Galanin messenger RNA during postnatal development of the rat brain: expression patterns in Purkinje cells differentiate anterior and posterior lobes of cerebellum

Following our initial mapping of preprogalanin messenger RNA in adult brain and its presence in a subpopulation of cerebellar Purkinje neurons [Ryan M. C. and Gundlach A. C. (1996) Neuroscience 70, 709-728], the present study examined the ontogenic expression of preprogalanin messenger RNA in the postnatal rat brain focussing on the Purkinje cells of the cerebellar cortex. Using in situ hybridization histochemistry, preprogalanin messenger RNA was detected in the developing forebrain and hindbrain from postnatal day 4 to day 60 (adult). On postnatal day 4 very light hybridization signal (labelling) was observed in cells of a number of nuclei including the central amygdaloid nucleus, the medial preoptic area, paraventricular nucleus and dorsomedial hypothalamic nucleus of the forebrain while lightly-labelled cells were detected in neurons of the nucleus of the solitary tract and locus coeruleus of the hindbrain. Hybridization signal was not apparent in other nuclei until later, with positively-labelled neurons first apparent in the dorsal cochlear nucleus at postnatal day 21. The abundance of preprogalanin messenger RNA-positive neurons and the intensity of the hybridization signal increased, in most regions, until postnatal day 28 when labelling resembled that of the mature rat. Preprogalanin messenger RNA was first detected in the cerebellum on postnatal day 10 only in Purkinje cells of lobule 10 of the posterior vermis and increased in distribution throughout Purkinje cell layers of the entire cerebellar cortex by postnatal day 13. The intensity of hybridization signal in Purkinje cells varied between lobules, with Purkinje cells in lobule 10 displaying a moderate to heavy degree of labelling, while lobules 6-9 and the more posterior lobules of the hemisphere including crus 2 of the ansiform lobule, the paramedian lobule and the copula pyramis, displayed only light labelling. The intensity of labelling in the anterior vermis and the remaining lobules of the hemisphere including crus 1 of the ansiform lobule, the simple lobule, the paraflocculus and the flocculus, was homogeneously weak. By postnatal day 21, Purkinje cell labelling reached maximum intensity in all lobules. Regional differences were still apparent, however, with labelling in the posterior vermis and hemisphere ranging from moderate to heavy, with only light to moderate labelling detected in the anterior vermis. The intensity of labelling in the posterior vermis and most lobules of the hemisphere was similar from postnatal day 21 to adulthood, while, in the anterior vermis, crus 1 of the ansiform lobule and the simple lobule, the intensity of hybridization decreased slightly by postnatal day 28 and was completely absent in Purkinje cells of the adult rat. Differential expression of preprogalanin messenger RNA in Purkinje cells of the developing rat cerebellum and transient expression in certain lobules suggests that galanin gene products may have a role in both the developing and mature rat brain and that galanin gene expression may represent a useful marker for differentiating the anterior and posterior cerebellar lobes.     

BMP1-related metalloproteinases promote the development of ventral mesoderm in early Xenopus embryos

Bone morphogenetic protein 1 (BMP1) is a metalloproteinase closely related to Drosophila Tolloid (Tld). Tld regulates dorsoventral patterning in early Drosophila embryos by enhancing the activity of Dpp, a member of the TGF-beta family most closely related to BMP2 and BMP4. In Xenopus BMP4 appears to play an essential role in dorsoventral patterning, promoting the development of ventral fates during gastrula stages. To determine if BMP1 has a role in regulating the activity of BMP4, we have isolated cDNAs for Xenopus BMP1 and a novel closely related gene that we have called xolloid (xld). Whereas xbmp1 is uniformly expressed at all stages tested, the initial uniform expression of xld becomes localized to two posterior ectodermal patches flanking the neural plate and later to the inner ectoderm of the developing tailbud. xld is also expressed in dorsal regions of the brain during tailbud stages and is especially abundant in the ventricular layer of the dorsal hindbrain caudal to the otic vesicle. Overexpression of either gene inhibits the development of dorsoanterior structures in whole embryos and ventralizes activin-induced dorsal mesoderm in animal caps. Since ventralization of activin-induced animal caps can be blocked by coinjecting a dominant-inhibitory receptor for BMP2 and BMP4, we suggest a role for BMP1 and Xld in regulating the ventralizing activity of these molecules.     

