Xenopus Neural Development Research Articles

FGF/MAPK/Ets signaling in Xenopus ectoderm contributes to neural induction and patterning in an autonomous and paracrine manner, respectively.

FGF and anti-BMP signals from the Spemann organizer of mesodermal origin are essential for Xenopus neural development from gastrula ectoderm. However, the detailed cellular and molecular mechanisms of signaling, especially those underlying the neural induction process, are still controversial. We show here that the expression of early neural marker genes such as sox2 and otx2 is suppressed both in vivo and in vitro, when ectoderm cells are loaded with a dominant-negative construct of Ets transcription factors or a translation-blocking antisense FGF2 MO or FGF8 MO, respectively. This indicates that the expression of these FGF signaling molecules in ectoderm cells contributes significantly to neural induction, in contrast to the "neural default model" that has been emphasized, in which anti-BMP signaling from the organizer plays a major role in the neural induction in Xenopus. Our results indicate that cell-autonomous FGF signaling between ectoderm cells, rather than paracrine signaling from organizer cells, directly induces the expression of sox2 and otx2 via the FGF2, FGF8/MAPK/Ets pathway at very low signaling levels. This is independent of inhibiting BMP signaling via the FGF/MAPK/Smad pathway, which has been proposed as the primary pathway for FGF signaling in Xenopus neural induction. Using cultured ectoderm cells, we also obtained results suggesting that the mode of contribution of FGF signaling to neural development is altered during the subsequent neural patterning stage. As we increase the amounts of FGF in the culture medium, anterior neural genes activated at low FGF signaling levels are suppressed, and instead position-specific, more posterior neural genes are activated in a dose-dependent manner via the FGF/MAPK/Ets pathway. These results support the claim that morphogenic FGFs from the organizer diffuse in a paracrine manner through the plane of the induced neuroectoderm, causing anteroposterior patterning of neural tissue.

The dual-specificity protein kinase Clk3 is essential for Xenopus neural development

During vertebrate development, the formation of the central nervous system (CNS) is initiated by neural induction and patterning of the embryonic ectoderm. We previously reported that Cdc2-like kinase 2 (Clk2) promotes neural development in Xenopus embryos by regulating morphogen signaling. However, the functions of other Clk family members and their roles in early embryonic development remain unknown. Here, we show that in addition to Clk2, Clk1 and Clk3 play a role in the formation of neural tissue in Xenopus. clk1 and clk3 are co-expressed in the developing neural tissue during early Xenopus embryogenesis. We found that overexpression of clk1 and clk3 increases the expression of neural marker genes in ectodermal explants. Furthermore, knockdown experiments showed that clk3 is required for the formation of neural tissues. These results suggest that Xenopus Clk3 plays an essential role in promoting neural development during early embryogenesis.

Dual roles of Akirin2 protein during Xenopus neural development

To ensure correct spatial and temporal patterning, embryos must maintain pluripotent cell populations and control when cells undergo commitment. The newly identified nucleoprotein Akirin has been shown to modulate the innate immune response through epigenetic regulation and to play important roles in other physiological processes, but its role in neural development remains unknown. Here we show that Akirin2 is required for neural development in Xenopus and that knockdown of Akirin2 expands the expression of the neural progenitor marker Sox2 and inhibits expression of the differentiated neuronal marker N-tubulin. Akirin2 acts antagonistically to Geminin, thus regulating Sox2 expression, and maintains the neural precursor state by participating in the Brg1/Brm-associated factor (BAF) complex mediated by BAF53a. Additionally, Akirin2 also modulates N-tubulin expression by acting upstream of neuronal differentiation 1 (NeuroD) and in parallel with neurogenin-related 1 (Ngnr1) during terminal neuronal differentiation. Thus, our results reveal a novel model in which Akirin2 precisely coordinates and temporally controls Xenopus neural development.

