Biomedical subjects

A W Neff

Publications and source records attributed to A W Neff.

At least 19 recordsLinked to original sources

PubMed2003

Effects of fluoride on Xenopus embryo development.

Fluoride was first associated with fetal malformation shortly after water fluoridation was initiated in the 1940s. Since many chemicals can interact directly with the embryo to cause malformation, the effects of fluoride on embryonic and fetal development were investigated. The effects of sodium fluoride on the development of frog embryos were studied under conditions described by the Frog Embryo Teratogenesis Assay-Xenopus (FETAX), a screening assay for teratogens. The most prominent malformations caused by sodium fluoride are reduction in the head-tail lengths and dysfunction of the neuromuscular system of the tadpoles. The values for LC50, EC50, and minimal concentration to inhibit growth (MCIG) of sodium fluoride met the limits established for a teratogen in frog embryos, showing that sodium fluoride is a direct acting teratogen on developing embryos. Since FETAX has a high degree of success in identifying mammalian teratogens, the observed teratogenic action of sodium fluoride on frog embryos would indicate a strong possibility that sodium fluoride may also act directly on developing mammalian fetuses to cause malformation.

Animals↗

PubMed2002

Evaluation of the developmental toxicity of crocetin on Xenopus.

Crocetin, a carotenoid isolated from the spice saffron, has been found to be effective in treating certain types of cancer treatable with all-trans retinoic acid (ATRA). ATRA, however, is a potent teratogen, and the possibility that crocetin may also be a teratogen becomes an important medical issue. The teratogenic potential of crocetin and ATRA were evaluated in frog (Xenopus) embryos. The data show that crocetin is a teratogen, but far less potent than ATRA. This suggests that crocetin may be a safer alternative to treat ATRA-sensitive cancers in women of childbearing age.

Animals↗

PubMed2001

Human truncated Smad 6 (Smad 6s) inhibits the BMP pathway in Xenopus laevis.

A previously identified truncated form of the human Smad 6 gene containing a unique 12 amino acid motif at its N-terminus was studied. We have named this truncated form of the gene Smad 6s, for 'short-form', to distinguish it from the full-length form (Smad 6fl). Reverse transcription-polymerase chain reaction and immunohistochemistry revealed that Smad 6s has a unique pattern of expression in human coronary tissue and is upregulated in diseased heart tissue. We used the expression of human Smad 6s in Xenopus laevis as a model system to assess Smad 6s function. Injection of Smad 6fl RNA (4-cell embryos, 2 x ventral) produced tadpoles with partial secondary axes. In contrast, Smad 6s RNA injected in a similar manner produced tadpoles with a severe 'head-only' phenotype with no morphological appearance of a secondary axis. Mutant Smad 6s RNA lacking the unique 12 amino acids at the N-terminus of the Smad 6s isoform produced no embryonic phenotype, suggesting that this region is important in conferring biological activity. Ectodermal explant assays show that Smad 6s has activity consistent with being a BMP antagonist and can synergize with and enhance the activities of the activin and fibroblast growth factor pathways, all of which are novel findings in this study.

Animals↗

PubMed1998

Anterior structural defects by misexpression of Xgbx-2 in early Xenopus embryos are associated with altered expression of cell adhesion molecules.

The RNA of the noncluster homeobox gene, Xgbx-2, is localized during neurulation to a narrow band of tissue at the midbrain hindbrain boundary (anterior hindbrain). The localized expression of Xgbx-2 within the nervous system prompted us to assess its function during early development by injection of synthetic Xgbx-2 RNA into the animal pole region of both dorsal blastomeres at the four-cell stage. Injection of Xgbx-2 RNA leads to dose-dependent alterations in anterior dorsal structures. These defects include abnormal eye development including reduced and missing eyes, reduced or missing cement glands, and abnormal brain development. Additionally, coinjection with lineage label (either beta-galactosidase or green fluorescent protein) shows there is a dose-dependent misplacement of cells. These misplaced cells can be found in such locations as the blastocoele, gastrocoele, or ventricles in the brain. In some spawnings, misplaced cells are expelled from the embryo into the periviteline space. In general, the phenotype of Xgbx-2 RNA-injected embryos is strikingly similar to the phenotypes observed when dominant-negative RNA constructs of Ca2+-dependent cell-adhesion molecules are injected into similar regions of early embryos. Xgbx-2 misexpression enhanced the dissociation of animal hemisphere cells, and inhibited Ca2+-dependent cell adhesion in dissociated animal hemisphere cells in vitro. Additionally, when the expression of various calcium-dependent cadherins was tested, it was shown that misexpression of Xgbx-2 prevents N-cadherin expression during early neurulation. These observations suggest that the transcription factor, Xgbx-2, functions normally in the regionalization of the neural tube (specifically the anterior hindbrain) by regulating differential cell adhesion and subsequently cell identity.

