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R M Harland

Publications and source records attributed to R M Harland.

17 recordsLinked to original sources
PubMed1992

Expression cloning of noggin, a new dorsalizing factor localized to the Spemann organizer in Xenopus embryos.

We have cloned a cDNA encoding a novel polypeptide capable of inducing dorsal development in Xenopus embryos. RNA transcripts from this clone rescue normal development when injected into ventralized embryos and result in excessive head development at high doses. Therefore, we have named the cDNA noggin, noggin cDNA contains a single reading frame encoding a 26 kd protein with a hydrophobic amino-terminal sequence, suggesting that it is secreted. In Northern blot analysis this cDNA hybridizes to two mRNAs that are expressed both maternally and zygotically. Although noggin transcript is not localized in the oocyte and cleavage stage embryo, zygotic transcripts are initially restricted to the presumptive dorsal mesoderm and reach their highest levels at the gastrula stage in the dorsal lip of the blastopore (Spemann organizer). In the neurula, noggin is transcribed in the notochord and prechordal mesoderm. The activity of exogenous noggin RNA in embryonic axis induction and the localized expression of endogenous noggin transcripts suggest that noggin plays a role in normal dorsal development.

Amino Acid Sequence↗
PubMed1992

A protein expressed in the growth cones of embryonic vertebrate neurons defines a new class of intermediate filament protein.

We have isolated and characterized cDNAs that encode a protein expressed in the axons and growth cones of a subset of Xenopus embryonic neurons. The protein is also expressed in a subset of cells of the brain, including cells in even-numbered rhombomeres, the eye, and the heart. The sequence of the cDNA suggests that the protein belongs to a new class of neural-specific intermediate filaments. Both the RNA and the protein are expressed in the neurula and persist during embryogenesis in the brain, cranial nerves, and spinal cord. Because of the predicted structure of the protein, we have named it tanabin (from the Persian word for rope).

Amino Acid Sequence↗
PubMed1992

Localized expression of a Xenopus POU gene depends on cell-autonomous transcriptional activation and induction-dependent inactivation.

We have cloned a cDNA encoding a Xenopus POU domain protein, XLPOU91, which is expressed at high levels in gastrula embryos. XLPOU91 transcription initiates at the midblastula transition, and declines to low levels by late neurula stages. In early neurula embryos, XLPOU91 transcripts are enriched 35-fold in the most ventroposterior versus anterior regions. Initial transcriptional activation of the gene is cell autonomous; the gene is activated in dissociated gastrula stage embryos as well as in animal cap explants. Cell-cell communication is needed for proper temporal down-regulation of XLPOU91 expression in late neurula embryos; cell dissociation during blastula stages or removal of explants from the embryo prevents normal transcriptional shunt down. Explants treated with peptide growth factors (PGFs) mimic the normal temporal and spatial shut down in whole embryos. This negative regulatory pathway may be important for determining cell fate or maintaining an inducible state in the ventroposterior region of the embryo.

Amino Acid Sequence↗
PubMed1992

Ventral ectoderm of Xenopus forms neural tissue, including hindbrain, in response to activin.

The peptide growth factor Activin A has been shown to induce complete axial structures in explanted blastula animal caps. However, it is not understood how much this response to activin depends upon early signals that prepattern the ectoderm. We have therefore asked what tissues can be induced in blastula animal caps by activin in the absence of early dorsal signals. Using whole-mount in situ hybridization, we compare the expression of three neural markers, N-CAM, En-2 and Krox-20 in activin-treated ectoderm from control and ventralized embryos. In response to activin, both normal and ventralized animal caps frequently form neural tissue (and express N-CAM) and express the hindbrain marker Krox-20. However, the more anterior marker, En-2, is expressed in only a small fraction of normal animal caps and rarely in ventralized animal caps; the frequency of expression does not increase with higher doses of activin. In all cases En-2 and Krox-20 are expressed in coherent patches or stripes in the induced caps. Although mesoderm is induced in both control and ventralized animal caps, notochord is found in response to activin at moderate frequency in control caps, but rarely in ventralized animal caps. These results support the idea that in the absence of other signals, activin treatment elicits hindbrain but not notochord or anterior neural tissue; and thus, the anterior and dorsal extent of tissues formed in response to activin depends on a prior prepatterning or previous inductions.

Activins↗
PubMed1991

Expression of GATA-binding proteins during embryonic development in Xenopus laevis.

Proteins that recognize the core sequence GATA are important regulators of hematopoietic-specific gene transcription. We have characterized cDNAs encoding the Xenopus laevis homologues of three related transcription factors, designated GATA-1, -2, and -3. Comparative sequence analysis reveals strong conservation of the zinc-finger DNA-binding domain among all vertebrate GATA-binding proteins. GATA-2 and GATA-3 polypeptides are homologous throughout their entire sequences, whereas GATA-1 sequence is conserved only in the region responsible for DNA binding. In Xenopus, RNAs encoding GATA-binding proteins are expressed in both larval and adult erythroid cells. GATA-1, -2, and -3 RNAs are first detectable in early gastrula (Nieuwkoop developmental stage 11). This is earlier than the appearance of the early larval alpha T1 globin RNA (stage 15), beta T1 globin RNA (stage 26), or blood island formation (stage 30). The expression of GATA-1, -2, and -3 in early development may signal an early commitment of mesoderm to form hematopoietic tissue.