40LoVe and Samba Are Involved in Xenopus Neural Development and Functionally Distinct from hnRNP AB

Heterogeneous nuclear ribonucleoproteins (hnRNPs) comprise a large group of modular RNA-binding proteins classified according to their conserved domains. This modular nature, coupled with a large choice of alternative splice variants generates functional diversity. Here, we investigate the biological differences between 40LoVe, its splice variant Samba and its pseudoallele hnRNP AB in neural development. Loss of function experiments lead to defects in neural development with reduction of eye size, which stem primarily from increased apoptosis and reduced proliferation in neural tissues. Despite very high homology between 40LoVe/Samba and hnRNP AB, these proteins display major differences in localization, which appear to be in part responsible for functional differences. Specifically, we show that the 40Love/Samba carboxy-terminal domain (GRD) enables nucleocytoplasmic shuttling behavior. This domain is slightly different in hnRNP AB, leading to nuclear-restricted localization. Finally, we show that shuttling is required for 40LoVe/Samba function in neural development.

Nuclear Envelope Phosphatase 1-Regulatory Subunit 1 (Formerly TMEM188) Is the Metazoan Spo7p Ortholog and Functions in the Lipin Activation Pathway

Lipin-1 catalyzes the formation of diacylglycerol from phosphatidic acid. Lipin-1 mutations cause lipodystrophy in mice and acute myopathy in humans. It is heavily phosphorylated, and the yeast ortholog Pah1p becomes membrane-associated and active upon dephosphorylation by the Nem1p-Spo7p membrane complex. A mammalian ortholog of Nem1p is the C-terminal domain nuclear envelope phosphatase 1 (CTDNEP1, formerly "dullard"), but its Spo7p-like partner is unknown, and the need for its existence is debated. Here, we identify the metazoan ortholog of Spo7p, TMEM188, renamed nuclear envelope phosphatase 1-regulatory subunit 1 (NEP1-R1). CTDNEP1 and NEP1-R1 together complement a nem1Δspo7Δ strain to block endoplasmic reticulum proliferation and restore triacylglycerol levels and lipid droplet number. The two human orthologs are in a complex in cells, and the amount of CTDNEP1 is increased in the presence of NEP1-R1. In the Caenorhabditis elegans embryo, expression of nematode CTDNEP1 and NEP1-R1, as well as lipin-1, is required for normal nuclear membrane breakdown after zygote formation. The expression pattern of NEP1-R1 and CTDNEP1 in human and mouse tissues closely mirrors that of lipin-1. CTDNEP1 can dephosphorylate lipins-1a, -1b, and -2 in human cells only in the presence of NEP1-R1. The nuclear fraction of lipin-1b is increased when CTDNEP1 and NEP1-R1 are co-expressed. Therefore, NEP1-R1 is functionally conserved from yeast to humans and functions in the lipin activation pathway.

Competition for ligands between FGFR1 and FGFR4 regulates Xenopus neural development

Cell-surface-localized receptors and their extracellular ligands usually comprise distinct families and promote diversity of signal transduction regulation. The number of available ligand molecules is often the limiting factor for receptor activation during interpretation of the signal by the responding cell. Limited ligand availability in a particular area of tissue should lead to local competition between different members of a receptor family for binding and subsequent activation. Fibroblast growth factor receptor (FGFR) 4 (FGFR4) is a less potent activator of downstream pathways than FGFR1, the major subtype of FGFR. Regional expression of Xenopus FGFR1 and FGFR4 (XFGFR1 and XFGFR4, respectively) overlap in the anterior part of prospective and developing neural tissue. In this paper we show that XFGFR1 and XFGFR4 have opposing effects on the positioning of expression domains of mid- and hindbrain markers when the expression levels of the receptors are altered. We present a line of evidence to support our hypothesis that competition between XFGFR1 and XFGFR4 for ligands is required for correct positioning of marker expression. Local competition between receptors with different potencies should provide an efficient means for a cell to interpret the ligand signal correctly, and may constitute a more general mechanism for regulating signal transduction.

Involvement of an inner nuclear membrane protein, Nemp1, in Xenopus neural development through an interaction with the chromatin protein BAF

