Visualization of endogenous BMP signaling during Xenopus development

Abstract The TGF-β superfamily of growth factors is known to transmit signals to the nucleus mainly through the Smads, intracellular signaling components that are highly conserved from nematodes to humans. The signaling activity of the Smads is regulated by their ligand-stimulated phosphorylation through Ser/Thr kinase receptors. Here, to examine the in vivo role of BMP, we investigated the spatio-temporal activation of BMP-regulated signals during Xenopus development, using a polyclonal antibody that specifically recognizes the phosphorylated form of BMP-regulated Smads. BMP signaling was observed uniformly in embryos as early as stage 7, but was restricted to the ventral side of the embryo at the late blastula stage, supporting the proposed role of BMP4 as a ventralizing factor in Xenopus embryos. In addition, localized staining was detected in several developing organs, consistent with the predicted function of BMP family members in organogenesis.     

Visualization of endogenous BMP signaling during Xenopus development

The TGF-beta superfamily of growth factors is known to transmit signals to the nucleus mainly through the Smads, intracellular signaling components that are highly conserved from nematodes to humans. The signaling activity of the Smads is regulated by their ligand-stimulated phosphorylation through Ser/Thr kinase receptors. Here, to examine the in vivo role of BMP, we investigated the spatio-temporal activation of BMP-regulated signals during Xenopus development, using a polyclonal antibody that specifically recognizes the phosphorylated form of BMP-regulated Smads. BMP signaling was observed uniformly in embryos as early as stage 7, but was restricted to the ventral side of the embryo at the late blastula stage, supporting the proposed role of BMP4 as a ventralizing factor in Xenopus embryos. In addition, localized staining was detected in several developing organs, consistent with the predicted function of BMP family members in organogenesis.     

Regulation of Raf-1-dependent signaling during early Xenopus development.

The Raf-1 gene product is activated in response to cellular stimulation by a variety of growth factors and hormones. Raf-1 activity has been implicated in both cellular differentiation and proliferation. We have examined the regulation of the Raf-1/MEK/MAP kinase (MAPK) pathway during embryonic development in the frog Xenopus laevis. We report that Raf-1, MEK, and MAPK activities are turned off following fertilization and remain undetectable up until blastula stages (stage 8), some 4 h later. Tight regulation of the Raf-1/MEK/MAPK pathway following fertilization is crucial for embryonic cell cycle progression. Inappropriate reactivation of MAPK activity by microinjection of oncogenic Raf-1 RNA results in metaphase cell cycle arrest and, consequently, embryonic lethality. Our findings demonstrate an absolute requirement, in vivo, for inactivation of the MAPK signaling pathway to allow normal cell cycle progression during the period of synchronous cell divisions which occur following fertilization. Further, we show that cytostatic factor effects are mediated through MEK and MAPK.     

Expression of intermediate filament proteins during development of Xenopus laevis. III. Identification of mRNAs encoding cytokeratins typical of complex epithelia

A Xenopus laevis mRNA encoding a cytokeratin of the basic (type II) subfamily that is expressed in postgastrulation embryos was cDNA-cloned and sequenced. Comparison of the deduced amino acid sequence of this polypeptide (513 residues, calculated mol. wt 55,454; Mr approximately 58,000 on SDS-PAGE) with those of other cytokeratins revealed its relationship to certain type II cytokeratins of the same and other species, but also remarkable differences. Using a subclone representing the 3'-untranslated portion of the 2.4 kb mRNA encoding this cytokeratin, designated XenCK55(5/6), in Northern blot experiments, we found that it differs from the only other Xenopus type II cytokeratin known, i.e. the simple epithelium-type component XenCK1(8), in that it is absent in unfertilized eggs and pregastrulation embryos. XenCK55(5/6) mRNA was first detected at gastrulation (stage 11) and found to rapidly increase during neurulation and further development. It was also identified in Xenopus laevis cultured kidney epithelial cells of the line A6 and in the adult animal where it is a major polypeptide in the oesophageal mucosa but absent in most other tissues examined. The pattern of XenCK55(5/6) expression during embryonic development was similar to that reported for the type I polypeptides of the 'XK81 subfamily' previously reported to be embryo-specific and absent in adult tissues. Therefore, we used a XK81 mRNA probe representing the 3'-untranslated region in Northern blots, S1 nuclease and hybrid-selection-translation assays and found the approximately 1.6 kb XK81 mRNA and the resulting protein of Mr approximately 48,000 not only in postgastrula embryos and tadpoles but also in the oesophagus of adult animals. Our results show that both these type II and type I cytokeratins are synthesized only on gastrulation and are very actively produced in early developmental stages but is continued in at least one epithelium of the adult organism. These observations raise doubts on the occurrence of Xenopus cytokeratins that are strictly specific for certain embryonic or larval stages and absent in the adult. They rather suggest that embryonically expressed cytokeratins are also produced in some adult tissues, although in a restricted pattern of tissue and cell type distribution.     

