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Experimental studies on a lethal gene (1) in the Mexican axolotl, Ambystoma mexicanum.

1. Gene ł is a recessive lethal factor found in the white strain of axolotls. Animals heterozygous for the gene are phenotypically normal. When mated with each other they give offspring 25% of which exhibit the lethal effects of the gene. 2. The ł/ł homozygotes develop normally to an advanced embryonic stage (Harrison stage 40) before the effects of the gene are first manifested. They then come to display a characteristic combination of abnormalities, including a disproportionately small head, small and poorly developed eyes, abnormal poorly developed gills, undifferentiated limb buds, and reduced overall growth rate. They may feed briefly, but soon stop and invariably die within a few weeks of the time of hatching. 3. The action of gene ł has been analyzed by parabiosing mutant and normal embryos, and by grafting various organ primordia reciprocally between mutant and normal embryos. Parabiosis to normal embryos fails to correct the abnormalities of the mutants, although their survival may be somewhat prolonged. Grafts of mutant organ primordia (eye, limb, gill, pronephros, gonad, head) also invariably fail to show improved development or to survive on normal hosts; normal organ primordia develop normally on mutant hosts so long as the mutant survives. These experiments indicate that gene ł is a recessive autonomous cell lethal affecting all of the organ systems during late embryonic and early larval development.

Ambystoma↗

The marginal zone of the 32-cell amphibian embryo contains all the information required for chordamesoderm development.

The formation of the amphibian organizer is evidenced by the ability of cells of the dorsal marginal zone (DMZ) to self-differentiate to form notochord and to induce the formation of other axial structures from neighboring regions of the embryo. We have attempted to determine when these abilities are acquired in the urodele, Ambystoma mexicanum (axolotl), and in the anuran, Xenopus laevis, by removing the mesodermalizing influence of the vegetal hemisphere at different stages of development and culturing the animal hemisphere isolate. This was possible, even at the 32 and 64-cell stage, through the use of embryos with rare cleavage patterns. Cultured isolates were analyzed for morphological differentiation of mesodermal and neural structures, and for biochemical differentiation of the tissue-specific enzyme, acetylcholinesterase (AChE). Large amounts of mesodermal and neural structures, and normal expression of AChE were found in isolates made as early as the 32-cell stage in both species. Only a small increase in the percentage of isolates developing mesoderm was detected when isolations were made at later cleavage or blastula stages. The amount of mesoderm formed did not depend on the stage of isolation. Mesoderm differentiation was usually limited to the notocord and muscle. The isolates rarely formed pronephros, mesothelium, or mesenchyme, derivatives of ventral mesoderm, during normal development. The results indicate that the marginal zone of the cleavage-stage embryo contains all of the information needed for the formation of the organizer. The formation of dorsal mesoderm does not require subsequent interaction with the cells of the vegetal hemisphere, although the presence of those cells is likely to play a role in normal pattern formation.

Acetylcholinesterase↗

Activation of Met tyrosine kinase by hepatocyte growth factor is essential for internal organogenesis in Xenopus embryo.

Hepatocyte growth factor (HGF) specifically activates Met tyrosine kinase receptor, leading to mitogenic, motogenic, and morphogenic responses in a wide variety of cells. To know a role of HGF in Xenopus embryogenesis, loss-of-function mutation was introduced by dominant expression of truncated tyrosine kinase-negative Met. When tyrosine kinase-negative Met mRNA was micro-injected into two-cell to eight-cell stages Xenopus embryos, the liver development was mostly impaired and structures of pronephros and the gut were grossly underdeveloped in the restricted, late stage of development. These results strongly suggest that functional coupling between HGF and Met is essential for the development of internal organs originated from primitive gut and possibly involved in embryonic skeletogenesis. Together with developmental abnormality in mice mutated with HGF or Met gene, essential role of HGF for liver development is highly conserved from amphibian to mammalian species.

Animals↗

Ectopic expression of negative ARNT2 factor disrupts fish development.