Amino Acid Sequence↗
PubMed1991

Injected Xwnt-8 RNA acts early in Xenopus embryos to promote formation of a vegetal dorsalizing center.

Expression cloning from a pool of gastrula cDNAs identified the Wnt family member Xwnt-8 as having dorsal axis-inducing activity in Xenopus embryos. Microinjected Xwnt-8 mRNA was able to rescue the development of a dorsally complete anterior-posterior axis in embryos ventralized by exposure to UV light. Axis induction was observed in embryos injected in either marginal or vegetal blastomeres at the 32-cell stage. Vegetal blastomeres receiving Xwnt-8 mRNA contributed progeny not to the induced dorsal axis, but to the endoderm, a result consistent with Xwnt-8 causing cells to act as a Nieuwkoop center (the vegetal-inducing component of normal dorsal axis formation), rather than as a Spemann organizer (the induced dorsal marginal zone component that directly forms the dorsal mesoderm). Xwnt-8, which is normally expressed ventrally in midgastrula and neurula embryos, appears to mimic, when injected, maternally encoded dorsal mesoderm-inducing factors that act early in development.

Animals↗
PubMed1991

Cephalic expression and molecular characterization of Xenopus En-2.

We have isolated and characterized cDNAs corresponding to the Xenopus En-2 gene. Comparison of amino acid sequences between the entire Xenopus En-2 and the Drosophila engrailed proteins confirms conservation of sequences inside as well as proximal to the homeobox and reveals a region of similarity towards the N terminus. Two transcripts encode the Xenopus En-2 protein. Both transcripts are regulated temporally in an identical fashion and are likely to be transcribed from two copies of the En-2 gene. We have also analyzed the distribution of the protein in the head tissue and in the dissected brain of tailbud stage embryos. In addition to the main band of expression at the midbrain-hindbrain boundary, we show that the protein is expressed in three novel areas: the mandibular arch, the optic tectum and the region of anterior pituitary.

Amino Acid Sequence↗
PubMed1991

Transient expression of XMyoD in non-somitic mesoderm of Xenopus gastrulae.

XMyoD is the earliest marker of muscle development in Xenopus embryos and is expressed in presumptive somites in the late gastrula. In the early gastrula, in situ hybridization showed XMyoD transcripts in precursors of both muscle and non-muscle mesoderm. Embryos ventralized by UV irradiation made no muscle, but expressed XMyoD transiently. Embryo explants that differentiated ventral mesoderm also expressed XMyoD transiently. These results show that the initiation of XMyoD expression is not sufficient to convert cells to muscle and suggest that XMyoD is expressed in response to a general mesodermalizing signal; expression is stabilized and enhanced only in muscle precursors that have received a dorsalizing signal.

Animals↗
PubMed1990

Region-specific neural induction of an engrailed protein by anterior notochord in Xenopus.

Anterior-specific neural induction can be assayed by means of an antibody that recognizes the Xenopus homeobox-containing protein En-2. The En-2 antigen is an excellent early marker, since it is present as a discrete band in the anterior neural plate of neurula embryos. Regional induction was assayed by combining dorsal mesoderm with competent ectoderm. Anterior notochord from the early neurula induced En-2 frequently, while posterior notochord induced En-2 less frequently. Presumptive somitic mesoderm and presumptive head mesoderm, though they induced neural tissue, were not strong inducers of En-2. Thus, anterior notochord may be the primary mesodermal tissue responsible for the patterning of the anterior neural plate.

Animals↗
PubMed1990

Endonucleolytic cleavage of a maternal homeo box mRNA in Xenopus oocytes.

We have identified a messenger RNA (mRNA) sequence from a Xenopus homeo box-containing gene that is the target for a sequence-specific endoribonuclease in vivo. Synthetic RNA transcribed from an allele of the maternal gene Xlhbox2B is efficiently cleaved when injected into Xenopus oocytes. The cleavage sequence lies between the protein-coding region and a 600-base 3'-untranslated region. Intermediates in degradation are readily observed: Both the 5' and 3' products of cleavage are recovered, thus showing that the cleavage activity is an endonuclease. When a 90-base region of the Xlhbox2B sequence is inserted into a second homeo box RNA that is normally stable, it is sufficient to confer an identical cleavage reaction on the hybrid RNA. The cleaved region contains a repeated sequence motif and is cut at multiple sites. Inhibition of translation does not affect the rate or extent of cleavage, while the coinjection of antisense RNA complementary to the 90-base region completely blocks the reaction. Because most mRNAs are not found on polysomes during oogenesis, translation-independent cleavage at such sites may provide a novel post-transcriptional mechanism to regulate the amount of mRNA available for embryogenesis.