Animals↗

PubMed1997

Autonomous neural axis formation by ectopic expression of the protooncogene c-ski.

The ski oncogene was originally isolated as an avian retroviral gene with the ability to induce quail embryonic cells to differentiate into muscle. Mice containing a chicken c-ski transgene exhibit postnatal hypertrophy of skeletal muscle. Xenopus ski (Xski) protein is maternal and present throughout early development. We show that overexpression of Xski RNA in Xenopus embryos results in the cell-autonomous induction of secondary neural axis formation. Injection of Xski RNA into prospective endodermal cells resulted in the formation of an ectopic neural tube-like structure and cells derived from the injected blastomeres populated the spinal cord. Injected Xski RNA was able to induce neural-specific gene expression directly in ectodermal explants in the absence of the expression of mesodermal markers. The widespread distribution of ski protein in the early gastrula embryo including the dorsal animal region supports a role for ski in neural axis formation in vivo.

Amino Acid Sequence↗

PubMed1996

Optomotor behaviour in Xenopus laevis tadpoles as a measure of the effect of gravity on visual and vestibular neural integration.

The ability of aquatic vertebrates to maintain their position requires integration of visual and vestibular sensory information. To understand better how aquatic animals integrate such information, we measured the optomotor behaviour of Xenopus laevis tadpoles raised in growth chambers in microgravity (< 10(-3)g), normal gravity (1 g), hypergravity (3 g) and on a slowly rotating clinostat (simulated microgravity). The goal of this research was to determine how development in an altered gravitational force field affects the visual- and vestibular-dependent behaviour of tadpoles. This research represents the first time that the optomotor behaviour of an organism raised from fertilization in microgravity has been tested. Significant differences were observed in the optomotor behaviour among the four gravity treatments. When first exposed to normal gravity, the microgravity-raised tadpoles exhibited the strongest (or most positive) optomotor behaviour, while the 3 g centrifuge tadpoles showed no optomotor response. Some abnormal behaviours (such as erratic swimming, lying motionless and abnormal swimming posture) were observed in the tadpoles raised in altered gravity on the initial day of testing. One day later, the tadpoles raised in hypergravity did not differ significantly in their optomotor behaviour from control tadpoles raised in normal gravity. However, tadpoles raised in microgravity still displayed an exaggerated optomotor response. One week after the tadpoles had been introduced to normal gravity, there was no longer a significant difference in optomotor behaviour among the different gravity treatments. This convergence of optomotor behaviour by tadpoles from the different treatment reflects the acclimation of their vestibular systems to normal gravity.

Animals↗

PubMed1996

Heart development and regeneration in urodeles.

Classical fate mapping and transplantation studies have yielded a rich embryological understanding of heart development in urodeles. Recent advances in understanding the molecular nature of many early developmental events can be applied to urodele heart development. In this review we examine urodele heart development from both morphological and molecular viewpoints. We focus primarily on cardiac induction, early cardiogenesis, and heart regeneration.

Animals↗

PubMed1995

Expression of the axolotl homologue of mouse chaperonin t-complex protein-1 during early development.