ARNT factors are a cluster of bHLH-PAS factors that heterodimerize with other specific bHLH-PAS factors to mediate a wide range of biological responses. Previously, we obtained a truncated form of ARNT2-like factor, ARNT2A, from zebrafish, which encompasses the basic-helix-loop-helix and PAS A/B domains, but lacks a transactivation domain at its carboxyl end. Herein, we report another truncated ARNT2-like factor, ARNT2X, in zebrafish, which differs from ARNT2A at its N-terminal region. In cultured ZLE cells, transiently expressed ARNT2X and ARNT2A inhibited 2,3,7,8-TCDD-activated cyp1a1 transcription with different efficiencies. In the developing embryo, arnt2X mRNA was consistently expressed in the retinal and neural tube regions until the hatching stages, but it exhibited a more specific pattern at larval stages, including expression in the brain, eyes, hypothalamus, pharyngeal skeleton, heart, liver, pronephros duct, pectoral fin, and epithelial cells of the swim bladder. In contrast, arnt2A transcription diminished after hatching. Microinjecting a recombinant arnt2X-expression vector into fertilized eggs before cleavage stages caused severe defects in brain, eyes, pectoral fin, heart, and gut development. This suggests that the ARNT-mediated signal transduction pathways play important roles in fish tissue development.

Amino Acid Sequence↗

A novel Xenopus acetyltransferase with a dynamic expression in early development.

We have isolated a novel acetyltransferase from Xenopus laevis, named Xat-1. Xat-1 cDNA encodes a predicted protein of 846 amino acids that contains tetratricopeptide repeat (TPR) domains mediating protein-protein interactions and a bipartite nuclear localization signal (NLS). Its apparent molecular mass of 98.8 kDa was determined by SDS-PAGE analysis of Xat-1 recombinant protein in vitro translated in rabbit reticulocyte lysate. Xat-1 is homologous to N-terminal acetyltransferase 1 (NAT1), a gene that was originally discovered in yeast. Furthermore, it has many orthologs from human, mouse, Drosophila, C. elegans, and even Arabidopsis, thereby suggesting that these constitute a novel acetyltransferase family whose functions have been not examined. Xat-1 transcripts are expressed at relatively constant levels throughout early embryonic stages. They also exhibit dynamic expression pattern in brain, somites, branchial arches, pronephros, and otic vesicles.

Acetyltransferases↗

The formation of the pronephric duct in Xenopus involves recruitment of posterior cells by migrating pronephric duct cells.

Cell migration and cell-cell interactions play important roles in numerous processes during embryogenesis. One of these is the formation of the pronephric (Wolffian) duct (PD), which connects the pronephros to the cloaca. It is currently accepted that in most amphibians the pronephric duct is formed by active migration of the pronephric duct rudiment (PDR) cells along a predetermined pathway. However, there is evidence that in Xenopus, the PD may be formed entirely by in situ segregation of cells out of the lateral mesoderm. In this study, we show, using PDR ablation and Xenopus laevis-Xenopus borealis chimeras, that PD elongation in Xenopus requires both active cell migration and an induced recruitment of cells from the posterior. We also show that PDR cell migration is limited to only a few stages during development and that this temporal control is due, at least in part, to changes in the competence of the PD pathway to support cell migration.

Animals↗

Superficial cells in the early gastrula of Rana pipiens contribute to mesodermal derivatives.

We have studied the fate of presumptive mesodermal cells in the early Rana pipiens gastrula. We labeled superficial cells of the early gastrula with 125I. We also labeled all cells in a gastrula with rhodamine-lysine-dextran cell lineage tracer and superficial cells with 125I and then grafted small pieces of the marginal zone orthotopically into unlabeled host embryos. Labeled progeny were identified in sectioned embryos at the tail bud stage. The use of double-labeled grafts allowed us to study the relative contributions by superficial and deep cells to different derivatives. We found that the notochord and somite regions are both derived from the superficial and deep portions of circumblastoporal regions. In contrast, pronephros, lateral plate, cardiac anlagen, and blood cells only arise from deep cells in circumblastoporal regions. Such observations indicate that the fate map for R. pipiens is different from that of Xenopus laevis, where mesodermal derivatives appear to be restricted to the deep layers, and from that of Pleurodeles waltl, where all mesodermal derivatives are formed both from superficial and deep layers. We also have shown that from the neurula stage there is substantial mixing between cells due to the ingression of cells in the dorsal region so that superficial labeled cells, initially located in the roof of the archenteron, contribute to ventral regions of both the somites and notochord.

