Xenopus Expression Research Articles

A Proton-mediated Conformational Shift Identifies a Mobile Pore-lining Cysteine Residue (Cys-561) in Human Concentrative Nucleoside Transporter 3

The concentrative nucleoside transporter (CNT) protein family in humans is represented by three members, hCNT1, hCNT2, and hCNT3. Belonging to a CNT subfamily phylogenetically distinct from hCNT1/2, hCNT3 mediates transport of a broad range of purine and pyrimidine nucleosides and nucleoside drugs, whereas hCNT1 and hCNT2 are pyrimidine and purine nucleoside-selective, respectively. All three hCNTs are Na(+)-coupled. Unlike hCNT1/2, however, hCNT3 is also capable of H(+)-mediated nucleoside cotransport. Using site-directed mutagenesis in combination with heterologous expression in Xenopus oocytes, we have identified a C-terminal intramembranous cysteine residue of hCNT3 (Cys-561) that reversibly binds the hydrophilic thiol-reactive reagent p-chloromercuribenzene sulfonate (PCMBS). Access of this membrane-impermeant probe to Cys-561, as determined by inhibition of hCNT3 transport activity, required H(+), but not Na(+), and was blocked by extracellular uridine. Although this cysteine residue is also present in hCNT1 and hCNT2, neither transporter was affected by PCMBS. We conclude that Cys-561 is located in the translocation pore in a mobile region within or closely adjacent to the nucleoside binding pocket and that access of PCMBS to this residue reports a specific H(+)-induced conformational state of the protein.

Functional Characterization of Pufferfish Slc26a6A and Slc26a6B

Euryhaline pufferfish (Takifugu obscurus, mefugu) can live in freshwater (FW) with ion concentration less than 1/100 of its body fluid or acclimate to seawater (SW) which has more than 3 times the ion concentration of its body fluid. However, the body‐fluid ion concentrations are maintained stable by the function of epithelial cells in the gill, intestine, and kidney which transport ions directionally and selectively. To understand the mechanisms of the fish body‐fluid homeostasis at the molecular level, there are several essentials: identification of transporters expressed in the epithelia, determination of transporter localization, and characterization of transporter activities. Analyses of various transporter mRNA levels in mefugu tissues identified increases for Slc26a6A and Slc26a6B in the intestine and the kidney of SW‐acclimated mefugu. To study their functions for SW acclimation, we subcloned the cDNAs into a Xenopus expression vector and assayed their functions using microelectrodes in oocytes.The electrophysiological analyses of the oocytes revealed that (a) Slc26a6A and Slc26a6B are electrogenic Cl−/nHCO3− exchangers; (b) Slc26a6A and Slc26a6B are also exchangers of Cl−/formate, Cl−/oxalate, and Cl−/SO42−; and (c) the slope of I–V relationship of Slc26a6A in the presence of SO42− is 70 times higher than that of Slc26a6B. These results suggest significant roles for Slc26a6A and Slc26a6B in intestinal HCO3− secretion and a role for Slc26a6A in renal SO42− excretion during SW acclimation.This work was supported by MEXT Japan, NIH (EY017732) and CFF grant (Romero‐06G0).

Partners in Crime

See related article, pages 12–15 Vertebrates have evolved 2 types of blood vessels—arteries and veins—that function to supply oxygen and nutrients and remove cellular waste. To efficiently accomplish these specialized functions, arteries and veins have developed distinct morphological and molecular differences. As a result, the endothelial cells that compose arteries and veins are vastly different in their biochemical and cellular properties. During development, the nascent endothelial cells generated from hemangioblasts or angioblasts1,2 undergo tightly regulated specification and differentiation processes to adopt either the arterial or venous endothelial fate. Differentiated endothelial cells migrate and aggregate to form either arteries or veins according to their adopted fate.3 Dysregulation of these regulated events often results in devastating consequences. For instance, failure to segregate arterial and venous endothelial cells causes potentially fatal clinical conditions such as arteriovenous malformation, hereditary hemorrhagic telangiectasia, and cerebral cavernous malformation.4 Significant progress has been made in understanding how nascent endothelial cells adopt the arterial fate, resulting in identification of several key signaling molecules and their intracellular transducers involved in the specification of arterial endothelial cells.5 However, transcription factors that function …