Molecular chaperones assist in the folding of proteins, but their role during development is not well understood. Here we report the temporal and spatial expression pattern of the axolotl homologue of mouse chaperonin TCP-1 during normal amphibian embryogenesis and in several models of abnormal embryogenesis. A partial axolotl TCP-1 cDNA (646 bp; 519 coding bp) isolated by 3' RACE PCR shows considerable homology to mouse TCP-1. Developmental Northerns and RT-PCR analyses of whole axolot1 embryos revealed a low level of maternal TCP-1 transcripts in fertilized eggs. The maternal transcripts were down-regulated to a non-detectable level in early gastrulae. Zygotic TCP-1 transcripts first appeared during gastrulation. They were mainly expressed in mid-neurula and later stage embryos. Whole-mount in situ hybridization studies showed abundant TCP-1 transcripts in the blastopore at the mid-gastrula stage and in the brain and spinal cord beginning at the neurula stage, and in the somites (myotomes) at the tailbud stage. RT-PCR analysis of TCP-1 expression in axolotl embryos treated with either high salt (causing exogastrulation) or ultraviolet (UV) irradiation (causing ventralization) substantiated the correlation between TCP-1 expression and neural and somitic development. In high salt-induced exogastrulated embryos TCP-1 mRNA was detectable in the ectoderm part (with neural tissues) but not in its exogastrulated endoderm part. Lower levels of TCP-1 expression were detected in UV-irradiated, ventralized embryos with smaller head and reduced neural and somitic tissues. Normal levels of TCP-1 expression were detected in embryos with double axes/heads. These studies provide strong evidence that at the transcript level axolotl chaperonin TCP-1 is regulated both temporally and spatially during embryogenesis, especially in neural and somitic development.

Ambystoma↗

PubMed1995

Cloning and expression of the axolotl proto-oncogene ski.

In vitro and in vivo overexpression studies have demonstrated that the c-ski proto-oncogene can influence proliferation, morphological transformation and myogenic differentiation. We report the isolation and expression of an axolotl (Ambystoma mexicanum) c-ski (aski) gene. Sequence analysis revealed a high degree of nucleotide and predicted amino acid (AA) homology with mammalian and anuran c-ski, showing the highest conservation to Xenopus laevis c-ski (74% nucleotide and 87% AA). Northern analysis showed that axolotl c-ski is expressed in unfertilized eggs and at increasing levels in embryos from blastula to tadpole stage. c-ski expression was also detected in larval limb muscle and in several stages of regenerating limb blastemas. These data indicate that axolotl c-ski is highly conserved among amphibians and mammals and suggests that it plays a role in urodele embryogenesis and limb regeneration.

Ambystoma↗

PubMed1995

Cardiac myosin heavy chain expression during heart development in Xenopus laevis.

Muscle-specific gene expression in the heart during Xenopus development was investigated using reverse transcription-polymerase chain reaction (RT-PCR) and whole-mount in situ hybridization to detect transcripts of the gene for the cardiac myosin heavy chain (CMHC). RT-PCR analysis determined that CMHC transcripts are present in the cardiac mesoderm at state 13, demonstrating that muscle-specific gene expression in the primitive myocardium has begun by the early neurula stage, approximately 30 h before the heart beat begins. Xenopus, therefore, is similar to amniotes and mammals in that cardiac precursor cells begin to express muscle-specific gene transcripts soon after commitment to the cardiac myocyte lineage. The earliest CMHC gene transcripts can be detected in the heart using whole-mount in situ hybridization is early tailbud stage 28, which coincides with the onset of heart tube morphogenesis. CMHC gene expression was also detected in skeletal muscle: RT-PCR analysis determined that CMHC transcripts are transiently expressed in the somite during the initial phases of skeletal muscle differentiation. Furthermore, CMHC mRNAs are expressed in a subset of head muscles of the feeding tadpole. CMHC gene expression is induced in ectodermal cells of the animal cap in blastula-stage embryos injected with synthetic MyoD or Myf5 RNA, suggesting that the CMHC gene contains regulatory elements that are responsive to the activity of those skeletal-muscle-specific transcription factors.

Amino Acid Sequence↗

PubMed1994

Overexpression of XMyoD or XMyf5 in Xenopus embryos induces the formation of enlarged myotomes through recruitment of cells of nonsomitic lineage.