Alleles↗
PubMed1990

Identification of a retinoic acid-sensitive period during primary axis formation in Xenopus laevis.

Retinoic acid (RA) is able to profoundly alter patterning of the primary body axis in embryos of the frog Xenopus laevis. The response to RA is dose-dependent, and leads to progressive truncation of the anteroposterior axis, with anterior structures most sensitive. Both mesodermal and ectodermal tissues are affected, and in vitro assays demonstrate that induced dorsal ectoderm is one direct target of RA. RA represses expression of anterior-specific genes and concomitantly induces expression of at least one posterior-specific gene. Resistance to RA is acquired gradually, during gastrula and early neurula stages, with posterior structures becoming resistant before anterior structures. These data demarcate in the embryo an anterior "domain," which may define the head rudiment and which transcends germ layers. RA can alter the axial pattern after its initial induction; thus, RA sensitivity defines a labile intermediate that occurs during axial patterning. These data suggest a possible role for RA in normal axis formation.

Animals↗
PubMed1990

Most of the homeobox-containing Xhox 36 transcripts in early Xenopus embryos cannot encode a homeodomain protein.

Multiple Xhox 36 transcripts accumulate in Xenopus embryos from gastrula to early tadpole stages. The transcripts were characterized by sequencing cDNA clones and by S1 protection and Northern (RNA) blotting of embryonic RNA with probes derived from the cDNAs. The Xhox 36 RNAs included unspliced precursor transcripts that accumulated in the embryonic nuclei, spliced transcripts that contained multiple stop codons in frame with the homeobox, and less abundant coding mRNAs. These transcripts were generated either by alternative splicing or multiple initiations from a single Xhox 36 gene. The sequence of a cDNA clone of the unspliced transcript showed that the intron contained a noncanonical 3' splice site. However, the intron was spliced efficiently when expressed from a plasmid injected into Xenopus embryos, suggesting that the inefficient splicing of the endogenous RNA is not due to the unusual 3' splice site. The accumulation of noncoding and unspliced transcripts suggests multiple levels of regulation in the embryonic expression of the Xhox 36 gene.

Animals↗
PubMed1990

Localization of specific mRNAs in Xenopus embryos by whole-mount in situ hybridization.

We have adapted a non-radioactive technique to detect localized mRNAs in whole-mount Xenopus embryos. Synthetic antisense RNA transcribed in the presence of digoxygenin-UTP is used as a probe and is detected via an anti-digoxygenin antibody. We show that localized mRNAs can be detected from late gastrula to tadpole stages and that high as well as low abundance RNAs can be detected. The method was tested on muscle actin and alpha-globin RNAs, whose localization has previously been characterized. In addition, we used the method to determine the distribution of XA-1 RNA, an anterior ectoderm-specific RNA, which we show is expressed in the periphery of the cement gland as well as in the region of the hatching gland. The sequence of an XA-1 cDNA predicts a protein rich in proline and histidine.

Alpha-Globulins↗
PubMed1989

Expression of an engrailed-related protein is induced in the anterior neural ectoderm of early Xenopus embryos.

We have used a monoclonal antibody directed against the C-terminus of the Drosophila invected homeodomain to detect a nuclear protein in brain cells of Xenopus laevis embryos. We refer to this antigen as the Xenopus EN protein. The EN protein is localized at midneurula stage to a band of cells in the anterior portion of the neural plate, on each side of the neural groove. Later in development, the expression coincides with the boundary of the midbrain and hindbrain, and persists at least to the swimming tadpole stage. These properties make the EN protein an excellent molecular marker for anterior neural structures. In embryos where inductive interactions between mesodermal and ectodermal tissues have been perturbed, the expression of the EN protein is altered; in embryos that have been anterodorsalized by LiCl treatment, the region that expresses the EN protein is expanded, but still well organized. In ventralized UV-irradiated embryos, the absence of the protein is correlated with the absence of anterior neural structures. In extreme exogastrulae, where the contacts between head mesoderm and prospective neurectoderm are lost, the EN protein is not expressed.

Animals↗
PubMed1987

Posterior expression of a homeobox gene in early Xenopus embryos.

The homeobox containing transcript Xhox-36 is expressed exclusively in the posterior mesoderm and ectoderm of early Xenopus embryos. Therefore, the transcript shows region-specific rather than tissue-specific expression in the gastrula and neurula, a time when cells are becoming committed to defined fates. Exposure of early embryos to LiCl, which shifts posterior cells to more anterior fates, reduces the abundance of this posterior-specific transcript. In contrast, embryos ventralized by u.v. treatment express normal levels of the transcript, implying that expression of the gene is not absolutely linked to dorsal cell identity. The sequence of a full-length cDNA corresponding to this transcript predicts a homeodomain-containing protein of 209 amino acids.

Amino Acid Sequence↗