Tissue-specific expression of an Ornithine decarboxylase paralogue, XODC2, in Xenopus laevis.

Ornithine decarboxylase (ODC) is involved in the biosynthesis of polyamines and hence has been found in almost all types of cells studied. Therefore it is frequently used as internal standard. We isolated a cDNA, XODC2, which is a paralogue to ubiquitous ODC and expressed in a spatial and temporal manner during the early embryogenesis of Xenopus laevis. Expression of XODC2was first detected at the animal pole at stage 9. During neurula stages the signals were found both in the extreme anterior and posterior part of the dorsal body axis. In tailbud stages the expression is further shifted to both the tail and head areas and gradually restricted to distinct tissues: forebrain, inner layer of epidermis of the head area, stomodeal-hypophyseal anlage, frontal gland, ear vesicle, branchial arches, the front tip of neural tube and proctodeum. In addition, signals were also found in the inner layer of epidermis underneath the cement gland during early tailbud stages while in later tailbud stages signals were detected at the apical zone of the cement gland. Comparative studies indeed could confirm that XODC1 in contrast to XODC2 is expressed ubiquitously throughout the whole embryos during early development of Xenopus laevis.

Xpitx3: a member of the Rieg/Pitx gene family expressed during pituitary and lens formation in Xenopus laevis.

Xpitx3 is the Xenopus homologue of the mouse Pitx3 gene and belongs to the family of RIEG/PITX homeobox genes. Here, we report on the embryonic expression of Xpitx3. It is transcribed in the presumptive pituitary already at the open neural tube stage. During further development Xpitx3 is strongly transcribed in the pituitary Anlage, the lens placodes and head mesenchyme, respectively.

Molecular cloning and expression analysis of the Hedgehog receptors XPtc1 and XSmo in Xenopus laevis.

Inductive signaling mediated by secreted factors of the Hedgehog (Hh) gene family regulates cellular proliferation and differentiation in many embryonic tissues. Two transmembrane proteins associated in a complex, Patched (Ptc) and Smoothened (Smo), are indispensable for the reception of Hh signals (Cell 86 (1996) 221; Nature 382 (1996) 547; Nature 384 (1996) 176; Nature 384 (1996) 129). Here, we report on the identification of Ptc and Smo homologues from Xenopus and analyze their spatio-temporal expression during embryogenesis. The intracellular response to Hh signals involves upregulation of Ptc transcription (Genes Dev. 10 (1996) 301; J. Biol. Chem. 271 (1996) 12125). In accordance with its putative function as Shh target gene, XPtc1 expression during early stages of Xenopus embryogenesis is detected in mesodermal and neuroectodermal tissues proximal to the notochord, a known expression domain of Shh. Although the expression pattern of XPtc1 was similar to that of other vertebrates, expression domains specific to Xenopus could be detected in the hypochord, dorsal mesencephalon, otic vesicles and pituitary anlage. Unlike other vertebrate Ptc1 homologues, somitic expression of XPtc1 is confined to a central cell layer. In contrast to the tissue-specific expression characteristics of XPtc1, XSmo expression appears to be ubiquitously activated in early embryonic stages but condenses in the terminal regions of the embryo at tailbud stage. In many tissues and organs of the adult, XPtc1 and XSmo are found to display similar expression levels.

Structure and expression of Xenopus karyopherin-beta3: definition of a novel synexpression group related to ribosome biogenesis.

Karyopherin-beta3 is a nuclear transport receptor that appears to be involved in nuclear import of ribosomal proteins. Here we report on sequence and expression of karyopherin-beta3 in Xenopus. The differential distribution of karyopherin-beta3 mRNA during Xenopus embryogenesis is similar to that of several other protein import factors and of ribosomal proteins. These genes thus define a novel synexpression group in the context of ribosome biogenesis.

Expanded retina territory by midbrain transformation upon overexpression of Six6 (Optx2) in Xenopus embryos.

During vertebrate eye development, the expression of the homeobox gene Six6 is restricted to the neural retina and is initiated later than Rx and Pax6 in the presumptive retina field. We show here that overexpression of mouse Six6 in Xenopus embryos can induce transformation of competent tissue of the anterior neural plate into retinal tissue. In Six6 injected embryos, the molecular identity of the presumptive midbrain and rostral hindbrain regions was lost, as shown by the absence of XEn-2 and Xpax2 expression, being replaced by the ectopic expression of the retinal markers Xpax6 and Xrx. When allowed to grow further, Six6 injected embryos developed ectopic eye-like structures in the rostral brain and showed a transformation of the midbrain into retina. Similar results were obtained upon overexpression of Six3 or Xsix3, revealing a possible redundance of Six3 and Six6 activities. Taken together, results obtained suggest that during normal retina development, the relatively late expressed Six6 gene becomes part of a network of retinal homeobox genes that are linked together by positive feedback loops. Furthermore, our results demonstrate that the primitive neural ectoderm of the future midbrain and rostral hindbrain is competent to form retinal tissue.