Animals↗

The epidermis is a source of directional information for the migrating pronephric duct in Ambystoma mexicanum embryos.

In the urodele Ambystoma mexicanum, the pronephric duct (PND) is formed from a coherent group of cells that migrate from the pronephros to the cloaca along a pathway immediately ventral to the developing somites. The guidance cues used by the migrating PND primordium to find the cloaca are a local property of the migration substratum, are temporally regulated, and are both polarized and oriented. Since the pronephric duct migrates between two tissues--the underlying lateral mesoderm and the overlying epidermis--we performed a study to identify the tissue(s) in which PND guidance cues originate. Through direct manipulation of the epidermis overlying the duct pathway, we show that the migrating PND reads epidermally derived cues (1) along the anterior-posterior axis that direct migration from anterior to posterior and (2) along the dorsal-ventral axis that constrain migration to the duct pathway. Heterochronic grafting experiments reveal that the ability to direct PND migration is a stable property of flank epidermis throughout the period of PND migration. Epidermal cues are, therefore, not responsible for the observed temporal restrictions on PND migration. Thus, the region of the embryo within which the advancing PND tip can migrate actually represents an area where two distinct but required sets of PND migration cues overlap. The epidermis overlying the duct pathway provides directional information; temporal restriction of duct migration is hypothesized to be a property of the flank mesoderm.

Ambystoma mexicanum↗

Expression of zebrafish rag genes during early development identifies the thymus.

Recent experiments have demonstrated that zebrafish is a vertebrate in which it is possible to carry out large-scale mutagenic screens to identify genes involved in specific developmental pathways. To follow development of the immune system in zebrafish, we have analyzed the expression of the recombination activating genes, rag1 and rag2, which we have previously isolated and characterized. These genes catalyze the rearrangement of immunoglobulin genes in immature B lymphocytes and of T cell receptor genes in immature T lymphocytes and are therefore appropriate markers to follow the development of organs containing these cells. By whole-mount in situ hybridization, we detected expression of both rag genes in a paired organ in the head, beginning on the fourth day after fertilization. Histological examination of this organ indicated that it corresponds to the thymus, as described for other fish, an organ that has not previously been identified in zebrafish. By histological analysis, the thymus primordium appears at 54 hr but does not enlarge significantly until 30 hr later. The thymus continues to enlarge and reaches its mature histological organization at 1 month. The pronephros, the major hematopoietic organ in the adult fish, begins to develop hematopoietic tissue about 2 weeks after fertilization. By 1 month, mature lymphocytes are distinguishable in the tissue surrounding renal tubules. Lymphocytes appear in the kidney too late for screening by whole-mount in situ hybridization; however, the pattern of rag1 expression in the thymus forms the basis of an assay for mutations affecting development of the thymus or its constituent lymphocytes.

Animals↗

Synergism between Pax-8 and lim-1 in embryonic kidney development.

