Biomedical subjects

R M Grainger

Publications and source records attributed to R M Grainger.

At least 19 recordsLinked to original sources

PubMed1997

Anteroposterior neural tissue specification by activin-induced mesoderm.

The transforming growth factor beta superfamily member, activin, is able to induce mesodermal tissues in animal cap explants from Xenopus laevis blastula stage embryos. Activin can act like a morphogen of the dorsoventral axis in that lower doses induce more ventral, and higher doses more dorsal, tissue types. Activin has also previously been reported to induce neural tissues in animal caps. From cell mixing experiments it was inferred that this might be an indirect effect of induced mesoderm signaling to uninduced ectoderm. Here we demonstrate directly that neural tissues do indeed arise by the action of induced mesoderm on uninduced ectoderm. Dorsal mesoderm is itself subdivided into posterior and anterior domains in vivo, but this had not been demonstrated for induced mesoderm. We therefore tested whether different concentrations of activin recreate these different anteroposterior properties as well. We show that the anteroposterior positional value of induced mesoderm, including its neuroinductive properties, depends on the dose of activin applied to the mesoderm, with lower doses inducing more posterior and higher doses giving more anterior markers. We discuss the implications of these results for patterning signals and the relationship between anteroposterior and dorsoventral axes.

Activins↗

PubMed1997

Dorsal-ventral patterning during neural induction in Xenopus: assessment of spinal cord regionalization with xHB9, a marker for the motor neuron region.

While the role of the notochord and floor plate in patterning the dorsal-ventral (D/V) axis of the neural tube is clearly established, relatively little is known about the earliest stages of D/V regionalization. In an effort to examine more closely the initial, preneural plate stages of regionalization along the prospective D/V neural axis, we have performed a series of explant experiments employing xHB9, a novel marker of the motor neuron region in Xenopus. Using tissue recombinants and Keller explants we show that direct mesodermal contact is both necessary and sufficient for the initial induction of xHB9 in the motor neuron region. We also show that presumptive neural plate explants removed as early as midgastrulation and cultured in isolation are already specified to express xHB9 but do so in an inappropriate spatial pattern while identical explants are specified to express the floor plate marker vhh-1 with correct spatial patterning. Our data suggest that, in addition to floor plate signaling, continued interactions with the underlying mesoderm through neural tube stages are essential for proper spatial patterning of the motor neuron region.

Amino Acid Sequence↗

PubMed1997

Defining intermediate stages in cell determination: acquisition of a lens-forming bias in head ectoderm during lens determination.

Cell determination in vertebrates requires integration of many events, although the mechanisms controlling the different stages in this process are poorly understood. While studies of lens determination have helped define some of these stages, we know very little about the intermediate steps involved in the commitment of ectoderm to lens formation. Lens determination begins during gastrulation when ectoderm is briefly competent to respond to lens-inducing signals and progresses to a point, at the neural tube stage, when the presumptive lens ectoderm is specified. Between these two stages important regulatory genes are activated in the presumptive lens ectoderm, including the transcription factor Pax-6, and transplantation experiments presented here suggest that the presumptive lens ectoderm is becoming "biased" toward lens formation. We show that competent ectoderm from Xenopus laevis embryos forms well-differentiated lenses in most cases when transplanted to the presumptive lens area of neural plate stage hosts, where the lens-inductive environment is strong. When placed into later, neural tube stage hosts, optimally competent ectoderm does form small lenses in about half the cases, but the overall response is much weaker. Even in this weakly inducing environment, however, lens ectoderm that is part way through the inductive process (at the neural plate stage) is shown to have a lens-forming bias, since it forms well differentiated lenses in nearly all cases. While we find that ectoderm surrounding the lens-forming area at neural plate stages does not have a lens-forming bias, non-lens head ectoderm at the neural tube stage does, suggesting that a large region of head ectoderm is biased during neurulation. Using Rana palustris embryos, a species used in the earliest lens induction studies, we were also able to show that the optic vesicle can induce lenses in non-lens head ectoderm at neural tube stages. These results lead us to refine the definition of lens cell determination and provide a context that should allow clarification of determination mechanisms.

Animals↗

PubMed1997

Neural induction and antero-posterior patterning in the amphibian embryo: past, present and future.