Cloning and expression of xSix3, the Xenopus homologue of murine Six3.

The vertebrate Six family of transcription factor genes are homologues of the fruitfly gene sine oculis (so). Human, murine, avian and fish (medaka, zebrafish) homologues have recently been cloned. We report the cloning and developmental pattern of expression of xSix3, the Xenopus laevis homologue of Six3. In addition, we have compared all the known sequences of vertebrate Six3 genes. xSix3 is very homologous to Six3 in other vertebrates in terms of amino acid sequence. The reported developmental pattern of expression of Six3 in chick and mouse includes not only the developing eyes and the ventral diencephalic tissue between them, but also a large, sagittally-oriented telencephalic region. The distribution of xSix3, however, is virtually restricted to the eyes and ventral diencephalon, showing only a very small territory of expression in the telencephalon.

Xnkx-2.1: a homeobox gene expressed during early forebrain, lung and thyroid development in Xenopus laevis.

Nkx-2.1 is a member of the vertebrate Nkx family of homeobox genes; it was originally identified as a tissue-specific regulator of thyroglobulin and thyroperoxidase gene transcription. Here we report on the embryonic expression of Xnkx-2.1, which is expressed in the presumptive forebrain from early neurulation onwards. In tadpole stage embryos Xnkx-2.1 transcripts are primarily detected in ventral forebrain, lung buds and thyroid anlage. Therefore, Xnkx-2.1 may be part of the genetic network that controls the early development of these organs.

Planar signalling is not sufficient to generate a specific anterior/posterior neural pattern in pseudoexogastrula explants from Xenopus and Triturus.

Early observations on the morphology of total exogastrulae from urodeles (Axolotl) had provided evidence for essential vertical signalling mechanisms in the process of neural induction. Conversely, more recent studies with anurans (Xenopus laevis) making use of molecular markers for neural-specific gene expression appear to support the idea of planar signalling as providing sufficient information for neural differentiation along the anterior-posterior axis. In an attempt to resolve this apparent contradiction, we report on the comparative analysis of morphology and gene expression characteristics with explants prepared from both urodeles (Triturus alpestris) and anurans (Xenopus laevis). For this purpose, we have made use of a refined experimental protocol for the preparation of exogastrulae that is intended to combine the advantages of the Holtfreter type exogastrula and the Keller sandwich techniques, and which we refer to as pseudoexogastrula explants. Analysis of histology and expression of several neural and ectodermal marker genes in such explants suggests that neural differentiation is induced in both species, but only within the intermediate zone between ectoderm and endomesoderm. Therefore, experiments with Xenopus and Triturus explants described in this communication argue against planar signalling events as being sufficient to generate a specific anterior/posterior neural pattern.

Vax1, a novel homeobox-containing gene, directs development of the basal forebrain and visual system.

The novel homeobox-containing gene Vax1, a member of the Emx/Not gene family, is specifically expressed in the developing basal forebrain and optic nerve. Here, we show that Vax1 is essential for normal development of these structures. Mice carrying a targeted mutation of Vax1 show dysgenesis of the optic nerve, coloboma, defects in the basal telencephalon, and lobar holoprosencephaly. With the help of molecular markers we determined that in the developing visual system, the absence of Vax1 results in a proximal expansion of the activity of Pax6 and Rx. This observation suggests that Vax1 may interfere negatively with the expression of Pax6 and Rx. In reciprocal gain-of-function experiments, injection of Xvax1 mRNA or Shh into Xenopus embryos primarily affects the brain at the level of the eye primordium. Consistent with the loss-of-function results, the injection of Xvax1 results in a down-regulation of Rx. Similarly, Shh injection expands the Vax1 and Pax2 territory at the expense of the Pax6 and Rx region. On the basis of these results, we propose a model for a molecular cascade involved in the establishment of structures of the visual system.

Characterization of a subfamily of related winged helix genes, XFD-12/12'/12" (XFLIP), during Xenopus embryogenesis.

The fork head domain family of genes defines a growing group of proteins that serve important regulatory functions in pattern-forming events of both invertebrates and vertebrates. Here we add three closely related, novel members to this family in Xenopus laevis, termed XFD-12, XFD-12' and XFD-12". All three genes reveal indistinguishable expression patterns during Xenopus embryogenesis. During gastrulation, XFD-12 type transcripts are detected exclusively in the superficial layer of cells within the Spemann organizer territory. In the open neural plate, XFD-12 type expression defines a row of cells located along the dorsal midline and destined to become the floor plate of the neural tube. After closure of the neural tube, XFD-12 type encoding mRNAs are only detected in the tailtip and a small area located at the midbrain/hindbrain boundary. Within the Spemann organizer and in the floor plate area, expression of XFD-12 type genes is only partially overlapping with XFD-1 expression.