Pax genes encode a family of highly conserved DNA-binding transcription factors. These proteins play key roles in regulating a number of vertebrate and invertebrate developmental processes. Mutations in Pax-6 result in eye defects in flies, mice, and humans, and ectopic expression of this gene can trigger the development of ectopic compound eyes in flies. Likewise, mutation of other Pax genes in vertebrates results in the failure of specific differentiation programs-Pax-1 causes skeletal defects; Pax-2, kidney defects; Pax-3 or Pax-7, neural crest defects; Pax-4, pancreatic beta-cell defects; Pax-5, B-cell defects; Pax-8, thyroid defects; and Pax-9, tooth defects. Although this class of genes is obviously required for the normal differentiation of a number of distinct organ systems, they have not previously been demonstrated to be capable of directing the embryonic development of organs in vertebrates. In this report, it is demonstrated that Pax-8 plays such a role in the establishment of the Xenopus embryonic kidney, the pronephros. However, in order to efficiently direct cells to form pronephric kidneys, XPax-8 requires cofactors, one of which may be the homeobox transcription factor Xlim-1. These two genes are initially expressed in overlapping domains in late gastrulae, and cells expressing both genes will go on to form the kidney. Ectopic expression of either gene alone has a moderate effect on pronephric patterning, while coexpression of XPax-8 plus Xlim-1 results in the development of embryonic kidneys of up to five times normal complexity and also leads to the development of ectopic pronephric tubules. This effect was synergistic rather than additive. XPax-2 can also synergize with Xlim-1, but the expression profile of this gene indicates that it normally functions later in pronephric development than does XPax-8. Together these data indicate that the interaction between XPax-8 and Xlim-1 is a key early step in the establishment of the pronephric primordium.

Amino Acid Sequence↗

Developmental basis of pronephric defects in Xenopus body plan phenotypes.

We have used monoclonal antibodies that recognize the pronephric tubules or pronephric duct to explore the induction of the embryonic kidney in developing Xenopus embryos. Morphogenesis of the pronephros was examined in UV-ventralized and lithium-dorsalized embryos. We find that the pronephric tubules are present in all but the strongest UV-induced phenotypes, but absent from relatively moderate lithium phenotypes. Interestingly the pronephric duct, which develops from the ventroposterior portion of the pronephric anlage, is missing from more of the mild UV phenotypes than are pronephric tubules. The loss of the capacity to form pronephroi in UV-ventralized embryos is caused by the loss of tissues capable of inducing the pronephric mesoderm, as marginal zone explants from ventralized embryos are still competent to respond to pronephric-inductive signals. Explant recombination experiments indicate that the tissue responsible for both the loss of pronephroi in UV-ventralized embryos and the induction of pronephroi during normal development is the anterior somites. The absence of pronephroi in relatively mild lithium phenotypes has a developmental basis different from that of the UV phenotype, as explants from lithium-treated embryos are effective inducers of pronephroi in recombinants with competent mesoderm, even though they themselves do not form pronephroi in isolation. Together these data indicate that dorsal tissues, especially the anterior somites, are responsible for the establishment of the intermediate mesoderm and the induction of the embryonic kidneys and that even mild dorsalization destroys the capacity to form cells competent to receive this signal.

Animals↗

The FGFR pathway is required for the trunk-inducing functions of Spemann's organizer.

Xenopus laevis embryogenesis is controlled by the inducing activities of Spemann's organizer. These inducing activities are separated into two distinct suborganizers: a trunk organizer and a head organizer. The trunk organizer induces the formation of posterior structures by emitting signals and directing morphogenesis. Here, we report that the fibroblast growth factor receptor (FGFR) signaling pathway, also known to regulate posterior development, performs critical functions within the cells of Spemann's organizer. Specifically, the FGFR pathway was required in the organizer cells in order for those cells to induce the formation of somitic muscle and the pronephros. Since the organizer influences the differentiation of these tissues by emitting signals that pattern the mesodermal germ layer, our data indicate that the FGFR regulates the production of these signals. In addition, the FGFR pathway was required for the expression of chordin, an organizer-specific protein required for the trunk-inducing activities of Spemann's organizer. Significantly, the FGFR pathway had a minimal effect on the function of the head organizer. We propose that the FGFR pathway is a defining molecular component that distinguishes the trunk organizer from the head organizer by controlling the expression of organizer-specific genes required to induce the formation of posterior structures and somitic muscle in neighboring cells. The implications of our findings for the evolutionarily conserved role of the FGFR pathway in the functions of Spemann's organizer and other vertebrate-signaling centers are discussed.

Animals↗

Axial protocadherin is a mediator of prenotochord cell sorting in Xenopus.

