Early Neurula Stage Research Articles

Expression and functions of FGF-3 in Xenopus development.

We have analyzed the expression pattern of the Xenopus FGF-3 gene during early development and examined its biological activity in three different bioassays using Xenopus embryos. We show that from the early gastrula stage there is a domain of expression around the blastopore which becomes a posterior domain as the blastopore closes. An anterior ectodermal domain becomes detectable from mid-gastrula stages in the prospective hind-brain, and there are several later domains of expression: the midbrain-hindbrain junction, the otocyst, the pharyngeal pouches and the tailbud region. By using double whole-mount in situ hybridizations we show that the XFGF-3 expression in the brain is dynamically regulated both in time and space during development. The anterior domain of early neurula stage embryos corresponds to the prospective rhombomeres 3-5. By the time the neural tube is closed, XFGF-3 expression is restricted to r4 and later a new domain of expression is established at the midbrain/hindbrain junction. In addition, we show that, despite its difference in receptor specificity, XFGF-3 can induce the formation of mesoderm from animal caps similarly to other FGFs. It also displays a posteriorizing activity on whole embryos similar to other FGFs. Although the absence of maternal expression makes it unlikely that XFGF-3 is involved in mesoderm induction in vivo, its posterior domain of expression during gastrulation and its posteriorizing activity suggests that it participates in the maintenance of mesodermal gene expression and in the FGF mediated patterning of the anteroposterior axis during gastrulation.

Evidence for non-axial A/P patterning in the nonneural ectoderm of Xenopus and zebrafish pregastrula embryos.

Recent studies in early Xenopus and zebrafish embryos have demonstrated that posteriorizing, non-axial signals arising from outside the organizer (or shield) contribute to A/P patterning of the neural axis, in contradiction to the classical Spemann model in which such signals were proposed to be solely organizer derived. Our studies on the early expression of the transcription factors GATA-2 and 3 in both Xenopus and zebrafish nonneural ectoderm lend support to the existence of such non-axial signaling in the A/P axis. Thus we find that the earliest expression of GATA-2 and 3 is located in nonneural ectoderm and is strongly patterned in a graded manner along the A/P axis, being high anteriorly and absent from the most posterior regions. This results by early neurula stages in three broad zones: an anterior region which is positive for both GATA-2 and 3, a middle region which is positive for GATA-2 alone and a posterior region in which neither gene is expressed. These regions correspond to head, trunk and tail ectoderm and may represent the beginnings of functional segmentation of nonneural ectoderm, as suggested in the concept of the 'ectomere'. We find that A/P patterning of GATA expression in nonneural ectoderm may occur as early as late blastula/early gastrula stages. We investigate which posteriorizing signals might contribute to such distinct non axial ectodermal patterning in the A/P axis and provide evidence that both FGF and a Wnt family member contribute towards the final A/P pattern of GATA expression in nonneural ectoderm.

Two phases of Hox gene regulation during early Xenopus development.

We have shown previously that fibroblast growth factor (FGF) signalling in posterior regions of the Xenopus embryo is required for the development of the trunk and tail via a molecular pathway that includes the caudal-related gene Xcad3 and the posterior Hox genes [1]. These results have been contested by the work of Kroll and Amaya [2], which shows that Xenopus embryos transgenic for a dominant-negative form of the FGF receptor (FGF-RI) express posterior Hox genes normally, leading these authors to suggest that the FGFs are not required for anteroposterior (A-P) patterning of the dorsal axis. In order to investigate the apparent discrepancy between these studies, we have produced Xenopus embryos transgenic for two inhibitors of the FGF/Caudal pathway: a kinase-deficient dominant-negative FGF receptor (XFD) [3]; and a domain-swapped form of Xcad3 (Xcad-EnR) in which the activation domain of Xcad3 is replaced by the repression domain of the Drosophila Engrailed protein. Both of these were introduced as fusions with the green fluorescent protein (GFP), which allows identification of non-mosaic transgenic embryos at early gastrula stages by simply looking for GFP fluorescence. Analysis of gene expression in embryos transgenic for these constructs indicated that the activation of posterior Hox genes during early neurula stages absolutely requires FGF signalling and transcriptional activation by Xcad3, while the maintenance of Hox gene expression in the trunk and tail during later development is independent of both FGF and Xcad.