Conserved Glutamate Residues Are Critically Involved in Na+/Nucleoside Cotransport by Human Concentrative Nucleoside Transporter 1 (hCNT1)

Human concentrative nucleoside transporter 1 (hCNT1), the first discovered of three human members of the SLC28 (CNT) protein family, is a Na+/nucleoside cotransporter with 650 amino acids. The potential functional roles of 10 conserved aspartate and glutamate residues in hCNT1 were investigated by site-directed mutagenesis and heterologous expression in Xenopus oocytes. Initially, each of the 10 residues was replaced by the corresponding neutral amino acid (asparagine or glutamine). Five of the resulting mutants showed unchanged Na+-dependent uridine transport activity (D172N, E338Q, E389Q, E413Q, and D565N) and were not investigated further. Three were retained in intracellular membranes (D482N, E498Q, and E532Q) and thus could not be assessed functionally. The remaining two (E308Q and E322Q) were present in normal quantities at cell surfaces but exhibited low intrinsic transport activities. Charge replacement with the alternate acidic amino acid enabled correct processing of D482E and E498D, but not of E532D, to cell surfaces and also yielded partially functional E308D and E322D. Relative to wild-type hCNT1, only D482E exhibited normal transport kinetics, whereas E308D, E308Q, E322D, E322Q, and E498D displayed increased K50(Na+) and/or Km(uridine) values and diminished Vmax(Na+) and Vmax(uridine) values. E322Q additionally exhibited uridine-gated uncoupled Na+ transport. Together, these findings demonstrate roles for Glu-308, Glu-322, and Glu-498 in Na+/nucleoside cotransport and suggest locations within a common cation/nucleoside translocation pore. Glu-322, the residue having the greatest influence on hCNT1 transport function, exhibited uridine-protected inhibition by p-chloromercuriphenyl sulfonate and 2-aminoethyl methanethiosulfonate when converted to cysteine.

Neural MMP‐28 expression precedes myelination during development and peripheral nerve repair

Mammalian matrix metalloproteinase 28 (MMP-28) is expressed in several normal adult tissues, and during cutaneous wound healing. We show that, in frog and mouse embryos, MMP-28 is expressed predominantly throughout the nervous system. Xenopus expression increases during neurulation and remains elevated through early limb development where it is expressed in nerves. In the mouse, neural expression peaks at embryonic day (E) 14 but remains detectable through E17. During frog hindlimb regeneration XMMP-28 is not initially expressed in the regenerating nerves but is detectable before myelination. Following hindlimb denervation, XMMP-28 expression is detectable along regenerating nerves before myelination. In embryonic rat neuron-glial co-cultures, MMP-28 decreases after the initiation of myelination. Incubation of embryonic brain tissue with purified MMP-28 leads to the degradation of multiple myelin proteins. These results suggest that MMP-28 plays an evolutionarily conserved role in neural development and is likely to modulate the axonal-glial extracellular microenvironment.

Screening for plant transporter function by expressing a normalized Arabidopsis full-length cDNA library in Xenopus oocytes

BackgroundWe have developed a functional genomics approach based on expression cloning in Xenopus oocytes to identify plant transporter function. We utilized the full-length cDNA databases to generate a normalized library consisting of 239 full-length Arabidopsis thaliana transporter cDNAs. The genes were arranged into a 96-well format and optimized for expression in Xenopus oocytes by cloning each coding sequence into a Xenopus expression vector.ResultsInjection of 96 in vitro transcribed cRNAs from the library in pools of columns and rows into oocytes and subsequent screening for glucose uptake activity identified three glucose transporters. One of these, AtSTP13, had not previously been experimentally characterized.ConclusionExpression of the library in Xenopus oocytes, combined with uptake assays, has great potential in assignment of plant transporter function and for identifying membrane transporters for the many plant metabolites where a transporter has not yet been identified.