Prenotochord cell sorting is regarded as one of the first cell sorting events in early chordate development. We recently demonstrated that this sorting event occurs in vitro, although the mediator of this activity remains unidentified. Herein, we report the isolation of a full-length cDNA clone of Axial protocadherin (AXPC), the homologue of human protocadherin-1 (PCD1). AXPC encodes a transmembrane protein (AXPC) that is expressed exclusively in the notochord at the neurula stage and in the pronephros, somites, heart, optic vesicle, otic vesicle, and distinct parts of the brain at the tailbud stage. Cell dissociation and reaggregation assays and in vivo microinjection experiments demonstrated that cells overexpressing a membrane-tethered form of AXPC (MT-AXPC) acquired the same adhesive properties as prenotochord cells. Moreover, microinjection of either mRNA encoding the dominant negative form of AXPC (DN-AXPC) or morpholino oligonucleotides interferes with the sorting activity of prenotochord cells and normal axis formation. This study suggests that AXPC is necessary and sufficient for prenotochord cell sorting in the gastrulating embryo, and may also mediate sorting events later in development.

Animals↗

Essential function of Wnt-4 for tubulogenesis in the Xenopus pronephric kidney.

In the vertebrate embryo, development of the excretory system is characterized by the successive formation of three distinct kidneys: the pronephros, mesonephros, and metanephros. While tubulogenesis in the metanephric kidney is critically dependent on the signaling molecule Wnt-4, it is unknown whether Wnt signaling is equally required for the formation of renal epithelia in the other embryonic kidney forms. We therefore investigated the expression of Wnt genes during the pronephric kidney development in Xenopus. Wnt4 was found to be associated with developing pronephric tubules, but was absent from the pronephric duct. Onset of pronephric Wnt-4 expression coincided with mesenchyme-to-epithelium transformation. To investigate Wnt-4 gene function, we performed gain- and loss-of-function experiments. Misexpression of Wnt4 in the intermediate and lateral mesoderm caused abnormal morphogenesis of the pronephric tubules, but was not sufficient to initiate ectopic tubule formation. We used a morpholino antisense oligonucleotide-based gene knockdown strategy to disrupt Wnt-4 gene function. Xenopus embryos injected with antisense Wnt-4 morpholinos developed normally, but marker gene and morphological analysis revealed a complete absence of pronephric tubules. Pronephric duct development was largely unaffected, indicating that ductogenesis may occur normally in the absence of pronephric tubules. Our results show that, as in the metanephric kidney, Wnt-4 is critically required for tubulogenesis in the pronephric kidney, indicating that a common, evolutionary conserved gene regulatory network may control tubulogenesis in different vertebrate excretory organs.

Animals↗

Patterning of the avian intermediate mesoderm by lateral plate and axial tissues.

Amniote kidney tissue is derived from the intermediate mesoderm (IM), a strip of mesoderm that lies between the somites and the lateral plate. While much has been learned concerning the later events which regulate the differentiation of IM into tubules and other types of kidney tissue, much less is known concerning the earlier events which regulate formation of the IM itself. In the current study, the chick pronephros was used as a model system to identify tissues that play a role in patterning the IM and the critical time periods during which such patterning events take place. Explant studies revealed that the prospective pronephric IM is already specified to express kidney genes by stage 6, shortly after its gastrulation through the primitive streak, and earlier than previously reported. Transplant and explant experiments revealed that the lateral plate contains an activity that can repress IM formation in tissues that are already specified to express IM genes. In contrast, Hensen's node can promote formation of IM in the lateral plate. Paraxial tissues (presomitic mesoderm plus neural plate and notochord) were found to influence the morphogenesis of the nephric duct, but did not induce IM tissue to an appreciable extent. Combining lateral plate and paraxial tissue in vivo or in vitro led to induction of IM genes in the paraxial mesoderm but not in the lateral plate mesoderm. Based on these results and those of others, we propose a two-step model for the patterning of the IM. While tissue is still in the primitive streak, the prospective IM is relatively uncommitted. By stage 6, shortly after cells leave the primitive streak, a field of cells is generate which is specified to give rise to IM (Step 1). Subsequently, competing signals from the lateral plate and axial tissues modulate the number of cells that commit to an IM fate (Step 2).