Structure of aldolase A (muscle-type) cDNA and its regulated expression in oocytes, embryos and adult tissues of Xenopus laevis

We obtained cDNA (XALDA; 1466 bp) for Xenopus laevis aldolase A gene (muscle-type), whose amino acid sequence had 88% similarity to those of mammalian aldolase A genes. XALDA mRNA occurred abundantly in skeletal muscle and at low levels also in other adult tissues, and such mRNA distribution was reflected in zymograms. In oocytes XALDA mRNA occurred at a relatively high level from stage I, and the mRNA level peaked at stage II, then decreased in later stages. XALDA mRNA in the full-grown oocyte was inherited as maternal mRNA throughout maturation and fertilization until midblastula stage, but its level became very low during gastrula and early neurula stages, and then increased greatly in later stages. While maternal XALDA mRNA was distributed uniformly in early embryos, mRNA zygotically expressed after late neurula stage occurred mainly in somites. In blastula animal caps XALDA mRNA occurred at a low level, but the expression was greatly enhanced by activin treatment. Thus, in Xenopus laevis aldolase A gene is actively transcribed in earlier phase of oogenesis, inherited as maternal mRNA in early embryos in a cell-type nonspecific way, then in later phases of embryogenesis, it is strongly expressed in somites with its concomitant ubiquitous expression at low levels in almost all the other cell types.

Specific cellular localization of tyrosinase mRNA during Ciona intestinalis larval development.

A Ciona intestinalis cDNA clone that encodes a protein highly homologous to other tyrosinases was isolated. Northern blot analysis showed that expression of Ciona tyrosinase starts at the early neurula stage and continues throughout the tail-bud and tadpole larval stages. The earliest tyrosinase expression was detected, by in situ hybridization, at the neural plate stage, in pigment precursor cells located along the two neural folds, in the animal region of the embryo. In the course of embryonic development the strong hybridization signal was always localized, within the rostral part of the developing brain, in the pigment precursor cells and was later detected in the otolith and ocellus. These results are discussed in relation to tyrosinase as an early marker of neural induction.

Topographic changes in nascent and early mesoderm in amphioxus embryos studied by DiI labeling and by in situ hybridization for a Brachyury gene.

In amphioxus embryos, the nascent and early mesoderm (including chorda-mesoderm) was visualized by expression of a Brachyury gene (AmBra-2). A band of mesoderm is first detected encircling the earliest (vegetal plate stage) gastrula sub-equatorially. Soon thereafter, the vegetal plate invaginates, resulting in a cap-shaped gastrula with the mesoderm localized at the blastoporal lip and completely encircling the blastopore. As the gastrula stage progresses, DiI (a vital dye) labeling demonstrates that the entire mesoderm is internalized by a slight involution of the epiblast into the hypoblast all around the perimeter of the blastopore. Subsequently, during the early neurula stage, the internalized mesoderm undergoes anterior extension mid-dorsally (as notochord) and dorsolaterally (in paraxial regions where segments will later form). By the late neurula stage, AmBra-2 is no longer transcribed throughout the mesoderm as a whole; instead, expression is detectable only in the posterior mesoderm and in the notochord, but not in paraxial mesoderm where definitive somites have formed.

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.

Molecular cloning of the Xenopus c-met/hepatocyte growth factor receptor and its regional expression during early development.

Hepatocyte growth factor (HGF), a ligand for c-Met receptor tyrosine kinase, regulates the cell growth, migration and morphogenesis of a wide variety of cells, and plays important roles in organogenesis in embryos as well as regeneration of organs. HGF is structurally conserved in Xenopus laevis, and the Xenopus HGF gene is expressed in neurula and tailbud embryos. In the present work, we cloned complementary DNA for the Xenopus Met/ HGF receptor and analyzed its expression during embryogenesis. The open reading frame of Xenopus c-met is 4,125 base pairs long and encodes a putative polypeptide of 1,375 amino acids. The deduced amino acid sequences of the Xenopus and human Met proteins are 63% identical, the tyrosine kinase domains in the intracellular regions showing particular homology, over 90% identity in the amino acid sequences between Xenopus and human. During Xenopus embryogenesis, c-met mRNA was expressed at a high level from the gastrula to the neurula stage, while HGF mRNA expression was seen from the early neurula stage. Whole mount in situ hybridization showed that c-met mRNA was specifically localized in the foregut region, mesenchymal tissue of the tailbud, and neural tissues in neurula embryos. Thus, c-Met is a highly conserved molecule in a wide range of species, as is its ligand, HGF. The HGF-Met system may be involved in early multiple organogenesis in Xenopus embryos.