Characterization of the Transport Mechanism and Permeant Binding Profile of the Uridine Permease Fui1p of Saccharomyces cerevisiae

The uptake of Urd into the yeast Saccharomyces cerevisiae is mediated by Fui1p, a Urd-specific nucleoside transporter encoded by the FUI1 gene and a member of the yeast Fur permease family, which also includes the uracil, allantoin, and thiamine permeases. When Fui1p was produced in a double-permease knock-out strain (fur4Deltafui1Delta) of yeast, Urd uptake was stimulated at acidic pH and sensitive to the protonophore carbonyl cyanide m-chlorophenylhydrazone. Electrophysiological analysis of recombinant Fui1p produced in Xenopus oocytes demonstrated that Fui1p-mediated Urd uptake was dependent on proton cotransport with a 1:1 stoichiometry. Mutagenesis analysis of three charged amino acids (Glu(259), Lys(288), and Asp(474) in putative transmembrane segments 3, 4, and 7, respectively) revealed that only Lys(288) was required for maintaining high Urd transport efficiency. Analysis of binding energies between Fui1p and different Urd analogs indicated that Fuip1 interacted with C(3')-OH, C(2')-OH, C(5)-H, and N(3)-H of Urd. Fui1p-mediated transport of Urd was inhibited by analogs with modifications at C-5', but was not inhibited significantly by analogs with modifications at C-3', C-5, and N-3 or inversions of configuration at C-2' and C-3'. This characterization of Fui1p contributes to the emerging knowledge of the structure and function of the Fur family of permeases, including the Fui1p orthologs of pathogenic fungi.

Effect of Human Carbonic Anhydrase II on the Activity of the Human Electrogenic Na/HCO3 Cotransporter NBCe1-A in Xenopus Oocytes

Others report that carbonic anhydrase II (CA II) binds to the C termini of the anion exchanger AE1 and the electrogenic Na/HCO3 cotransporter NBCe1-A, enhancing transport. After injecting oocytes with NBCe1-A cRNA (Day 0), we measured NBC current (I(NBC)) by two-electrode voltage clamp (Day 3), injected CA II protein + Tris or just Tris (Day 3), measured I(NBC) or the initial rate at which the intracellular pH fell (dpH(i)/dt) upon applying 5% CO2 (Day 4), exposed oocytes to the permeant CA inhibitor ethoxzolamide (EZA), and measured I(NBC) or dpH(i)/dt (Day 4). Because dpH(i)/dt was greater in CA II than Tris oocytes, and EZA eliminated the difference, injected CA II was functional. I(NBC) slope conductance was unaffected by injecting CA II. Moreover, EZA had identical effects in CA II versus Tris oocytes. Thus, injected CA II does not enhance NBC activity. In a second protocol, we made a fusion protein with enhanced green fluorescent protein (EGFP) at the 5' end of NBCe1-A and CA II at the 3' end (EGFP-e1-CAII). We measured I(NBC) or dpH(i)/dt (days 3-4), exposed oocytes to EZA, and measured I(NBC) or dpH(i)/dt (Day 3-4). dpH(i)/dt was greater in oocytes expressing EGFP-e1-CA II versus EGFP-e1, and EZA eliminated the difference. Thus, fused CA II was functional. Slope conductances of EGFP-e1-CAII versus EGFP-e1 oocytes were indistinguishable, and EZA had no effect. Thus, even when fused to NBCe1-A, CA II does not enhance NBCe1-A activity.

Cloning and developmental characterization ofXenopus laevismembrane type-3 matrix metalloproteinase (MT3-MMP)