BMP regulates vegetal pole induction centres in early xenopus development

BACKGROUND: Bone morphogenetic protein (BMP) plays an important role in mesoderm patterning in Xenopus. The ectopic expression of BMP-4 protein hyperventralizes embryos, whereas embryos expressing a BMP-2/4 dominant-negative receptor (DNR) are hyperdorsalized. Mesoderm is initially induced in the marginal zone by cells in the underlying vegetal pole. While much is known about BMP's expression and role in patterning the marginal zone, little is known about its early role in regulating vegetal mesoderm induction centre formation. RESULTS: The role of BMP in regulating formation of vegetal mesoderm inducing centres during early Xenopus development was examined. Ectopic BMP-4 expression in vegetal pole cells inhibited dorsal mesoderm induction but increased ventral mesoderm induction when recombined with animal cap ectoderm in Nieuwkoop explants. 32-cell embryos injected with BMP-4 RNA in the most vegetal blastomere tier were not hyperdorsalized by LiCl treatment. The ectopic expression of Smad or Mix.1 proteins in the vegetal pole also inhibited dorsal mesoderm induction in explants and embryos. Expression of the BMP 2/4 DNR in the vegetal pole increased dorsal mesoderm induction and inhibited ventral mesoderm induction in explants and embryos. CONCLUSIONS: These results support a role for BMP signalling in regulating ventral vegetal and dorsal vegetal mesoderm induction centre formation during early Xenopus development.     

Structure of the zebrafish snail1 gene and its expression in wild-type, spadetail and no tail mutant embryos

Mesoderm formation is critical for the establishment of the animal body plan and in Drosophila requires the snail gene. This report concerns the cloning and expression pattern of the structurally similar gene snail1 from zebrafish. In situ hybridization shows that the quantity of snail1 RNA increases at the margin of the blastoderm in cells that involute during gastrulation. As gastrulation begins, snail1 RNA disappears from the dorsal axial mesoderm and becomes restricted to the paraxial mesoderm and the tail bud. snail1 RNA increases in cells that define the posterior border of each somite and then disappears when somitic cells differentiate. Later in development, expression appears in cephalic neural crest derivatives. Many snail1-expressing cells were missing from mutant spadetail embryos and the quantity of snail1 RNA was greatly reduced in mutant no tail embryos. The work presented here suggests that snail1 is involved in morphogenetic events during gastrulation, somitogenesis and development of the cephalic neural crest, and that no tail may act as a positive regulator of snail1.     

The vegetal determinants required for the Spemann organizer move equatorially during the first cell cycle

Embryos with no dorsal axis were obtained when more than 15% of the egg surface was deleted from the vegetal pole of the early 1-cell embryo of Xenopus laevis. The timing of the deletion in the first cell cycle was critical: dorsal-deficient embryos were obtained when the deletion began before time 0.5 (50% of the first cell cycle) whereas normal dorsal axis usually formed when the deletion was done later than time 0.8. The axis deficiency could be restored by lithium treatment and the injection of vegetal but not animal cytoplasm. Bisection of the embryo at the 2-cell stage, which is known to restore the dorsal structures in the UV-ventralized embryos, had no effect on the vegetal-deleted embryos. These results show clearly that, in Xenopus, (1) the dorsal determinants (DDs) localized in the vegetal pole region at the onset of development are necessary for dorsal axis development and (2) the DDs move from the vegetal pole to a subequatorial region where they are incorporated into gastrulating cells to form the future organizing center. A model for the early axis formation process in Xenopus is proposed.     

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.     

Domains of retinoid signalling and neurectodermal expression of zebrafish otx1 and goosecoid are mutually exclusive

Retinoid signalling plays an important role in embryonic pattern formation. Excess of retinoic acid during gastrulation results in axial defects in vertebrate embryos, suggesting that retinoids are involved in early anteroposterior patterning. To study retinoid signalling in zebrafish embryos, we developed a novel method to detect endogenous retinoids in situ in embryos, using a fusion protein of the ligand inducible transactivation domain of a retinoic acid receptor and a heterologous DNA binding domain. Using this method, we show that retinoid signalling is localized in zebrafish embryos in the region of the embryonic shield, and towards the end of gastrulation in a posterior dorsal domain. To investigate the relationships between the spatial distribution of retinoid signalling and the regulation of retinoid target genes, we studied the downregulation by retinoic acid of two genes expressed in anterior regions of the embryo, goosecoid and otx1. These experiments show that expression of both genes is strongly downregulated in the anterior neurectoderm of zebrafish embryos treated with retinoic acid, whereas mesendodermal expression is only mildly affected. Interestingly, a significant downregulation of goosecoid expression by retinoic acid was observed only during midgastrulation but not in earlier stages. In agreement with these results, spatial expression of goosecoid and otx1 does not overlap with the region of retinoid signalling in the late gastrula. Our data support the hypothesis that a localized retinoid signal is involved in axial patterning during early development, at least in part through the repression of anterior genes in posterior regions of the embryo. Furthermore, our data suggest that the action of retinoids is spatially as well as temporally regulated in the developing embryo.     