What mechanisms drive neural induction and neural determination in urodeles?

In our laboratory we use Urodeles (Pleurodeles waltl) and Anurans (Xenopus laevis) to perform comparative studies on neural determination. Urodeles are a good embryological system to study early events in ontogenesis since they present several advantages: slow development, external localization of chordamesoderm at the beginning of gastrulation, large size of cells, diploid genome, etc. I have focused this overview-report on the main findings on Pleurodeles neurogenesis. The determination of the two neural lineages (neuronal and astroglial) appears during gastrulation as a consequence of (a) permissive event(s) activated through a Ca(++)-dependent transducing pathway. This signaling-pathway involves L-type Ca++ channels. The activation of this Ca++ transduction route is sufficient to activate both neuronal and glial structural specific genes, via direct activation of "immediate early genes". The specification of neuronal functional differentiation depends on additional factors of chordamesoderm origin acting during gastrulation and later on. At the early neurula stage, in the neural plate, 20% of progenitor cells present a neuronal fate, 80% are at least bipotential and generate mixed clones (neurons and astrogliocytes). The issue of the state of "commitment" of the precursor cells (competent ectoderm) and the identification of specifying molecules (from Spemann organizer) are underway in Pleurodeles and Xenopus.

Maternal and zygotic expression of mRNA for S-adenosylmethionine decarboxylase and its relevance to the unique polyamine composition in Xenopus oocytes and embryos

From Xenopus tailbud cDNA library, we isolated the cDNA for S-adenosylmethionine decarboxylase (SAMDC), an enzyme which provides putrescine and spermidine with the aminopropyl group to form spermidine and spermine, respectively. The cDNA coded for 335 amino acids whose sequence had high homology (ca. 83%) to other vertebrate SAMDCs, preserving the sequences reportedly essential for enzyme activity, proenzyme processing, and putrescine stimulation of the enzyme activity. Northern blot analysis showed one major mRNA signal of ca. 3.5 kb, with a minor signal of ca. 2.0 kb which may probably be due to cross-hybridization. In oocytes the SAMDC mRNA occurred from stage I, and its amount peaked at stage II, then gradually decreased from stage III to VI. The decreased level of the mRNA was maintained during oocyte maturation, further decreased from the cleavage to early neurula stage, and then increased greatly due to the zygotic expression during late neurula stages (stage 21–25), reaching a plateau level at the late tailbud stage (stage 28). Enzyme assays showed that the changing level of the SAMDC mRNA was reflected in the level of the functional enzyme, suggesting strongly that the zygotic expression of the mRNA leads to a large increase in the amount of SAMDC, albeit in the pre-neurula embryo the amount of the enzyme is very small. We found that the relative composition of polyamines is the eukaryote-type (high-level spermine) at the beginning of oogenesis, but it changes to the prokaryote-type, or more appropriately Escherichia coli-type (high-level putrescine but background level spermine) during oocyte maturation, and remains E. coli-type throughout embryogenesis. We assume that the E. coli-type polyamine composition is a necessary factor for the normal embryogenic development in Xenopus and its maintenance, especially that in pre-neurula stages, can be explained by the low level of both SAMDC mRNA and SAMDC.