To clarify the molecular mechanisms of neural development in vertebrates, we analyzed a novel gene, termed nemp1 ( nuclear envelope integral membrane protein 1), which is expressed in the Xenopus anterior neuroectoderm at the neurula stage. Nemp1 has a putative signal peptide and five transmembrane domains, but does not have any other known domains. We show that Nemp1 is localized to the inner nuclear membrane (INM) with its evolutionarily conserved C-terminal region facing the nucleoplasm. Both overexpression and knockdown of Nemp1 in Xenopus embryos reduced the expression of early eye marker genes, rax, tbx3, and pax6, and later resulted mainly in severe eye defects at the tailbud stage. In contrast, the expression of a forebrain/midbrain marker, otx2, and a pan-neural marker, sox2, was largely unaffected. Deletion analysis of Nemp1 showed that nuclear envelope-localization of the C-terminal region is necessary for its eye-reducing activity. Furthermore, nemp1 is coexpressed with baf (barrier-to-autointegration factor) in the eye anlagen, and that Nemp1 interacts with BAF through the BAF-binding site in the C-terminal region and this site is required for Nemp1 activity. These data suggest that Nemp1 is involved in the expression of eye marker genes by functioning at the INM at least partly through BAF.

Identification and expression of XRTN1‐A and XRTN1‐C in Xenopus laevis

Reticulon1, also known as neuroendocrine-specific protein, belongs to the reticulon (RTN) family, whose members possess a conserved reticulon domain and are associated with the endoplasmic reticulum (ER) membrane. Here, we report cloning and expression of Xenopus homologues of Reticulon1-A (XRTN1-A) and -C (XRTN1-C). XRTN1-A and -C contain an open reading frame of 752 and 207 amino acids, respectively, each containing a conserved reticulon domain. Sequence analysis shows that XRTN1 proteins have an ER membrane retention signal and four putative membrane-spanning domains. Reverse transcription-polymerase chain reaction and whole-mount in situ hybridization showed that XRTN1-A is expressed in early neural precursors and differentiating neuronal populations, including the trigeminal placode, olfactory placode, lateral line placode, and otic vesicle. XRTN1-C is expressed in the developing brain and spinal cord. We found that XRTN1-C protein is localized to the ER of Xenopus and mammalian cells and the granules in neurites of primary neurons of the Xenopus spinal cord and rat hippocampus. We also showed that XRTN1-C protein is detected in the heavy membrane fraction, which contains lysosomal and ER-resident proteins, as well as in the nucleus and polysomal fractions of the Xenopus embryo. Finally, we showed that thyroid hormone specifically down-regulates XRTN1-A mRNA in the head of premetamorphic Xenopus tadpoles. Our work characterizes the intracellular roles of XRTN1 during Xenopus neural development.

Fibroblast Growth Factor 13 Is Essential for Neural Differentiation in Xenopus Early Embryonic Development

In Xenopus embryonic development, the MEK5-ERK5 pathway, one of the MAPK pathways, lies downstream of SoxD and upstream of Xngnr1 in a signaling pathway regulating neural differentiation. It remains unclear, however, how the MEK5-ERK5 pathway is regulated in Xenopus neural development. As SoxD is a transcription factor, we hypothesized that some growth factor should be induced by SoxD and activate the MEK5-ERK5 pathway. As the expression level of fibroblast growth factor 13 (FGF13) is increased by SoxD, we analyzed the function of FGF13 in neural development. Knockdown of FGF13 with antisense morpholino-oligonucleotides (MOs) results in the reduced head structure and inhibition of neural differentiation. FGF13 MOs inhibit the SoxD-induced expression of Xngnr1 and the Xngnr1-induced expression of NeuroD, suggesting that FGF13 is necessary both upstream and downstream of Xngnr1 in neural differentiation. In addition, FGF13 MOs inhibit the activation of the MEK5-ERK5 pathway by dominant-negative bone morphogenetic protein receptor, a mimicker of neural inducers, indicating that FGF13 is involved in the activation of the MEK5-ERK5 pathway. Together, these results identify a role of FGF13 in Xenopus neural differentiation.

Balancing BMP Signaling through Integrated Inputs into the Smad1 Linker

FGF and other Ras/MAPK pathway activators counterbalance BMP action during neurogenesis, bone formation, and other aspects of vertebrate development and homeostasis. BMP receptors signal through C-terminal phosphorylation and nuclear translocation of the transcription factor Smad1, whereas MAPKs catalyze inhibitory phosphorylation in the Smad1 linker region. Here we show that linker phosphorylation restricts Smad1 activity by enabling Smad1 recognition by the HECT-domain ubiquitin ligase Smurf1. Besides causing Smad1 polyubiquitination, Smurf1 binding inhibits the interaction of Smad1 with the nuclear translocation factor Nup214. Consequently, MAPK-dependent Smurf1 binding leads Smad1 alternatively to degradation or cytoplasmic retention. Smad1 linker phosphorylation and Smurf1 act as interdependent inputs to control BMP signaling during mouse osteoblast differentiation and Xenopus neural development. Linker phosphorylation is triggered also by BMP, providing feedback control. The interplay between linker phosphorylation, Smurf-dependent ubiquitination, and nucleoporin exclusion enables regulation of BMP action by diverse signals and biological contexts.