Poly(A) metabolism and polysomal recruitment of maternal mRNAs during early Xenopus development

Up to the midblastula stage, cell division in Xenopus is directed by maternal mRNAs and proteins that are inherited by the fertilized egg. We have isolated cDNA clones for mRNAs that undergo either adenylation or deadenylation during this developmental period. Coincidental with this poly(A) metabolism are changes in polysomal localization. The relationship between polyadenylation and translation is discussed.     

Ribosomal protein mRNAs increase dramatically during Xenopus development

The amount of messenger RNA per microgram of rRNA increases three- to fourfold during Xenopus early development. This increase is the same when measured by stimulation of in vitro protein synthesis or by poly(U) hybridization. The increase in mRNA per embryo therefore is approximately six- to eightfold since the ribosome content doubles between fertilization and the stage 41 tadpole. The amount of ribosomal protein mRNA, as assayed by in vitro protein synthesis, also increases dramatically during early development. This increase is much more pronounced than the general increase in mRNA content, i.e., there is a dramatic increase in the abundance as well as the amount of the ribosomal protein mRNA. Since ribosomal protein mRNAs are predominantly small mRNAs, the increase in ribosomal protein mRNA abundance contributes to the general decrease in the average size of pA⁺ RNA that occurs during early development in Xenopus.     

Regulation of three beta-tubulin mRNAs during rat brain development.

The nucleotide sequence of a complete rat brain beta-tubulin T beta 15 has been determined from three overlapping cDNA clones. The overall length of the T beta 15 sequence is 1589 bp and shows between 84.5% and 88.6% homology within the coding region as compared with chick and human beta-tubulin sequences. On the other hand, the 3'-non-coding region is highly divergent. Comparison of the derived amino acid sequences from different species demonstrates that the amino acid changes are not randomly distributed, but rather there are several conserved and two highly variable regions common to beta-tubulin polypeptides from various sources. The T beta 15 sequence encodes a dominant neuronal 1.8-kb beta-tubulin mRNA species. Two other minor beta-tubulin mRNA species of 2.6 and 2.9 kb are present in rat brain. By using two synthetic oligonucleotide probes complementary to the carboxyl-terminal divergent region and to the amino-terminal conserved region, we have shown that the three mRNAs are distinct species, which are developmentally regulated. The level of the 1.8-kb mRNA species increases till the age of 12 days thereafter its level decreases. The 2.9-kb mRNA is an early neuronal mRNA species, while the 2.6-kb mRNA is a late neuronal species which is detected at 30 days of rat brain development. The data illustrate that there is a differential expression of the beta-tubulin multigene family during rat brain development which may suggest different functions for the various beta-tubulin isotopes.     

Role of TAK1 and TAB1 in BMP signaling in early Xenopus development.

Transforming growth factor-beta (TGF-beta) superfamily members elicit signals through stimulation of serine/threonine kinase receptors. Recent studies of this signaling pathway have identified two types of novel mediating molecules, the Smads and TGF-beta activated kinase 1 (TAK1). Smads were shown to mimic the effects of bone morphogenetic protein (BMP), activin and TGF-beta. TAK1 and TAB1 were identified as a MAPKKK and its activator, respectively, which might be involved in the up-regulation of TGF-beta superfamily-induced gene expression, but their biological role is poorly understood. Here, we have examined the role of TAK1 and TAB1 in the dorsoventral patterning of early Xenopus embryos. Ectopic expression of Xenopus TAK1 (xTAK1) in early embryos induced cell death. Interestingly, however, concomitant overexpression of bcl-2 with the activated form of xTAK1 or both xTAK1 and xTAB1 in dorsal blastomeres not only rescued the cells but also caused the ventralization of the embryos. In addition, a kinase-negative form of xTAK1 (xTAK1KN) which is known to inhibit endogenous signaling could partially rescue phenotypes generated by the expression of a constitutively active BMP-2/4 type IA receptor (BMPR-IA). Moreover, xTAK1KN could block the expression of ventral mesoderm marker genes induced by Smad1 or 5. These results thus suggest that xTAK1 and xTAB1 function in the BMP signal transduction pathway in Xenopus embryos in a cooperative manner.     