Enzymatischer Abbau der Embryonalhüllen des Lanzettfischchens, Branchiostoma lanceolatum, während des Schlüpfens / Hatching through Enzymatic Breakdown of the Embryonic Coats in the Lancelet, Branchiostoma lanceolatum (Cephalochordata)

Abstract A study was made of the hatching process of the lancelet, Branchiostoma lanceolatum (Amphioxus), under laboratory conditions. Embryos in the early neurula stage (5 to 6 pairs of somites) escape through a hole made in the fertilization envelope, following intensive rotatory movements and partial digestion of the envelope. Using the chromogenic substrate Hide Powder Azure (HPA) at pH 8.0, proteolytic activity was detected in the hatching medium. The effect of various protease inhibitors was investigated. Whereas the activity was only slightly affected by inhibitors of trypsin, chymotrypsin or thiol proteases, chelating agents such as EDTA and 1,10-phenanthroline prevented the degradation of HPA, as well as the breakdown of the vitelline membrane of alcohol fixed oocytes by the protease from hatching fluid. These data suggest the presence of a metalloprotease in the hatching medium. How far this enzyme can be compared with hatching enzymes found in echinoderms and fishes (where they have been identified as matrix degrading metalloproteases), awaits further study of the lancelet enzyme, including its isolation and purification.

Experimental analyses of the rearrangement of ectodermal cells during gastrulation and neurulation in avian embryos.

The rearrangement of ectodermal cells was studied in chimeras in which grafts were transplanted during late gastrula and early neurula stages to heterotopic locations in avian embryos. Three types of experiments were done. In all experiments, Hensen's node was extirpated completely and replaced with an epithelial plug derived from 1 of 3 regions of the prospective ectoderm. In type-1 experiments, Hensen's node was replaced with a plug consisting of precursor cells of the floor plate of the neural tube. In type-2 experiments, Hensen's node was replaced with a plug consisting of precursor cells of the lateral wall of the neural tube. In type-3 experiments, Hensen's node was replaced with a plug consisting of precursor cells of the epidermal ectoderm. In all experiments, the amount and direction of cell rearrangement that occurred in the transplanted ectodermal plug was essentially typical for prospective ectodermal cells normally residing within Hensen's node. That is, transplanted ectodermal cells underwent lateral-to-medial cell-cell intercalation and contributed to the ventral midline of the neural tube along its entire rostrocaudal extent. In most embryos, a notochord was reconstituted from host cells, despite the fact that Hensen's node--the prime source of prospective notochordal cells in intact embryos--was extirpated completely; however, a few embryos had long notochordal gaps. In such essentially notochordless embryos, the ventral midline of the neural tube still derived from grafted cells, but it failed to form a floor plate, providing further confirmation of the results of several previous studies that the notochord is required to induce the floor plate.(ABSTRACT TRUNCATED AT 250 WORDS)

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 transripts are present in the cardiac mesoderm at stage 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.

XIdx, A dominant negative regulator of bHLH function in early Xenopus embryos

We have identified a divergent member of the Xenopus Id family, XIdx, which disrupts binding of myogenic factor/E-protein complexes to DNA in vitro and inhibits transactivation of the E-box regulated cardiac actin gene by MyoD in embryonic tissue. XIdx transcripts accumulate from the early neurula stage in discrete domains of the anterior neural plate and subsequently identify regions of the developing nervous system, including the eye rudiments and the rhombencephalon. These results suggest that bHLH proteins and their Id protein regulators may participate in patterning of embryonic neural tissue. Phylogenetic analysis indicates that XIdx is the product of a novel Id gene and is distinct from XId2, which is expressed primarily in the developing pronephros.

Mesodermal patterning during avian gastrulation and neurulation: Experimental induction of notochord from non‐notochordal precursor cells

The cells that are normally fated to form notochord occupy a region at the rostral tip of the primitive streak at late gastrula/early neurula stages of avian and mammalian development. If these cells are surgically removed from avian embryos in culture, a notochord will nonetheless form in the majority of cases. The origin of this reconstituted notochord previously had not been investigated and was the objective of this study. Chick embryos at late gastrulal early neurula stages were cultured, and the rostral tip of the primitive streak including Hensen's node was removed and replaced with non-node cells from quail epiblast to ensure that the cells normally fated to be notochord would be absent and that healing of the blastoderm would occur. Embryos were allowed to develop for 24 hr, and the presence and origin (host or graft) of the notochord were assessed using antibodies against notochord or quail cells. Two notochords typically developed; both were almost exclusively of host origin. The primitive streak, and in some cases adjacent tissues, was removed from another group of embryos in an attempt to estimate the mediolateral position and extent of the cells required to form reconstituted notochord. Additional experimental embryos with and without grafts were transected at various rostrocaudal levels in an attempt to estimate the rostrocaudal extent of the cells required to form reconstituted notochord. Finally, various levels of the primitive streak either were placed in a neutral environment (the germ cell crescent) or were grafted in place of the node. Collective results from all experiments indicate that the areas lateral to the rostral portion of the primitive streak, estimated to have a rostrocaudal span of less than 500 microns and a mediolateral extent of less than 250 microns, are critical for formation of the reconstituted notochord. Fate mapping and histological examination of this region identify 4 possible precursor cell populations. Further studies are underway to determine which of the 4 possible precursor cell types forms or induces the reconstituted notochord, and which tissue interactions underlie this change in cell fate.