Secretion and mesoderm-inducing activity of the TGF-beta-related domain of Xenopus Vg1

Vg1 is a maternal mRNA localized to the vegetal hemisphere of Xenopus embryos during blastula stages, a region responsible for the induction of mesoderm in the adjacent marginal zone. Its homology to the transforming growth factor-beta family, which includes several proteins with mesoderm-inducing activity, suggests a role for Vg1 as an endogenous mesoderm-inducing factor. However, expression of Vg1 protein in the animal hemisphere, following injection of synthetic mRNA, has no effect on development, and isolated animal caps are not mesodermalized. It is shown that Vg1 protein fails to form dimers and is not processed to release the putative bioactive domain. Furthermore it is shown that the N-terminal signal peptide of Vg1 is not cleaved following translocation into the ER, which may explain the failure of this protein to dimerize. To explore the role of Vg1 in amphibian development, a fusion protein has been made of the preproregion of Xenopus bone morphogenetic protein-4 and the putative bioactive C-terminal domain of Vg1. This fusion protein forms dimers and the C-terminal domain of Vg1 is secreted. Injection of this construct into Xenopus embryos induces the formation of a second dorsal axis and isolated animal caps are mesodermalized. The results are consistent with a role for Vg1 in mesoderm induction during Xenopus development.     

Bridging the GAP in inositol 1,3,4,5-tetrakisphosphate signalling

Recent advances in analyzing wnt signaling have provided evidence that frizzled proteins can function as wnt receptors. We have identified Xfz3, a Xenopus frizzled family member. The amino acid sequence is 89% identical to the product of the murine gene Mfz3, and is predicted to be a serpentine receptor with seven transmembrane domains. Xfz3 is a maternal mRNA with low levels of expression until the end of gastrulation. The expression level increases significantly from neurulation onward. Whole-mount in situ hybridization analysis shows that expression of Xfz3 is highly restricted to the central nervous system. High levels of expression are detected in the anterior neural folds. Low levels of expression are also detected in the optic and otic vesicles, as well as in the pronephros anlage. In addition, Xfz3 mRNA is concentrated in a large band in the midbrain. Overexpression of Xfz3 blocks neural tube closure, resulting in embryos with either bent and strongly reduced anteroposterior axis in a dose-dependent manner. However, it does not affect gastrulation, the expression and localization of organizer-specific genes such as goosecoid, chordin and noggin. Therefore, Xfz3 is not involved in early mesodermal patterning. Injection of RNA encoding GFP-tagged Xfz3 shows that overexpressed proteins can be detected on the cell surface until at least late neurula stage, suggesting that they can exert an effect after gastrulation. Our expression data and functional analyses suggest that the Xfz3 gene product has an antagonizing activity in the morphogenesis during Xenopus development.     

Induction of a secondary body axis in Xenopus by antibodies to beta-catenin

We have obtained evidence that a known intracellular component of the cadherin cell-cell adhesion machinery, beta-catenin, contributes to the development of the body axis in the frog Xenopus laevis. Vertebrate beta-catenin is homologous to the Drosophila segment polarity gene product armadillo, and to vertebrate plakoglobin (McCrea, P. D., C. W. Turck, and B. Gumbiner. 1991. Science (Wash. DC). 254: 1359-1361.). Beta-Catenin was found present in all Xenopus embryonic stages examined, and associated with C-cadherin, the major cadherin present in early Xenopus embryos. To test beta-catenin's function, affinity purified Fab fragments were injected into ventral blastomeres of developing four-cell Xenopus embryos. A dramatic phenotype, the duplication of the dorsoanterior embryonic axis, was observed. Furthermore, Fab injections were capable of rescuing dorsal features in UV-ventralized embryos. Similar phenotypes have been observed in misexpression studies of the Wnt and other gene products, suggesting that beta-catenin participates in a signaling pathway which specifies embryonic patterning.