Neural induction and patterning in competent ectoderm occurs during gastrula and early neurula stages in response to signals from dorsal mesoderm. The earliest views of antero-posterior (A-P) patterning were modified beginning in the 1930s, as complexities concerning the timing of the pattern-forming process and potential sources of the patterning signals were revealed. In the 1950s and 1960s several different models for A-P patterning were proposed, all of which, however, bear a number of similarities, including a two-component system for generating A-P axial information in the embryo. Early attempts to identify neural-inducing molecules were largely unsuccessful due to technical limitations in biochemical analyses and concerns about assaying neural responses. The advent of modern molecular genetic technology has permitted more precise tests of a number of classic observations about the timing of A-P patterning and the sources of patterning signals. While some early observations have been confirmed, a number of new concepts have emerged in recent years, particularly concerning the source of patterning signals in the embryo. Striking progress has been made in identifying putative neural-inducing molecules, and recent experiments have begun to suggest how these might contribute to A-P patterning. While the successes in recent years have been revealing, many of the classic issues concerning neural induction and patterning remain essentially as they were when first defined many decades ago. The power of modern molecular genetics, however, should permit many of these issues to be significantly clarified in the decades to come.

Amphibians↗

PubMed1996

Inductive processes leading to inner ear formation during Xenopus development.

This study examines the spatial and temporal attributes of inner ear induction in Xenopus embryos. These results are compared to recent experiments concerning lens induction to assess whether head sensory structures share common ontogenetic features. Ectoderm from different regions and stages was transplanted to the presumptive ear region of hosts of either early (neural plate) or late (neural tube) stages. Explants of the presumptive ear ectoderm were also taken from embryos at these stages to establish the time of otic ectoderm specification. We find that ectodermal competence for otic vesicle formation extends through neural plate stages, far longer than for lens formation. Otic vesicle specification occurs substantially earlier, at neural plate stages, than lens specification. Competent ectoderm forms otic vesicles in a high fraction of cases when exposed to the ear-inducing environment of either neural plate stages or neural tube stages, a result which contrasts with lens induction where the neural tube stage embryo provides a much weaker inducing environment than earlier stages. Otic vesicles induced in neural tube stage hosts are primarily in contact with presumptive hindbrain, suggesting that this neural tissue may be sufficient for otic vesicle induction. These studies reveal overall similarities between lens and inner ear induction, but sufficient differences to propose that some facets of determination of these sensory tissues may occur by independent mechanisms and not via a common developmental state.

Animals↗

PubMed1996

Lens induction in axolotls: comparison with inductive signaling mechanisms in Xenopus laevis.

Amphibian lens induction is an embryonic process whose broad outlines are conserved between anurans and urodeles; however, it has been argued that some aspects of this process differ significantly between even closely related species. Classical embryologists concluded that in some species direct contact between the optic vesicle and ectoderm was both necessary and sufficient to induce the ectoderm to form a lens, while in other species tissues other than the optic vesicle induce lens formation. Recent studies of lens induction in Xenopus have argued that lens induction may be more conserved evolutionarily than was previously thought and that the different conclusions reached in the classical literature may be due more to experimental methodology than to actual differences in the process of lens induction. We have tested this hypothesis by examining the timing of lens induction in the axolotl and the ability of various tissues to induce lenses in explant cultures. We find that, despite the evolutionary divergence between Xenopus and Ambystoma, the mechanism of lens specification is substantially similar in the two species. These results support the hypothesis that the mechanism of lens induction is evolutionarily conserved among amphibians.

Ambystoma↗

PubMed1994

Xenopus gamma-crystallin gene expression: evidence that the gamma-crystallin gene family is transcribed in lens and nonlens tissues.

Crystallins, the major gene products of the lens, accumulate to high levels during the differentiation of the vertebrate lens. Although crystallins were traditionally thought to be lens specific, it has recently been shown that some are also expressed at very low levels in nonlens tissues. We have examined the embryonic expression pattern of gamma-crystallins, the most abundant crystallins of the embryonic lens in Xenopus laevis. The expression profile of five Xenopus gamma-crystallin genes mirrors the pattern of lens differentiation in X. laevis, exhibiting on average a 100-fold increase between tailbud and tadpole stages. Four of these genes are also ubiquitously expressed outside the lens at a very low level, the first demonstration of nonlens expression of any gamma-crystallin gene; expression of the remaining gene was not detected outside the head region, thus suggesting that there may be two classes of gamma-crystallin genes in X. laevis. Predictions regarding control mechanisms responsible for this dual mode of expression are discussed. This study raises the question of whether any crystallin, on stringent examination, will be found exclusively in the lens.