The myogenic regulatory factors (MRFs) MyoD and Myf5 are the earliest described muscle-specific genes to be expressed in Xenopus development. To study the in vivo effects of overexpressing Xenopus MyoD and Myf5, synthetic RNAs were microinjected into single blastomeres of 2- to 32-cell stage Xenopus embryos. In vivo overexpression of these MRFs initiates the precocious and ectopic expression of actin and myosin. The effects of unilateral injection of either mRNA were indistinguishable; embryos injected at the 2-cell stage showed ipsilaterally enlarged cranial and anterior trunk myotomes composed of increased numbers of primary myotome myocytes. In addition, formation of ectopic muscle in lateral plate and neural tissue was observed. The MRF-induced effects persist through secondary myogenesis, with the enlarged cranial myotomes failing to undergo the normal program of degeneration. Experiments combining MRF RNA and lineage tracer injections showed that myotomal enlargement is due in part to the contribution of cells of nonsomitic lineage to the myotome, rather than to an increase in muscle precursor cell division. Overexpression of XMyoD and XMyf5 also affected the morphogenesis of the skin and the nervous system. These results reveal that overexpression of XMyoD or XMyf5 in vivo clearly influences the regulation of early myogenesis and the morphogenesis of skin and nervous tissue.

Animals↗

PubMed1994

Early development of Xenopus embryos is affected by simulated gravity.

Early amphibian (Xenopus laevis) development under clinostat-simulated weightlessness and centrifuge-simulated hypergravity was studied. The results revealed significant effects on (i) "morphological patterning" such as the cleavage furrow pattern in the vegetal hemisphere at the eight-cell stage and the shape of the dorsal lip in early gastrulae and (ii) "the timing of embryonic events" such as the third cleavage furrow completion and the dorsal lip appearance. Substantial variations in sensitivity to simulated force fields were observed, which should be considered in interpreting spaceflight data.

Animals↗

PubMed1994

The location of the third cleavage plane of Xenopus embryos partitions morphogenetic information in animal quartets.

Analysis of the developmental potential of animal quartets (the set of four animal blastomeres isolated from the 8-cell stage Xenopus embryo) provided insight into the manner in which morphogenetic information is distributed along the animal-vegetal axis. Gravity treatments were employed to alter the partitioning plane. Animal quartets isolated from embryos exposed to simulated weightlessness had larger animal blastomeres, and they formed structures such as a groove and a protrusion more often than 1g-control animal quartets. Animal quartets with an unusual non-horizontal third cleavage plane were also found to have a higher frequency of protrusion formation than animal quartets with a typical horizontal cleavage plane. The increase in the frequency seen in simulated weightlessness animal quartets was not due to their increased size. Fusing two animal quartets isolated from hypergravity (3g) exposed embryos (small blastomeres and low incidence of protrusions) did not affect the frequency of protrusion formation. Molecular analyses revealed that a partial induction was associated with the protrusion formation. Transcripts of the dorsal lip specific homeobox gene, goosecoid, and alpha-cardiac actin were detectable by PCR amplification in the animal quartet with a protrusion, and alpha-cardiac actin mRNA was found by whole-mount in situ hybridization to be localized in the protrusion. Taken together, all these results are consistent with the notion that both animal and vegetal information is necessary for normal development and the partitioning of morphogenetic information into animal quartets results in gravity-dependent differential morphogenesis and gene regulation.

Actins↗

PubMed1993

Early amphibian (anuran) morphogenesis is sensitive to novel gravitational fields.

Anuran amphibian embryos (Xenopus laevis and Rana dybowskii) are sensitive to novel gravitational fields. Under simulated weightlessness, (i) the location of the first horizontal cleavage furrow was shifted toward the vegetal pole at the eight-cell stage; (ii) the position of the blastocoel was more centered, and the number of cell layers in the blastocoel roof was increased at the blastula stage; (iii) the dorsal lip appeared closer to the vegetal pole at the gastrula stage; and (iv) head and eye dimensions were enlarged at the hatching tadpole stage. Effects of simulated hypergravity were opposite to those of simulated weightlessness, except that hypergravity, unlike simulated weightlessness, reduced the number of primordial germ cells in feeding tadpoles. Despite those dramatic differences in the early embryogenesis, tadpoles at the feeding stage are largely indistinguishable from controls.