Proper extracellular matrix (ECM) remodeling, mediated by matrix metalloproteinases (MMPs), is crucial for the development and survival of multicellular organisms. Full-length Xenopus laevis membrane type-3 matrix metallo proteinase (MT3-MMP) was amplified by PCR and cloned from a stage 28 Xenopus head cDNA library. A comparison of the derived Xenopus MT3-MMP protein sequence to that of other vertebrates revealed 86% identity with human and mouse and 85% identity with chicken. The expression profile of MT3-MMP was examined during Xenopus embryogenesis: MT3-MMP transcripts were first detected at the later stages of development and were localized to dorsal and anterior structures. During metamorphosis and in the adult frog, MT3-MMP expression was restricted to specific tissues and organs. Treatment of Xenopus embryos with lithium chloride (LiCl), ultraviolet irradiation (UV), or retinoic acid (RA) revealed that MT3-MMP levels increased with LiCl-dorsalizing treatments and decreased with UV-ventralizing and RA-anterior neural truncating treatments. Overexpression of MT3-MMP through RNA injections led to dose-dependent developmental abnormalities and death. Moreover, MT3-MMP overexpression resulted in neural and head structure abnormalities, as well as truncated axes. Taken together, these results indicate that MT3-MMP expression in Xenopus is spatially and temporally restricted. Furthermore, deregulation of MT3-MMP during early embryogenesis has detrimental effects on development.

A new method to remove hybridization bias for interspecies comparison of global gene expression profiles uncovers an association between mRNA sequence divergence and differential gene expression in Xenopus

The recent sequencing of a large number of Xenopus tropicalis expressed sequences has allowed development of a high-throughput approach to study Xenopus global RNA gene expression. We examined the global gene expression similarities and differences between the historically significant Xenopus laevis model system and the increasingly used X.tropicalis model system and assessed whether an X.tropicalis microarray platform can be used for X.laevis. These closely related species were also used to investigate a more general question: is there an association between mRNA sequence divergence and differences in gene expression levels? We carried out a comprehensive comparison of global gene expression profiles using microarrays of different tissues and developmental stages of X.laevis and X.tropicalis. We (i) show that the X.tropicalis probes provide an efficacious microarray platform for X.laevis, (ii) describe methods to compare interspecies mRNA profiles that correct differences in hybridization efficiency and (iii) show independently of hybridization bias that as mRNA sequence divergence increases between X.laevis and X.tropicalis differences in mRNA expression levels also increase.

Examination of the stress-induced expression of the collagen binding heat shock protein, hsp47, in Xenopus laevis cultured cells and embryos

HSP47 is an endoplasmic reticulum (ER)-resident molecular chaperone involved in collagen production. This study examined the stress-induced pattern of hsp47 gene expression in Xenopus cultured cells and embryos. Sequence analysis revealed that protein encoded by the hsp47 cDNA exhibited 70–77% identity with fish, avian and mammalian HSP47. In A6 kidney epithelial cells hsp47 mRNA and HSP47 were present constitutively and inducible by heat shock but not ER stressors including tunicamycin and A23187, both of which enhanced BiP mRNA. Furthermore A23187 treatment inhibited constitutive accumulation of hsp47 mRNA and retarded heat-induced accumulation of hsp47 and hsp70 mRNA. Interestingly, hsp47 gene expression but not hsp70 or BiP mRNA accumulation was enhanced by treatment with a procollagen-specific stressor, β-aminopropionitrile. In Xenopus embryos hsp47 mRNA was present constitutively throughout development. In tailbud embryos hsp47 mRNA was enriched in tissues associated with collagen production including notochord, somites and head region. Heat shock-induced accumulation of hsp47 mRNA was enhanced primarily in embryonic tissues already exhibiting hsp47 mRNA accumulation. These studies suggest that the pattern of Xenopus hsp47 gene expression is similar to hsp70 in response to heat shock but also displays unique features including a response to a procollagen-specific stressor and preferential expression in collagen-containing tissues.