Xpitx-1: a homeobox gene expressed during pituitary and cement gland formation of Xenopus embryos.

Pitx-1 is a member of the family of bicoid-related vertebrate homeobox genes; it was originally identified as a tissue-specific transcriptional regulator of the proopiomelacortin gene. Here we report on the embryonic expression of Xpitx-1, which is expressed in the anterior neural ridge and in the cement gland Anlage during late gastrulation/early neurulation. In tadpole stage embryos Xpitx-1 transcripts are primarily detected in the cement gland, stomodeal-hypophyseal Anlage, oral epithelia and lens placode. Therefore, Xpitx-1 may be part of the genetic network that controls the early development of these structures.

Characterization of the Ets-type protein ER81 in Xenopus embryos.

A function for FGF-type peptide growth factors has been implied for early mesodermal patterning events in Xenopus laevis. FGF signalling operates via the MAP kinase cascade that can directly activate the transcription of organizer-expressed genes, such as Xbra and Xegr-1. We have recently provided evidence for a critical role of Ets-type transcription factors in FGF mediated Xegr-1 transcription activation. Here, we report on the identification of the Xenopus Ets-type protein ER81 that is expressed in a pattern overlapping with the ones of Xegr-1 and Xbra during gastrulation. Microinjection in XER81 encoding mRNA into ventral blastomeres of Xenopus embryos results in the induction of ectopic, tail-like protrusions, whereas dorsal overexpression results in disturbed eye development. In the animal cap assay, ectopic expression of XER81 is found to interfere with activin mediated induction of Xegr-1 and gsc, but not with the Xbra response to activin.

Regionalized metabolic activity establishes boundaries of retinoic acid signalling.

The competence of a cell to respond to the signalling molecule retinoic acid (RA) is thought to depend largely on its repertoire of cognate zinc finger nuclear receptors. XCYP26 is an RA hydroxylase that is expressed differentially during early Xenopus development. In Xenopus embryos, XCYP26 can rescue developmental defects induced by application of exogenous RA, suggesting that the enzymatic modifications introduced inhibit RA signalling activities in vivo. Alterations in the expression pattern of a number of different molecular markers for neural development induced upon ectopic expression of XCYP26 reflect a primary function of RA signalling in hindbrain development. Progressive inactivation of RA signalling results in a stepwise anteriorization of the molecular identity of individual rhombomeres. The expression pattern of XCYP26 during gastrulation appears to define areas within the prospective neural plate that develop in response to different concentrations of RA. Taken together, these observations appear to reflect an important regulatory function of XCYP26 for RA signalling; XCYP26-mediated modification of RA modulates its signalling activity and helps to establish boundaries of differentially responsive and non-responsive territories.

The Spemann organizer-expressed zinc finger gene Xegr-1 responds to the MAP kinase/Ets-SRF signal transduction pathway.

The transcriptional activity of a set of genes, which are all expressed in overlapping spatial and temporal patterns within the Spemann organizer of Xenopus embryos, can be modulated by peptide growth factors. We identify Xegr-1, a zinc finger protein-encoding gene, as a novel member of this group of genes. The spatial expression characteristics of Xegr-1 during gastrulation are most similar to those of Xbra. Making use of animal cap explants, analysis of the regulatory events that govern induction of Xegr-1 gene activity reveals that, in sharp contrast to transcriptional regulation of Xbra, activation of Ets-serum response factor (SRF) transcription factor complexes is required and sufficient for Xegr-1 gene expression. This finding provides the first indication for Ets-SRF complexes bound to serum response elements to be activated during gastrulation. MAP kinase signalling cascades can induce and sustain expression of both Xegr-1 and Xbra. Ectopic Xbra can induce Xegr-1 transcription by an indirect mechanism that appears to operate via primary activation of fibroblast growth factor secretion. These findings define a cascade of events that links Xbra activity to the signal-regulated control of Xegr-1 transcription in the context of early mesoderm induction in Xenopus laevis.

The Xenopus homologue of the Drosophila gene tailless has a function in early eye development.