Animals↗

PubMed1992

Understanding the organization of the amphibian egg cytoplasm: gravitational force as a probe.

A combination of hypergravity (centrifugation) and hypogravity (clinostat) studies have been carried out on amphibian (frog, Xenopus) eggs. The results reveal that the twinning caused by centrifugation exhibits substantial spawning to spawning variation. That variation can be attributed to the apparent viscosity of the egg's internal cytoplasm. Simulated hypogravity results in a relocation of the egg's third (horizontal) cleavage furrow, towards the equator. Substantial egg-to-egg variation is also observed in this "cleavage effect". For interpreting spaceflight data and for using G-forces as probes for understanding the egg's architecture the egg variation documented herein should be considered.

Animals↗

PubMed1992

Altering the position of the first horizontal cleavage furrow of the amphibian (Xenopus) egg reduces embryonic survival.

The animal/vegetal cleavage ratio (AVCR), defined as the ratio of the height of the animal blastomere to the height of the Xenopus embryo at the 8 cell stage, can be shifted by placing embryos in novel gravitational fields: clinostating (microgravity simulation) increases AVCR, and centrifugation (hypergravity simulation) reduces AVCR. This report contributes to an understanding of the subcellular mechanism responsible for the furrow relocation and assesses its significance. Embryo inversion and D2O immersion were found to increase AVCR, and cold shock was found to reduce AVCR. Based on the additive or antagonistic effects of combined treatments, it is postulated that the primary cause of AVCR changes is an alteration in the distribution of yolk platelets and the rearrangement of microtubule arrays. Embryos with a decreased AVCR exhibited reduced survival in early developmental stages, indicating serious difficulties in cleavage, blastulation and/or gastrulation. Cold-shocked embryos with a reduced AVCR could be rescued by D2O pretreatment or clinostating, an observation which supports the notion that changes accompanying AVCR modifications represent the primary cause of the reduction in percent survival.

Animals↗

PubMed1990

Bifurcation of the amphibian embryo's axis: analysis of variation in response to egg centrifugation.

Xenopus embryos have been reported to vary widely in their developmental response to centrifugation. Variation in response to centrifugation, as measured by embryo survival and twinning of axial structures, was monitored different spawnings of Xenopus laevis eggs. A convenient method for quantifying the egg cytoplasm's potential for displacement in a centrifugal field was employed. It involved testing small batches of eggs from each spawn under carefully controlled conditions for displacement of the cytoplasm while held in an inverted orientation. The cytoplasmic immobility (CIM) values thus measured in samples from each spawn were correlated with the spawning's developmental success (survival of embryos) and the twinning frequency after centrifugation. Those spawnings with high CIM values (i.e. a rigid or stiff cytoplasm) had the highest survival rates and the lowest frequency and severity of twinning in centrifuged eggs. Variations in CIM account for the broad variation in response to centrifugation previously noted in several reports and further emphasize the role cytoplasmic compartments play vis-à-vis egg organization and early embryonic pattern formation.

Animals↗

PubMed1989

Autonomous death of amphibian (Xenopus laevis) cranial myotomes.

The death of cranial myotomes during Xenopus laevis embryogenesis is employed as a model system to study programmed cell death. The first primary myotomes to differentiate and functionally develop are in the occipital region of the embryonic head. Between stages 27 (tailbud) and 48 (feeding tadpole), they degenerate and disappear in a craniocaudal sequence. Descriptive and experimental studies were undertaken to establish whether this apparent cell (myotome) death program is autonomous or whether it depends on interactions with surrounding tissues (e.g., otic vesicle). Removal of the adjacent otic vesicle did not affect cranial myotome death. Likewise, grafting the otic vesicle to a novel location along the somite file did not induce local myotome degeneration (death). Cranial myotome primordia grafted into the trunk region degenerated on schedule. Trunk myotome primordia grafted to the cranial myotome location did not degenerate. It is therefore concluded that the cranial myotome death program has become autonomous by the time the cranial myotomes reach the developmental stage of segmentation.

Animals↗