Ventral mesodermal patterning in Xenopus embryos: Expression patterns and activities of BMP‐2 and BMP‐4

We provide a comparative analysis of the expression patterns and ventral mesoderm-inducing properties of Xenopus BMP-2 and BMP-4. Transcripts for BMP-2 and BMP-4 are maternally stored in eggs, and zygotic expression of these genes is uniform in the ectoderm and mesoderm in late blastulae. During gastrulation, BMP-2 is expressed at a low level throughout the ectoderm and marginal zone, but at early neurula stages a patch of dorso-anterior cells displays enhanced expression. In contrast, BMP-4 transcripts are restricted to the ventrolateral marginal zone during gastrulation, and in late gastrula and early neurula BMP-4 is expressed in the epidermis but not the neural plate. At post-neurula stages, BMP-2 and BMP-4 transcripts are associated with a variety of mesodermal structures, including the pharyngeal pouches, heart, blood island, and blastopore. At tailbud stages, BMP-2 and BMP-4 are expressed in neural tissues including the neural tube and brain. In mesoderm induction assays, BMP-2 and BMP-4 induce Xhox3, an early ventral-posterior mesoderm marker, and larval alpha Tl globin, a marker for red blood cells. Induction of red blood cells in response to BMP-4 was demonstrated by staining with a hemoglobin-specific reagent. Little is known about factors that induce hematopoietic lineages in vertebrates, and these results provide evidence linking BMP activity and blood differentiation. Globin induction by BMP-2 and BMP-4 is blocked by co-expression of a dominant-negative activin receptor, suggesting that either endogenous activin signals are required for BMP-mediated induction, or that the truncated activin receptor interferes with signaling by BMP receptors. In assays on marginal zone explants, we demonstrate that BMP-4 respecifies dorsal mesoderm to form ventral mesoderm, consistent with its ability to induce blood and to ventralize embryos. BMP-2, however, does not display such activity. The findings extend and support evidence that BMP-2 and BMP-4 function in ventral mesoderm induction and patterning in Xenopus. Our data furthermore highlight the multiple functions these factors fulfill during early vertebrate embryogenesis.

Gr Transcripts Are Localized during early Xenopus laevis Embryogenesis and Overexpression of GR Inhibits Differentiation After Dexamethasone Treatment

We have studied the spatial expression of the Xenopus GR in early embryos by whole-mount in situ hybridization. At the gastrula stage, GR mRNA is localized in the dorsal ectoderm. By the early neurula stage, GR transcripts were detected along the notoplate. Between mid and late neurula stages, GR mRNA was not detectable. At the tailbud stage, GR mRNA was found in the anterior part of the embryo, including the cement gland, eyes, brain, the foregut, stomodeal-hypophyseal anlage, the olfactory placodes, head mesenchyme and somites. Injection of Xenopus GR RNA into zygotes followed by treatment with dexamethasone from the blastula stage onwards inhibits early differentiation. Expression of Xbra, gsc and histone H3 genes in these embryos is not inhibited, indicating that the GR effects are not due to a general squelching effect on transcription.