Animals↗

PubMed1993

Characterization of Xenopus laevis gamma-crystallin-encoding genes.

In order to gain insight into crystallin (Cry)-encoding gene (cry) evolution and developmental function, we have determined the gene structure and sequence of several Xenopus laevis gamma-cry. These encode the most abundant Cry in the embryonic lens. Four of the X. laevis gamma-cry, which are part of a multigene family, were isolated from a X. laevis genomic library and demonstrated to have the same gene structure as gamma-cry from other vertebrates, thereby providing further evidence that the split between beta and gamma members of the beta gamma cry family occurred relatively early in evolution. Sequence comparisons indicate that these X. laevis genes share 88-90% nucleotide sequence identity in the protein coding regions, which is slightly higher than the identity observed between gamma-cry of other species. The 5' upstream regions of X. laevis gamma-cry contain a few short stretches of homology and one putative promoter element conserved among all cry genes but lack other regions common to gamma-cry promoters from other organisms. The deduced amino acid sequences of all four genes and one cDNA suggest that the structure of X. laevis gamma-Cry is highly conserved with that of other vertebrate gamma-Cry, as deduced from the known three-dimensional structure of bovine gamma B Cry.

Amino Acid Sequence↗

PubMed1993

Early opsin expression in Xenopus embryos precedes photoreceptor differentiation.

The visual pigment which serves as the first step in the phototransduction cycle in vertebrate rod cells consists of a retinal chromophore which is linked to the transmembrane protein, opsin. Opsin genes have been isolated from a number of different organisms and studies have shown opsin to be developmentally regulated with both mRNA and protein expression associated with the morphological differentiation of photoreceptor cells. Due to its potential utility as a marker for rod photoreceptor determination in studies of retinal tissue interactions, and because no amphibian opsin genes have as yet been cloned, we isolated cDNA clones of the Xenopus laevis opsin gene. Sequence analysis shows that within the coding region Xenopus opsin shares a high degree of identity with other rod opsin genes, except at the C-terminal where it more closely resembles the mammalian color opsins. A developmental analysis, on the other hand, reveals that Xenopus opsin transcripts are detectable in a retina-specific fashion early in retinal development. Using in situ hybridization we find that Xenopus opsin mRNA is initially restricted to a few isolated cells in the presumptive photoreceptor layer which express the gene at relatively high levels. This suggests that rod photoreceptor determination occurs in single cells, and that the mechanisms controlling opsin expression in Xenopus are initiated well before any evidence of morphological differentiation.

Amino Acid Sequence↗

PubMed1993

A Xenopus homebox gene defines dorsal-ventral domains in the developing brain.

One of the distinguishing features of vertebrate development is the elaboration of the anterior neural plate into forebrain and midbrain, yet little is known about the early tissue interactions that regulate pattern formation in this region or the genes that mediate these interactions. As an initial step toward analyzing the process of regionalization in the anterior-most region of the brain, we have screened an anterior neural cDNA library for homeobox clones and have identified one which we have called XeNK-2 (Xenopus NK-2) because of its homology to the NK-2 family of homeobox genes. From neurula stages, when XeNK-2 is first detectable, through hatching stages, XeNK-2 mRNA is expressed primarily in the anterior region of the brain. By swimming tadpole stages, XeNK-2 expression resolves into a set of bands positioned at the forebrain-midbrain and the midbrain-hindbrain boundaries, after which XeNK-2 transcripts are no longer detectable. In addition to localized expression along the anterior-posterior axis, XeNK-2 may also play a role in the process of regionalization along the dorsal-ventral axis of the developing brain. At all stages examined, XeNK-2 mRNA is restricted to a pair of stripes that are bilaterally symmetrical in the ventral-lateral region of the brain. To begin to identify the tissue interactions that are required for the proper spatial and temporal localization of XeNK-2, we have performed a series of explant experiments. Consistent with earlier work showing that the A/P axis is not fixed at mid-gastrula stages, we show that XeNK-2 expression is activated when assayed in gastrula stage explants taken from any region along the entire A/P axis and that the tissue interactions necessary to localize XeNK-2 along the A/P axis are not completed until later neurula stages.

Amino Acid Sequence↗

PubMed1993

Interphotoreceptor retinoid-binding protein (IRBP), a major 124 kDa glycoprotein in the interphotoreceptor matrix of Xenopus laevis. Characterization, molecular cloning and biosynthesis.