Regulation of somitogenesis by Ena/VASP proteins and FAK during Xenopus development

The metameric organization of the vertebrate body plan is established during somitogenesis as somite pairs sequentially form along the anteroposterior axis. Coordinated regulation of cell shape, motility and adhesion are crucial for directing the morphological segmentation of somites. We show that members of the Ena/VASP family of actin regulatory proteins are required for somitogenesis in Xenopus. Xenopus Ena (Xena) localizes to the cell periphery in the presomitic mesoderm (PSM), and is enriched at intersomitic junctions and at myotendinous junctions in somites and the myotome, where it co-localizes with beta1-integrin, vinculin and FAK. Inhibition of Ena/VASP function with dominant-negative mutants results in abnormal somite formation that correlates with later defects in intermyotomal junctions. Neutralization of Ena/VASP activity disrupts cell rearrangements during somite rotation and leads to defects in the fibronectin (FN) matrix surrounding somites. Furthermore, inhibition of Ena/VASP function impairs FN matrix assembly, spreading of somitic cells on FN and autophosphorylation of FAK, suggesting a role for Ena/VASP proteins in the modulation of integrin-mediated processes. We also show that inhibition of FAK results in defects in somite formation, blocks FN matrix deposition and alters Xena localization. Together, these results provide evidence that Ena/VASP proteins and FAK are required for somite formation in Xenopus and support the idea that Ena/VASP and FAK function in a common pathway to regulate integrin-dependent migration and adhesion during somitogenesis.     

BMP and Hedgehog signaling during the development of scleral ossicles

Bone development is a complex process, involving multiple tissues and hierarchical inductive interactions. The study of skeletal development has largely focused on endochondral bones while intramembranous bones, such as the scleral ossicles within the avian eye, have received less attention. Our previous research directly demonstrated the involvement of sonic hedgehog and suggested the involvement of bmp2 and 4 during the development of scleral ossicles. The bones of the sclerotic ring are induced by overlying conjunctival papillae at HH 35 and 36. Here, we examine the spatial and temporal expression patterns of ptc1, ihh, bmp2, bmp4 and bmp7. We show that the cells of conjunctival papillae express ptc1, ihh and bmp2 at these stages; coincident with shh expression previously described. Interestingly, both ihh and ptc1 are also expressed in the mesenchyme underlying the papillae unlike shh and bmp2. Bmp4 and bmp7 are not expressed in these regions at any stages examined. Furthermore, using Noggin soaked beads implanted adjacent to papillae, we provide direct evidence that the BMP family of genes are important factors in the development of scleral ossicles. Localized inhibition of BMPs in this way causes a reduced expression of ihh in the surrounding tissue demonstrating that the BMP and Hedgehog pathways interact. Our data also demonstrates that the sclerotic ring has an intrinsic ability to compensate for missing elements. The scleral ossicle system provides a unique opportunity to investigate the epithelial-mesenchymal induction of intramembranous bones of the vertebrate skull.     

Endogenous patterns of BMP signaling during early chick development

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor beta superfamily signaling molecules that play important roles in a wide variety of developmental processes. In this study, we have used an antibody specific for the phosphorylated and activated form of Smad1 to examine endogenous patterns of BMP signaling in chick embryos during early development. We find complex spatial and temporal distributions of BMP signaling that elucidate how BMPs may function in multiple patterning events in the early chick embryo. In the pregastrula embryo, we find that BMP signaling is initially ubiquitous and is extinguished in the epiblast at the onset of primitive streak formation. At the head process stage, BMP signaling is inactivated in prospective neural plate, while it is strongly activated at the neural plate border, a region which is populated by cells that will give rise to neural crest. During later development, we find a dynamic spatiotemporal activation of BMP signaling along the rostrocaudal axis, in the dorsal neural tube, in the notochord, and in the somites during their maturation process. We discuss the implication of our results for endogenous functions of BMP signaling during chick development.     