Genetic circuits responsible for the development of photoreceptive organs appear to be evolutionarily conserved. Here, the Xenopus homologue Xtll of the Drosophila gene tailless (tll), which we find to be expressed during early eye development, is characterized with respect to its relationship to vertebrate regulators of eye morphogenesis, such as Pax6 and Rx. Expression of all three genes is first detected in the area corresponding to the eye anlagen within the open neural plate in partially overlapping, but not identical, patterns. During the evagination of the optic vesicle, Xtll expression is most prominent in the optic stalk, as well as in the distal tip of the forming vesicle. In tadpole-stage embryos, Xtll gene transcription is most prominent in the ciliary margin of the optic cup. Inhibition of Xtll function in Xenopus embryos interferes specifically with the evagination of the eye vesicle and, in consequence, Xpax6 gene expression is severely reduced in such manipulated embryos. These findings suggest that Xtll serves an important regulatory function in the earliest phases of vertebrate eye development.

Vax1 is a novel homeobox-containing gene expressed in the developing anterior ventral forebrain.

The vertebrate forebrain is formed at the rostral end of the neural plate under the regulation of local and specific signals emanating from both the endomesoderm and neuroectoderm. The development of the rostral and ventral forebrain in particular was difficult to study, mainly because no specific markers are available to date. Here, we report the identification of Vax1, a novel homeobox-containing gene identified in mouse, Xenopus and human. It is closely related to members of the Not and Emx gene families, all of which are required for the formation of structures where they are expressed. In mouse and Xenopus, Vax1 expression first occurs in the rostral neural plate, in the medial anterior neural ridge and adjacent ectoderm. Later, at midgestation in the mouse and tadpole stage in Xenopus, the expression remains confined in the derivatives of this territory which differentiate into rostromedial olfactory placode, optic nerve and disc, and anterior ventral forebrain. Together, these observations suggest that Vax1 could have an early evolutionary origin and could participate in the specification and formation of the rostral and ventral forebrain in vertebrates. Comparison of the limits of the expression territory of Vax1 with that of Dlx1, Pax6 and Emx1 indicates that the corticostriatal ridge is a complex structure with distinct identifiable genetic compartments. Besides, the study of Vax1 expression in Pax6-deficient homozygous brains indicates that its regulation is independent of Pax6, although the expression patterns of these two genes appear complementary in wild-type animals. Vax1 chromosomal location is mapped at the distal end of the mouse chromosome 19, linked with that of Emx2. These two genes may have arisen by tandem duplication. The Vax1 gene is thus an interesting new tool to study the rostral ventral forebrain patterning, morphogenesis and evolution as well as the terminal differentiation of the forebrain in mouse and Xenopus.

X-MyT1, a Xenopus C2HC-type zinc finger protein with a regulatory function in neuronal differentiation.

X-MyT1 is a C2HC-type zinc finger protein that we find to be involved in the primary selection of neuronal precursor cells in Xenopus. Expression of this gene is positively regulated by the bHLH protein X-NGNR-1 and negatively regulated by the Notch/Delta signal transduction pathway. X-MyT1 is able to promote ectopic neuronal differentiation and to confer insensitivity to lateral inhibition, but only in cooperation with bHLH transcription factors. Inhibition of X-MyT1 function inhibits normal neurogenesis as well as ectopic neurogenesis caused by overexpression of X-NGNR-1. On the basis of these findings, we suggest that X-MyT1 is a novel, essential element in the cascade of events that allows cells to escape lateral inhibition and to enter the pathway that leads to terminal neuronal differentiation.

Xenopus Xsal-1, a vertebrate homolog of the region specific homeotic gene spalt of Drosophila.

We have isolated an amphibian homolog of the homeotic gene spalt of Drosophila. Like its Drosophila counterpart the Xenopus Xsal-1 gene encodes a protein that contains three widely separated sets of sequence related double zinc finger motifs of the CC/HH-type as well as a single CC/HH zinc finger. The Xenopus gene encodes a fourth double zinc finger and a single CC/HC zinc finger motif that have no counterpart in the fly protein. Alternative splicing of Xsal-1 transcripts gives rise to RNAs coding for either four, three or two double zinc fingers, respectively. The main expression domains of Xsal-1 in early development are confined to distinct regions along the lateral axon tracts within the midbrain, hindbrain, and spinal cord. Outside the central nervous system Xsal-1 is expressed in the facio-acoustic ganglion and in the developing limb buds. The pattern of expression suggests that Xsal-1 might be under control of signals emanating from the notochord and/or the floor plate and that it might function in neuronal cell specification.

Zinc finger proteins in early Xenopus development.

The C2H2-type zinc finger motif defines a large super family of specific DNA and specific RNA binding proteins. Individual members of this protein family have been demonstrated to carry important regulatory functions in embryogenesis. We have isolated a large collection of C2H2-type zinc finger proteins from Xenopus laevis. Some of these proteins are highly conserved in evolution and found to be differentially expressed during embryonic development of the central nervous system. We also summarize our recent findings on the biochemical characterization of RNA and DNA binding activities in vitro for other Xenopus zinc finger proteins, which fall into structurally defined, distinct subfamilies.