A Concentration Gradient of Retinoids in the Early Xenopus laevis Embryo

Previous studies have postulated that Xenopus embryos contain an endogenous retinoic acid (RA) concentration gradient from posterior to anterior during the process of primary axis formation, since RA is able to alter profoundly primary axis formation in Xenopus embryos, to increase the expression of some posterior markers, and to inhibit the expression of some anterior markers, including homeobox-containing genes. Here, we provide direct evidence for this hypothesis. By using a reporter cell system, we demonstrate that the endogenous biologically active retinoid concentration in whole Xenopus embryos increases 3-fold from the two-cell stage to the neurula stage, and that the active retinoid concentration in the dorsal marginal zone, a region wherein the Spemann's organizer is located, increases about 5-fold from the early gastrula to late gastrula stages, suggesting the developmental regulation of the retinoid levels. In the early neurula stage (stage 13-14), endogenous active retinoids are present in a concentration gradient with the highest level at the posterior end, about 10-fold higher than that at the anterior end, of the embryo. This concentration gradient may be established during gastrulation and may provide positional cues for primary axis formation.

Retinoic acid affects central nervous system development of Xenopus by changing cell fate

Retinoic acid (RA) may play a role in anterior-posterior (A-P) patterning in the central nervous system (CNS) of vertebrates. To understand this role, Xenopus embryos were treated with increasing doses of all- trans RA at the late gastrula to early neurula stage, and changes in the brain were assessed. When embryos were treated with a low dose of 10 −8 M RA, alterations of the brain were observed: a 20% increase in the expression of a neural-specific marker, XIPOU 1, in the brain and eye with a concurrent loss of the forebrain. Higher doses of RA led to progressively more severe truncations in the brain and a loss of XLPOU 1 expression. Most importantly, after observing changes in the RA-treated embryos, we determined that the lineage of cells that contribute to the brain of these embryos do not die but change their fate. With higher doses of RA (≥10 −7 M), the normal cell fate of the A1 lineage was changed from a mostly neuronal phenotype to an epidermal one. Our data suggest that exogenous RA or a closely related derivative causes changes in cell fate of the A1 lineage which may in part be responsible for alterations in the developing CNS.

The role of noradrenaline in the differentiation of amphibian embryonic neurons.

The possibility that monoamines might act as signalling molecules during the early development of the nervous system has been examined in embryos of the amphibian Xenopus laevis. The distributions of 5-hydroxytryptamine, dopamine, noradrenaline and their precursor, dopa, were determined from the fertilized egg up to the late neurula stages using High Performance Liquid Chromatography, formaldehyde-induced fluorescence and antibody staining. 5-hydroxytryptamine was not detected until the tail bud stage. The fertilized egg contained significant concentrations of dopa (10(-6) M) and dopamine (10(-7) M). Both monoamines persisted with little change in concentration up to the late neurula stage. Early neurula stage embryos contained very low levels of noradrenaline. Aldehyde-induced fluorescence showed that monoamines are localized in dorsal regions of the embryo, in ectoderm and mesoderm cells. Monoamines were not present in endoderm cells. Immunocytochemical staining showed dopamine predominantly in the ectoderm, except in future neural regions where it was found also in the mesoderm. Dopamine staining was always most intense in dorsal regions of the embryo. The consequences for subsequent neuronal differentiation of interfering with the biosynthesis and receptor binding of monoamines during neurulation was assayed. Neuronal differentiation was monitored quantitatively in cultures set up as the neural tube closed and qualitatively in intact tadpoles that were left to develop for two days after washout of test reagent. The number of neurons, the number of muscle cells and the total number of differentiated cells were counted after 18-24 hours of culture. Comparison of the number of neurons that differentiated from control and treated embryos showed that inhibition of dopamine beta-hydroxylase, the enzyme catalysing the conversion of dopamine to noradrenaline, during the neural plate stages reduced substantially subsequent neuronal differentiation. The differentiation of myocytes and the total number of differentiated cells were not affected. Exogenous noradrenaline (10(-6) M) or dopamine (10(-6) M) could increase the number of neurons that differentiated subsequently in culture. Interfering with noradrenaline binding to receptors with receptor antagonists during neurulation showed that alpha-adrenergic receptor antagonists reduced substantially the subsequent differentiation of neurons. The differentiation of myocytes and the total number of differentiated cells were not affected. The effect of alpha-adrenergic receptor antagonists was overcome by the simultaneous inclusion of noradrenaline or alpha-receptor agonists, but not agonists at beta-adrenergic receptors. The quantitative reduction in the differentiation of neurons was paralleled by defects in the Central Nervous System of intact tadpoles.(ABSTRACT TRUNCATED AT 400 WORDS)