Expression of intermediate filament proteins during development of Xenopus laevis. I. cDNA clones encoding different forms of vimentin

To provide a basis for studies of the expression of genes encoding the diverse kinds of intermediate-filament (IF) proteins during embryogenesis of Xenopus laevis we have isolated and characterized IF protein cDNA clones. Here we report the identification of two types of Xenopus vimentin, Vim1 and Vim4, with their complete amino acid sequences as deduced from the cloned cDNAs, both of which are expressed during early embryogenesis. In addition, we have obtained two further vimentin cDNAs (Vim2 and 3) which are sequence variants of closely related Vim1. The high evolutionary conservation of the amino acid sequences (Vim1: 458 residues; Mr approximately 52,800; Vim4: 463 residues; Mr approximately 53,500) to avian and mammalian vimentin and, to a lesser degree, to desmin from the same and higher vertebrate species, is emphasized, including conserved oligopeptide motifs in their head domains. Using these cDNAs in RNA blot and ribonuclease protection assays of various embryonic stages, we observed a dramatic increase of vimentin RNA at stage 14, in agreement with immunocytochemical results obtained with antibody VIM-3B4. The significance of very weak mRNA signals detected in earlier stages is discussed in relation to negative immunocytochemical results obtained in these stages. The first appearance of vimentin has been localized to a distinct mesenchymal cell layer underlying the neural plate or tube, respectively. The results are discussed in relation to programs of de novo synthesis of other cytoskeletal proteins in amphibian and mammalian development.     

Regulation of neural stem cell by bone morphogenetic protein (BMP) signaling during brain development

Neurogenesis is the process in which neurons are generated from neural stem/progenitor cells (NSCs/NPCs). It involves the proliferation and neuronal fate specification/differentiation of NSCs, as well as migration, maturation and functional integration of the neuronal progeny into neuronal network. NSCs exhibit the two essential properties of stem cells: self-renewal and multipotency. Contrary to previous dogma that neurogenesis happens only during development, it is generally accepted now that neurogenesis can take place throughout life in mammalian brains. This raises a new therapeutic potential of applying stem cell therapy for stroke, neurodegenerative diseases and other diseases. However, the maintenance and differentiation of NSCs/NPCs are tightly controlled by the extremely intricate molecular networks. Uncovering the underlying mechanisms that drive the differentiation, migration and maturation of specific neuronal lineages for use in regenerative medicine is, therefore, crucial for the application of stem cell for clinical therapy as well as for providing insight into the mechanisms of human neurogenesis. Here, we focus on the role of bone morphogenetic protein (BMP) signaling in NSCs during mammalian brain development.     

Transgenic Xenopus embryos reveal that anterior neural development requires continued suppression of BMP signaling after gastrulation.

In vertebrates, BMP signaling before gastrulation suppresses neural development. Later in development, BMP signaling specifies a dorsal and ventral fate in the forebrain and dorsal fate in the spinal cord. It is therefore possible that a change in the competence of the ectoderm to respond to BMP signaling occurs at some point in development. We report that exposure of the anterior neural plate to BMP4 before gastrulation causes suppression of all neural markers tested. To determine the effects of BMP4 after gastrulation, we misexpressed BMP4 using a Pax-6 promoter fragment in transgenic frog embryos and implanted beads soaked in BMP4 in the anterior neural plate. Suppression of most anterior neural markers was observed. We conclude that most neural genes continue to require suppression of BMP signaling into the neurula stages. Additionally, we report that BMP4 and BMP7 are abundantly expressed in the prechordal mesoderm of the neurula stage embryo. This poses the paradox of how the expression of most neural genes is maintained if they can be inhibited by BMP signaling. We show that at least one gene in the anterior neural plate suppresses the response of the ectoderm to BMP signaling. We propose that the suppressive effect of BMP signaling on the expression of neural genes coupled with localized suppressors of BMP signaling result in the fine-tuning of gene expression in the anterior neural plate.