Cloning and characterization of Xenopus Id4 reveals differing roles for Id genes

We have identified Xenopus Id4, a member of the Id (inhibitor of differentiation/DNA binding) class of helix–loop–helix proteins. Id factors dimerize with general bHLH factors, preventing their interaction with tissue-specific bHLH factors, to inhibit premature differentiation. The presence of several Id proteins could reflect simple redundancy in function, or more interestingly, might suggest different activities for these proteins. During embryonic development, Xenopus Id4 is expressed in a number of neural tissues, including Rohon–Beard neurons, olfactory placode, eye primordia, and the trigeminal ganglia. It is also expressed in other organs, such as the pronephros and liver primordium. As embryogenesis progresses, it is expressed in the migrating melanocytes and lateral line structures. We compare the expression of Id4 mRNA with that of Id2 and Id3 and find that the Id genes are expressed in complementary patterns during neurogenesis, myogenesis, kidney development, in the tailbud, and in the migrating neural crest. To examine the regulation of Id gene expression during Xenopus neural development, we show that expression of Id3 and Id4 can be induced by overexpression of BMP4 in the whole embryo and in ectodermal explants. Expression of Id2, Id3, and Id4 in these explants is unaffected by the expression of FGF-8 or a dominant-negative Ras (N17ras), suggesting that Id genes are not regulated by the FGF signaling pathway in naive ectoderm. We also show that Notch signaling can activate Id2 and Id3 expression in the whole embryo. In contrast, Id4 expression in the Rohon–Beard cells is inhibited by activated Notch and increased by a dominant-negative Delta. This may reflect an increase in Rohon–Beard cells in response to inhibition of Notch signaling rather than transcriptional regulation of Id4. Finally, to compare the activities of Id2, Id3, and Id4, we use animal cap explants and in vivo overexpression to show that Id proteins can differentially inhibit the activities of neurogenin and neuroD, both neurogenic bHLH molecules and MyoD, a myogenic bHLH protein. Id4 is able to inhibit the activity all these bHLH molecules, Id2 inhibits MyoD and neuroD, while Id3 blocks only neuroD activity in our assays.

Promotore analysis of XKROX-20 gene expression during xenopus neural development

Promotore analysis of XKROX-20 gene expression during xenopus neural development

Transcriptional regulation of the XCAD-3 gene during xenopus neural development is mediated by elements in the 5′ flanking sequence and the first nitron

Transcriptional regulation of the XCAD-3 gene during xenopus neural development is mediated by elements in the 5′ flanking sequence and the first nitron

Thyroid Hormone-dependent Gene Expression Program for Xenopus Neural Development

Although thyroid hormone (TH) plays a significant role in vertebrate neural development, the molecular basis of TH action on the brain is poorly understood. Using polymerase chain reaction-based subtractive hybridization we isolated 34 cDNAs for TH-regulated genes in the diencephalon of Xenopus tadpoles. Northern blots verified that the mRNAs are regulated by TH and are expressed during metamorphosis. Kinetic analyses showed that most of the genes are up-regulated by TH within 4-8 h and 13 are regulated by TH only in the brain. All cDNA fragments were sequenced and the identities of seven were determined through homology with known genes; an additional five TH-regulated genes were identified by hybridization with known cDNA clones. These include five transcription factors (including two members of the steroid receptor superfamily), a TH-converting deiodinase, two metabolic enzymes, a protein disulfide isomerase-like protein that may bind TH, a neural-specific cytoskeletal protein, and two hypophysiotropic neuropeptides. This is the first successful attempt to isolate a large number of TH-target genes in the developing vertebrate brain. The gene identities allow predictions about the gene regulatory networks underlying TH action on the brain, and the cloned cDNAs provide tools for understanding the basic molecular mechanisms underlying neural cell differentiation.