A C-terminal Domain in KCC2 Confers Constitutive K+-Cl- Cotransport

The neuron-specific K(+)-Cl(-) cotransporter KCC2 plays a crucial role in determining intracellular chloride activity and thus the neuronal response to gamma-aminobutyric acid and glycine. Of the four KCCs, KCC2 is unique in mediating constitutive K(+)-Cl(-) cotransport under isotonic conditions; the other three KCCs are exclusively swelling-activated, with no isotonic activity. We have utilized a series of chimeric cDNAs to localize the determinant of isotonic transport in KCC2. Two generations of chimeric KCC4-KCC2 cDNAs initially localized this characteristic to within a KCC2-specific expansion of the cytoplasmic C terminus, between residues 929 and 1043. This region of KCC2 is rich in prolines, serines, and charged residues and encompasses two predicted PEST sequences. Substitution of this region in KCC2 with the equivalent sequence of KCC4 resulted in a chimeric KCC that was devoid of isotonic activity, with intact swelling-activated transport. A third generation of chimeras demonstrated that a domain just distal to the PEST sequences confers isotonic transport on KCC4. Mutagenesis of this region revealed that residues 1021-1035 of KCC2 are sufficient for isotonic transport. Swelling-activated K(+)-Cl(-) cotransport is abrogated by calyculin A, whereas isotonic transport mediated by KCC chimeras and KCC2 is completely resistant to this serine-threonine phosphatase inhibitor. In summary, a 15-residue C-terminal domain in KCC2 is both necessary and sufficient for constitutive K(+)-Cl(-) cotransport under isotonic conditions. Furthermore, unlike swelling-activated transport, constitutive K(+)-Cl(-) cotransport mediated by KCC2 is completely independent of serine-threonine phosphatase activity, suggesting that these two modes of transport are activated by distinct mechanisms.

Serotonin Signaling Is a Very Early Step in Patterning of the Left-Right Axis in Chick and Frog Embryos

Consistent left-right (LR) asymmetry is a fascinating problem in developmental and evolutionary biology. Conservation of early LR patterning steps among vertebrates as well as involvement of nonprotein small-molecule messengers are very poorly understood. Serotonin (5-HT) is a key neurotransmitter with crucial roles in physiology and cognition. We tested the hypothesis that LR patterning required prenervous serotonin signaling and characterized the 5-HT pathway in chick and frog embryos. A pharmacological screen implicated endogenous signaling through receptors R3 and R4 and the activity of monoamine oxidase (MAO) in the establishment of correct sidedness of asymmetric gene expression and of the viscera in Xenopus embryos. HPLC and immunohistochemistry analysis indicates that Xenopus eggs contain a maternal supply of serotonin that is progressively degraded during cleavage stages. Serotonin's dynamic localization in frog embryos requires gap junctional communication and H,K-ATPase function. Microinjection of loss- and gain-of-function constructs into the right ventral blastomere randomizes asymmetry. In chick embryos, R3 and R4 activity is upstream of the asymmetry of Sonic hedgehog expression. MAO is asymmetrically expressed in the node. Serotonin is present in very early chick and frog embryos. 5-HT pathway function is required for normal asymmetry and is upstream of asymmetric gene expression. The microinjection data reveal asymmetry existing in frog embryos by the 4-cell stage and suggest novel intracellular 5-HT mechanisms. These functional and localization data identify a novel role for the neurotransmitter serotonin and implicate prenervous serotonergic signaling as an obligate aspect of very early left-right patterning conserved to two vertebrate species.

Transdifferentiation of the retinal pigment epithelia to the neural retina by transfer of the Pax6 transcriptional factor

The Pax6 gene plays an important role in eye morphogenesis throughout the animal kingdom. The Pax6 gene and its homologue could form ectopic eyes by targeted expression in Drosophila and Xenopus. Thus, this gene is a master gene for the eye morphogenesis at least in these animals. In the early development of the vertebrate eye, Pax6 is required for the instruction of multipotential progenitor cells of the neural retina (NR). Primitive retinal pigment epithelial (RPE) cells are able to switch their phenotype and differentiate into NR under exogenous intervention, including treatment with fibroblast growth factors (FGFs), and surgical removal of endogenous NR. However, the molecular basis of phenotypic switching is still controversial. Here, we show that Pax6 alone is sufficient to induce transdifferentiation of ectopic NR from RPE cells without addition of FGFs or surgical manipulation. Pax6-mediated transdifferentiation can be induced even at later stages of development. Both in vivo and in vitro studies show that the Pax6 lies downstream of FGF signaling, highlighting the central roles of Pax6 in NR transdifferentiation. Our results provide an evidence of retinogenic potential of nearly mature RPE and a cue for new therapeutic approaches to regenerate functional NR in patients with a visual loss.