We have demonstrated that the neural retina of Xenopus laevis secretes into the extracellular matrix surrounding the inner and outer segments of its photoreceptors a glycoprotein containing hydrophobic domains conserved in mammalian interphotoreceptor retinoid-binding proteins (IRBPs). The soluble extract of the interphotoreceptor matrix contains a 124 kDa protein that cross-reacts with anti-bovine IRBP immunoglobulins. In vitro [3H]fucose incorporation studies combined with in vivo light and electron microscopic autoradiographic analysis, showed that the IRBP-like glycoprotein is synthesized by the neural retina and secreted into the interphotoreceptor matrix. A 1.2 kb Xenopus IRBP cDNA was isolated by screening a stage 42 (swimming tadpole) lambda Zap II library with a human IRBP cDNA under low-stringency conditions. The cDNA hybridizes with a 4.2 kb mRNA in adult Xenopus neural retina, tadpole heads as well as a less-abundant mRNA of the same size in brain. During development, IRBP and opsin mRNA expression correlates with photoreceptor differentiation. The translated amino acid sequence of the Xenopus IRBP clone has an overall 70% identity with the fourth repeat of the human protein. Sequence alignment with the four repeats of human IRBP showed three highly conserved regions, rich in hydrophobic residues. This focal conservation predicts domains important to the protein's function, which presumably is to facilitate the exchange of 11-cis retinal and all-trans retinol between the pigment epithelium and photoreceptors, and to the transport of fatty acids through the hydrophilic interphotoreceptor matrix.

Amino Acid Sequence↗

PubMed1992

Embryonic lens induction: shedding light on vertebrate tissue determination.

The principle of embryonic induction was defined by early studies of lens determination, and because of the relative simplicity of the developing lens and its interaction with presumptive retinal tissue it has been a favored system for examining mechanisms of induction. Recent studies have led to substantial alterations of the classic model for this process, introducing several elements that significantly refine our view of vertebrate tissue determination.

Animals↗

PubMed1992

A labile period in the determination of the anterior-posterior axis during early neural development in Xenopus.

The process by which the vertebrate central nervous system acquires its regional properties remains a central problem in developmental biology. It is generally argued that at early gastrula stages the dorsal mesoderm possesses precise anterior-posterior positional information, which is subsequently imparted to the overlying ectoderm. However, using regionally specific gene probes to monitor regional responses in Xenopus embryos, we find that anterior-posterior properties are not fixed until early neurula stages. During gastrulation the regional inducing capacities of the dorsal mesoderm as well as the regional responses of the presumptive neural ectoderm are activated along the entire anterior-posterior axis when these properties are assayed in recombinant and explant experiments, respectively. Restriction of regional inducing capacity in the mesoderm and responsiveness in the neural ectoderm occur only at neural plate stages.

Animals↗

PubMed1992

Vertebrate eye development.

Vertebrate eye determination is mediated by a series of inductive interactions that have now been more precisely defined with the use of regional markers. Analyses of the genes responsible for eye mutations and the cloning of genes delimiting spatial domains within the developing eye have begun to elucidate the molecular basis of this process.

Animals↗

PubMed1992

Recent progress on the mechanisms of embryonic lens formation.

Formation of the lens during embryonic development depends on tissue interactions as shown clearly both from teratological data and from extensive experimental analysis. Recent work has, however, altered our view of the importance of particular tissue interactions for lens formation. While earlier work emphasises the role of the optic vesicle in lens induction, more recent studies argue that lens-inducing signals important for determination act before optic vesicle formation. Evidence is given for a four stage model in which ectoderm first becomes competent to respond to lens inducers. It then receives inductive signals, at least in part emanating from the anterior neural plate, so that it gains a lens-forming bias and subsequently becomes specified for lens formation. Complete lens differentiation does require signals from the optic vesicle, and in addition an inhibitory signal from head neural crest may suppress any residual lens-forming bias in head ectoderm adjacent to the lens.

Animals↗

PubMed1991

Delta-crystallin gene expression and patterns of hypomethylation demonstrate two levels of regulation for the delta-crystallin genes in embryonic chick tissues.

