiological disorders within the CNS and the activation of inappropriate programmed cell death.
In a large scale screen for genetic defects in zebrafish embryogenesis we identified 49 mutations affecting development of the retina. Based on analysis of living embryos as well as histological sections, we grouped the isolated mutations into six phenotypic categories. (1) Mutations in three loci result in a loss of wild-type laminar pattern of the neural retina. (2) Defects in four loci lead to an abnormal specification of the eye anlagen. Only one eye frequently forms in this class of mutants. (3) Seven loci predominantly affect development of the outer retinal layers. Mutants in this category display cell loss mainly in the photoreceptor cell layer. (4) Nine mutations cause retardation of eye growth without any other obvious abnormalities in the retina. (5) A group of twelve mutations is characterized by nonspecific retinal degeneration. (6) Four mutations display retinal degeneration associated with a pigmentation defect. Finally, two mutations, one with absence of the ventral retina and one with an eye-specific pigmentation defect, are not classified in any of the above groups. The identified mutations affect numerous aspects of eye development, including: specification of the eye anlage, growth rate of the optic cup, establishment of retinal stratification, specification or differentiation of retinal neurons and formation of the dorsoventral axis in the developing eye.
In a large scale screen for genetic defects in zebrafish embryogenesis we identified mutations affecting several aspects of ear development, including: specification of the otic placode, growth of the otic vesicle (otocyst), otolith formation, morphogenesis of the semicircular canals and differentiation of the otic capsule. Here we report initial phenotypic and genetic characterization of 20 of these mutations defining 13 independent loci. Embryos mutant at the quadro locus display abnormal specification of the otic placode. As revealed by dlx-3 expression, the otic field in the mutant embryos is smaller or split into two fields. At later stages of development the ear of quadro mutants is frequently divided into two smaller, incomplete units. Four loci affect ear shape shortly after formation of the otic vesicle. All of them also display abnormal brain morphology. Mutations in five loci result in the absence of otolith formation; two of these also produce changes of ear morphology. Two loci, little richard and golas, affect morphology of the otic vesicle shortly before formation of the semicircular canals. In both cases the morphogenesis of the semicircular canals is disrupted. Finally, the antytalent locus is involved in late expansion of the ear structure. Analysis of mutations presented here will strengthen our understanding of vertebrate ear morphogenesis and provide novel entry points to its genetic analysis.
As part of a large-scale mutagenesis screen of the zebrafish genome, we have identified 58 mutations that affect the formation and function of the cardiovascular system. The cardiovascular system is particularly amenable for screening in the transparent zebrafish embryo because the heart and blood vessels are prominent and their function easily examined. We have classified the mutations affecting the heart into those that affect primarily either morphogenesis or function. Nine mutations clearly disrupt the formation of the heart. cloche deletes the endocardium. In cloche mutants, the myocardial layer forms in the absence of the endocardium but is dysmorphic and exhibits a weak contractility. Two loci, miles apart and bonnie and clyde, play a critical role in the fusion of the bilateral tubular primordia. Three mutations lead to an abnormally large heart and one to the formation of a diminutive, dysmorphic heart. We have found no mutation that deletes the myocardial cells altogether, but one, pandora, appears to eliminate the ventricle selectively. Seven mutations interfere with vascular integrity, as indicated by hemorrhage at particular sites. In terms of cardiac function, one large group exhibits a weak beat. In this group, five loci affect both chambers and seven a specific chamber (the atrium or ventricle). For example, the weak atrium mutation exhibits an atrium that becomes silent but has a normally beating ventricle. Seven mutations affect the rhythm of the heart causing, for example, a slow rate, a fibrillating pattern or an apparent block to conduction. In several other mutants, regurgitation of blood flow from ventricle to atrium is the most prominent abnormality, due either to the absence of valves or to poor coordination between the chambers with regard to the timing of contraction. The mutations identified in this screen point to discrete and critical steps in the formation and function of the heart and vasculature.
We have identified mutations that perturb the formation or differentiation of the first embryonic blood cells in the zebrafish embryo. These 'primitive' red blood cells originate in the intermediate cell mass of the trunk, a derivative of the dorsal lateral plate mesoderm. By transfusion of blood between embryos we demonstrate that this cohort of cells provides the embryo with all, or nearly all, of its blood cells until at least day 5 postfertilization. Larval lethal mutations generated by ENU mutagenesis affect different steps in the development of these cells. Some cause defects in precursor generation, others defects in differentiation, and others an increase in cellular photosensitivity.