Multiple Retropseudogenes from Pluripotent Cell-specific Gene Expression Indicates a Potential Signature for Novel Gene Identification

Oct4, Nanog, and Stella are transcription factors specifically expressed in embryonic stem (ES) cells and germ lineage cells that impart critical functions in the maintenance of pluripotency. Here, we report the excessive frequency and apparent selectivity of retrotransposition of ES cell-specific genes. Six highly homologous pseudogenes for Oct4, 10 for Nanog, and 16 for Stella were identified by nucleotide BLAST (basic local alignment sequence tool) searches against the respective gene mRNA transcripts. Of 15 non-ES cell-specific transcription factor genes, only one had a single pseudogene hit in our screen, emphasizing the apparent selectivity. We present a hypothesis whereby retrotransposition of ES or germ cell-specific genes may reflect an innate predisposition. This is based on the increased probability of germ-line transmission when retrotransposition occurs at a very early stage of development within cells known to contribute to the germ cell lineage. The parental genes for Nanog, Stella, and another embryonic gene, GDF3 are all located on chromosome 12p13 of the human genome, and on chromosome 6 in mouse. Here, we identified an Oct4 pseudogene at the same respective loci in both human and mouse genomes, suggesting functional relevance and indicative of epigenetic regulation. We tested whether the apparent susceptibility for ES cell-specific gene retrotransposition may be extrapolated to a more unified phenomenon, such that a bioinformatic approach may represent a potentially novel strategy for identification of genes with embryonic cell-specific functionality. A preliminary investigation indeed revealed a single gene, previously demonstrated to be responsible for multiple retropseudogenes via germ cell-specific expression in Xenopus.

Myocardin is sufficient and necessary for cardiac gene expression in Xenopus

Myocardin is a cardiac- and smooth muscle-specific cofactor for the ubiquitous transcription factor serum response factor (SRF). Using gain-of-function approaches in the Xenopus embryo, we show that myocardin is sufficient to activate transcription of a wide range of cardiac and smooth muscle differentiation markers in non-muscle cell types. We also demonstrate that, for the myosin light chain 2 gene (MLC2), myocardin cooperates with the zinc-finger transcription factor Gata4 to activate expression. Inhibition of myocardin activity in Xenopus embryos using morpholino knockdown methods results in inhibition of cardiac development and the absence of expression of cardiac differentiation markers and severe disruption of cardiac morphological processes. We conclude that myocardin is an essential component of the regulatory pathway for myocardial differentiation.

Congenital Stationary Night Blindness Type 2 Mutations S229P, G369D, L1068P, and W1440X Alter Channel Gating or Functional Expression of Cav1.4 L-type Ca2+Channels

Mutations in the CACNA1F gene (voltage-dependent L-type calcium channel alpha1F subunit) encoding retinal Ca(v)1.4 L-type Ca2+ channels cause X-linked recessive congenital stationary night blindness type 2 (CSNB2). Many of them are predicted to yield nonfunctional channels. Complete loss of Ca(v)1.4 function is therefore regarded as a pathogenetic mechanism for the impaired signaling from photoreceptors to second-order retinal neurons. We investigated the functional consequences of CSNB2 missense mutations S229P, G369D, and L1068P and the C-terminal truncation mutant W1440X. After expression in Xenopus laevis oocytes or human embryonic kidney tsA-201 cells, inward Ca2+ current (I(Ca)) and inward Ba2+ current (I(Ba)) could be recorded from mutations G369D and L1068P. G369D shifted the half-maximal voltage for channel activation (V(0.5,act)) significantly to more negative potentials (>11 mV), slowed inactivation, and removed Ca2+-dependent inactivation. The L1068P mutant yielded currents only in the presence of the channel activator BayK8644. Currents (I(Ba)) inactivated faster than wild type (WT) and recovered more slowly from inactivation (I(Ba) and I(Ca)). No channel activity could be measured for mutants S229P and W1440X after oocyte expression. No W1440X alpha1 protein was detected after expression in tsA-201 cells, whereas S229P (as well as G369D and L1068P) alpha1 subunits were expressed at levels indistinguishable from WT (n = 3). Our data provide unequivocal evidence that CSNB2 missense mutations can induce severe changes in Ca(v)1.4 function, which may decrease (L1068P and S229P) or even increase (G369D) channel activity. The lower activation range of G369D can explain the reduced dynamic range of photoreceptor signaling. Moreover, we demonstrate that loss of channel function of one (L1068P) CSNB2 mutation can be rescued by a Ca2+ channel activator.