In this study we address two questions regarding the control of delta-crystallin gene expression in chick embryos. First we have determined whether delta-crystallin mRNA is found outside of the developing lens, in which it is the predominant mRNA. We find that this mRNA can be detected, although at relatively low levels, in all embryonic tissues we have examined (from the definitive streak stage onward). This low level of transcription may be related to a second function for one or both of the delta-crystallin genes: both genes have a high degree of sequence identity to the enzyme argininosuccinate lyase. This result led us to a second set of experiments in which we reevaluated the possible role of hypomethylation in the expression of the delta-crystallin genes. Previous work showed that particular HhaI and HpaII sites in the crystallin genes undergo hypomethylation early in the process of lens differentiation when there is a burst of delta-crystallin mRNA accumulation. We not find that these sites remain methylated in nonlens tissues, implying that they cannot be required for the delta-crystallin gene activity found in these tissues. Other sites are constitutively hypomethylated, however, and may be functionally linked to this low level of gene activity. From an analysis of the kinetics of the developmentally regulated hypomethylation of HhaI and HpaII sites we also find that complete hypomethylation of these sites is not required for activating high levels of delta-crystallin transcription during lens differentiation. We do find, however, that these sites approach a fully hypomethylated state later in the lens differentiation process. Our analyses of mRNA levels and hypomethylation together lead us to propose that the delta-crystallin genes are regulated by two different mechanisms, one that leads to high levels of expression in the lens and the other which is responsible for low level expression in all other tissues in the chick embryo.

Animals↗

PubMed1991

Homeogenetic neural induction in Xenopus.

Neural induction is known to involve an interaction of ectoderm with dorsal mesoderm during gastrulation, but several kinds of studies have argued that competent ectoderm can also be neutralized via an interaction with previously neuralized tissue, a process termed homeogenetic neural induction. Although homeogenetic neural induction has been proposed to play an important role in the normal induction of neural tissue, this process has not been subjected to detailed study using tissue recombinants and molecular markers. We have examined the question of homeogenetic neural induction in Xenopus embryos, both in transplant and recombinant experiments, using the expression of two neural antigens to assay the response. When ectoderm that is competent to be neuralized is transplanted to the region adjacent to the neural plate of early neurula embryos, it forms neural tissue, as assayed by staining with antibodies against the neural cell adhesion molecule, N-CAM. Transplants to the ventral region, far from the neural plate, do not express N-CAM, indicating that neuralization is not occurring as a result of the transplantation procedure itself. Because this response might be occurring as a result of interactions of ectoderm with either adjacent neural plate tissue, or with underlying dorsolateral mesoderm, recombinant experiments were performed to determine the source of the neuralizing signal. Ectoderm cultured in combination with neural plate tissue alone expresses neural markers, while ectoderm cultured in combination with dorsolateral mesoderm does not. We conclude that neural tissue can homeogenetically induce competent ectoderm to form neural tissue and argue that this induction occurs via planar signaling within the ectoderm, a mechanism that, in normal development, may be involved in interactions within presumptive neural ectoderm or in specifying structures that lie near the neural plate.

Animals↗

PubMed1991

Changes in neural and lens competence in Xenopus ectoderm: evidence for an autonomous developmental timer.

The ability of a tissue to respond to induction, termed its competence, is often critical in determining both the timing of inductive interactions and the extent of induced tissue. We have examined the lens-forming competence of Xenopus embryonic ectoderm by transplanting it into the presumptive lens region of open neural plate stage embryos. We find that early gastrula ectoderm has little lens-forming competence, but instead forms neural tissue, despite its location outside the neural plate; we believe that the transplants are being neuralized by a signal originating in the host neural plate. This neural competence is not localized to a particular region within the ectoderm since both dorsal and ventral portions of early gastrula ectoderm show the same response. As ectoderm is taken from gastrulae of increasing age, its neural competence is gradually lost, while lens competence appears and then rapidly disappears during later gastrula stages. To determine whether these developmental changes in competence result from tissue interactions during gastrulation, or are due to autonomous changes within the ectoderm itself, ectoderm was removed from early gastrulae and cultured for various periods of time before transplantation. The loss of neural competence, and the gain and loss of lens competence, all occur in ectoderm cultured in vitro with approximately the same time course as seen in ectoderm in vitro. Thus, at least from the beginning of gastrulation onwards, changes in competence occur autonomously within ectoderm. We propose that there is a developmental timing mechanism in embryonic ectoderm that specifies a sequence of competences solely on the basis of the age of the ectoderm.

Animals↗