Animals↗

Effects of chromium on humoral and cell-mediated immune responses and host resistance to disease in a freshwater catfish, Saccobranchus fossilis (Bloch).

The effects of subtoxic levels of Cr on humoral and cell-mediated immune responses, blood parameters, susceptibility to bacterial (Aeromonas hydrophila) infection, and macrophage activity in the freshwater air-breathing Asian catfish, Saccobranchus fossilis, during a 28-day exposure were examined by a static bioassay test procedure. At 0.1, 1.0, and 3.2 mg / liter Cr, dose-dependent Cr accumulation in kidney, liver, and spleen was observed at the end of the experiment. Chromium exposure caused a significant change in spleen to body weight ratio. Fish exposed to Cr concentrations had lower antibody titer values, reduced numbers of splenic and kidney plaque-forming cells, and higher counts of splenic lymphocytes but reduced counts of kidney cells when compared with the control group. At 0.1, 1.0, and 3.2 mg /liter Cr, dose-dependent decreases in red blood cell counts, hemoglobin content, and packed cell volume were observed. Differential leukocyte counts revealed that Cr exposure caused a significant decrease in large and small lymphocytes, whereas neutrophils and thrombocytes increased. Effects of Cr exposure to mitogen (Con A) on proliferation of splenic and pronephric lymphocytes suggests a decrease in mitogenic response. The eye-allograft rejection time, as a parameter of cell-mediated immunity, was statistically increased at 1.0 and 3.2 mg/liter Cr. Fish exposed to Cr for 28 days exhibited higher susceptibility to A. hydrophila infection than control fish. The results suggest that Cr exposure reduced the resistance of catfish to bacterial infections. The phagocytic activity of splenic and pronephros macrophages was examined in vitro and found to be significantly decreased.

Animals↗

The source of reactive cells during central Wallerian degeneration in the goldfish: a differential irradiation protocol.

We have used a partial irradiation paradigm to examine the provenance of cells that participate in Wallerian cellular responses in the goldfish visual system. Animals which received 50 Gy whole-body gamma-irradiation showed virtually complete inhibition of the proliferative burst usually seen after optic nerve section. These animals did, however, show a robust hyperplastic response in the optic tract that we believe represents the migration of nearby microglial cells into the affected tract. When only the postcephalic body was irradiated, proliferating cells in the major hematopoietic organs of the fish, the kidney and pronephros, were substantially inhibited. Despite this, the Wallerian cellular response in the visual paths was essentially normal. Thus, there is no obligate requirement for peripheral proliferative cells to participate in central Wallerian degeneration in the fish. However, when only the head was irradiated, and the hematopoietic organs were spared, there was a proliferative response in the visual system. We believe this represents the invasion of the visual pathways by peripheral blood cells through the optic nerve lesion and blood vessels in the nerve itself. This invasion, however, is not sufficient to generate substantial hyperplasia. In summary, although we find evidence for a small contribution by exogenous cells, the major source of reactive cells during central Wallerian degeneration in the fish is the endogenous microglia. Our data underscore the importance of elucidating the mechanisms by which microglial cells are activated and the role that they play in regeneration.

Animals↗

Rainbow trout neutrophils are responsible for non-specific cytotoxicity.

This is the first report that rainbow trout (Oncorhynchus mykiss) neutrophils are responsible for non-specific cytotoxicity. A monoclonal antibody (mab) for rainbow trout leucocytes was produced. Using this mab (TTL-5E9), neutrophils (5E9-positive cells) were isolated from the pronephros by a panning technique. The isolated neutrophils showed high viability (approximately 95%) and purity (92-95%), and were functional in cytotoxic activity assays. The neutrophils demonstrated significantly higher cytotoxic activities against YAC-1 target cells than the other cells (5E9-negative cells, predominantly lymphocytes). The number of neutrophils contaminating the 5E9-negative fraction and their non-specific cytotoxicities were positively correlated. These findings demonstrate that rainbow trout neutrophils possess non-specific cytotoxic activities.

Animals↗