Myocardin is sufficient and necessary for cardiac gene expression in Xenopus

Myocardin is sufficient and necessary for cardiac gene expression in Xenopus

SOX7 is an immediate-early target of VegT and regulates Nodal-related gene expression in Xenopus

In zebrafish, the divergent F-type SOX casanova acts downstream of Nodal signaling to specify endoderm. While no casanova orthologs have been identified in tetrapods, the F-type SOX, SOX7, is supplied maternally in Xenopus (Fawcett and Klymkowsky, 2004. GER 4, 29). Subsequent RT-PCR and section-based in situ hybridization analyses indicate that SOX7 mRNA is localized to the vegetal region of the blastula-stage embryo. Overexpression and maternal depletion studies reveal that the T-box transcription factor VegT, which initiates mesoendodermal differentiation, directly regulates SOX7 expression. SOX7, but not SOX17 (another F-type SOX), binds to sites within the Xnr5 promoter and SOX7, but not SOX17, induces expression of the Nodal-related genes Xnr1, Xnr2, Xnr4, Xnr5, and Xnr6, the homeodomain transcription factor Mixer, and the endodermal marker SOX17β; both SOX7 and SOX17 induce expression of the pan-endodermal marker endodermin. SOX7's induction of Xnr expression in animal caps is independent of Mixer and Nodal signaling. In animal caps, VegT's ability to induce Mixer and Edd appears to depend upon SOX7 activity. Whole embryo experiments suggests that vegetal factors partially compensate for the absence of SOX7. Based on the antagonistic effects of SOX7 and SOX3 (Zhang et al., 2004. Dev. Biol. 273, 23) and their common binding sites in the Xnr5 promoter, we propose a model in which competitive interactions between these two proteins are involved in refining the domain of endodermal differentiation.

XMAN1, an inner nuclear membrane protein, antagonizes BMP signaling by interacting with Smad1 in Xenopus embryos.

A family of inner nuclear membrane proteins is implicated in gene regulation by interacting with chromatin, nuclear lamina and intranuclear proteins; however, the physiological functions of these proteins are largely unknown. Using a Xenopus expression screening approach with an anterior neuroectoderm cDNA library, we have identified an inner nuclear membrane protein, XMAN1, as a novel neuralizing factor that is encoded by the Xenopus ortholog of human MAN1. XMAN1 mRNA is expressed maternally, and appears to be restricted to the entire ectoderm at the early gastrula stage, then to the anterior neuroectoderm at the neurula stage. XMAN1 induces anterior neural markers without mesoderm induction in ectodermal explants, and a partial secondary axis when expressed ventrally by dorsalizing the ventral mesoderm. Importantly, XMAN1 antagonizes bone morphogenetic protein (BMP) signaling downstream of its receptor Alk3, as judged by animal cap assays, in which XMAN1 blocks expression of downstream targets of BMP signaling (Xhox3 and Msx1), and by luciferase reporter assays, in which XMAN1 suppresses BMP-dependent activation of the Xvent2 promoter. Deletion mutant analyses reveal that the neuralizing and BMP-antagonizing activities of XMAN1 reside in the C-terminal region, and that the C-terminal region binds to Smad1, Smad5 and Smad8, which are intracellular mediators of the BMP pathway. Interference with endogenous XMAN1 functions with antisense morpholino oligos leads to the reduction of anterior neuroectoderm. These results provide the first evidence that the nuclear envelope protein XMAN1 acts as a Smad-interacting protein to antagonize BMP signaling during Xenopus embryogenesis.