Mechanical Tensions Regulate Gene Expression in the Xenopus laevis Axial Tissues.

During gastrulation and neurulation, the chordamesoderm and overlying neuroectoderm of vertebrate embryos converge under the control of a specific genetic programme to the dorsal midline, simultaneously extending along it. However, whether mechanical tensions resulting from these morphogenetic movements play a role in long-range feedback signaling that in turn regulates gene expression in the chordamesoderm and neuroectoderm is unclear. In the present work, by using a model of artificially stretched explants of Xenopus midgastrula embryos and full-transcriptome sequencing, we identified genes with altered expression in response to external mechanical stretching. Importantly, mechanically activated genes appeared to be expressed during normal development in the trunk, i.e., in the stretched region only. By contrast, genes inhibited by mechanical stretching were normally expressed in the anterior neuroectoderm, where mechanical stress is low. These results indicate that mechanical tensions may play the role of a long-range signaling factor that regulates patterning of the embryo, serving as a link coupling morphogenesis and cell differentiation.

Insulin Receptor-Related Receptor Regulates the Rate of Early Development in Xenopus laevis.

The orphan insulin receptor-related receptor (IRR) encoded by insrr gene is the third member of the insulin receptor family, also including the insulin receptor (IR) and the insulin-like growth factor receptor (IGF-1R). IRR is the extracellular alkaline medium sensor. In mice, insrr is expressed only in small populations of cells in specific tissues, which contain extracorporeal liquids of extreme pH. In particular, IRR regulates the metabolic bicarbonate excess in the kidney. In contrast, the role of IRR during Xenopus laevis embryogenesis is unknown, although insrr is highly expressed in frog embryos. Here, we examined the insrr function during the Xenopus laevis early development by the morpholino-induced knockdown. We demonstrated that insrr downregulation leads to development retardation, which can be restored by the incubation of embryos in an alkaline medium. Using bulk RNA-seq of embryos at the middle neurula stage, we showed that insrr downregulation elicited a general shift of expression towards genes specifically expressed before and at the onset of gastrulation. At the same time, alkali treatment partially restored the expression of the neurula-specific genes. Thus, our results demonstrate the critical role of insrr in the regulation of the early development rate in Xenopus laevis.

Cytoskeletal Protein Zyxin Inhibits the Activity of Genes Responsible for Embryonic Stem Cell Status.

Zyxin is a cytoskeletal LIM-domain protein that regulates actin cytoskeleton assembly and gene expression. In the present work, we find that zyxin downregulation in Xenopus laevis embryos reduces the expression of numerous genes that regulate cell differentiation, but it enhances that of several genes responsible for embryonic stem cell status, specifically klf4, pou5f3.1, pou5f3.2, pou5f3.3, and vent2.1/2. For pou5f3 family genes (mammalian POU5F1/OCT4 homologs), we show that this effect is the result of mRNA stabilization due to complex formation with the Y-box protein Ybx1. When bound to Ybx1, zyxin interferes with the formation of these complexes, thereby stimulating pou5f3 mRNA degradation. In addition, in zebrafish embryos and human HEK293 cells, zyxin downregulation increases mRNA levels of the pluripotency genes KLF4, NANOG, and POU5F1/OCT4. Our findings indicate that zyxin may play a role as a switch among morphogenetic cell movement, differentiation, and embryonic stem cell status.

Agr2-interacting Prod1-like protein Tfp4 from Xenopus laevis is necessary for early forebrain and eye development as well as for the tadpole appendage regeneration.

The Agr family genes, Ag1, Agr2, and Agr3, encode for the thioredoxin domain containing secreted proteins and are specific only for vertebrates. These proteins are attracting increasing attention due to their involvement in many physiological and pathological processes, including exocrine secretion, cancer, regeneration of the body appendages, and the early brain development. At the same time, the mode by which Agrs regulate intracellular processes are poorly understood. Despite that the receptor to Agr2, the membrane anchored protein Prod1, has been firstly discovered in Urodeles, and it has been shown to interact with Agr2 in the regenerating limb, no functional homologs of Prod1 were identified in other vertebrates. This raises the question of the mechanisms by which Agrs can regulate regeneration in other lower vertebrates. Recently, we have identified that Tfp4 (three-fingers Protein 4), the structural and functional homolog of Prod1 in Anurans, interacts with Agr2 in Xenopus laevis embryos. In the present work we show by several methods that the activity of Tfp4 is essential for the tadpole tail regeneration as well as for the early eye and forebrain development during embryogenesis. These data show for the first time the common molecular mechanism of regeneration regulation in amphibians by interaction of Prod1 and Agr2 proteins.

Genetically encodable bioluminescent system from fungi.

Bioluminescence is found across the entire tree of life, conferring a spectacular set of visually oriented functions from attracting mates to scaring off predators. Half a dozen different luciferins, molecules that emit light when enzymatically oxidized, are known. However, just one biochemical pathway for luciferin biosynthesis has been described in full, which is found only in bacteria. Here, we report identification of the fungal luciferase and three other key enzymes that together form the biosynthetic cycle of the fungal luciferin from caffeic acid, a simple and widespread metabolite. Introduction of the identified genes into the genome of the yeast Pichia pastoris along with caffeic acid biosynthesis genes resulted in a strain that is autoluminescent in standard media. We analyzed evolution of the enzymes of the luciferin biosynthesis cycle and found that fungal bioluminescence emerged through a series of events that included two independent gene duplications. The retention of the duplicated enzymes of the luciferin pathway in nonluminescent fungi shows that the gene duplication was followed by functional sequence divergence of enzymes of at least one gene in the biosynthetic pathway and suggests that the evolution of fungal bioluminescence proceeded through several closely related stepping stone nonluminescent biochemical reactions with adaptive roles. The availability of a complete eukaryotic luciferin biosynthesis pathway provides several applications in biomedicine and bioengineering.

HMG-box factor SoxD/Sox15 and homeodomain-containing factor Xanf1/Hesx1 directly interact and regulate the expression of Xanf1/Hesx1 during early forebrain development in Xenopus laevis.

The homeodomain-containing transcription factor Anf (also known as Rpx/Hesx1 in mammals) plays an important role during the forebrain development in vertebrates. Here we demonstrate the ability of the Xenopus laevis Anf, Xanf1/Hesx1, to directly bind SRY-related HMG-box-containing transcription factor SoxD/Sox15 and to cooperate with the latter during regulating of the expression of Xanf1/Hesx1 own gene. As we have shown by GST pull-down, EMSA and the luciferase reporter assays, Xanf1/Hesx1 and SoxD/Sox15 bind the Xanf1/Hesx1 promoter region counteracting the inhibitory effect of Xanf1/Hesx1 alone. This finding explains how Xanf1/Hesx1 could escape the repressive activity of its own protein during early patterning of the forebrain rudiment.

Mechano-sensitive regulation of gene expression during the embryonic development.

Cell movements during embryogenesis produce mechanical tensions that shape the embryo and can also regulate gene expression, thereby affecting cell differentiation. Increasing evidence indicates that mechanosensitive regulation of gene expression plays important roles during embryogenesis by coupling the processes of morphogenesis and differentiation. However, the molecular mechanisms of this phenomenon remain poorly understood. This review focuses on the molecular mechanisms that "translate" mechanical stimuli into gene expression.

The presence of Anf/Hesx1 homeobox gene in lampreys suggests that it could play an important role in emergence of telencephalon.

Accumulated evidence indicates that the core genetic mechanisms regulating early patterning of the brain rudiment in vertebrates are very similar to those operating during development of the anterior region of invertebrate embryos. However, the mechanisms underlying the morphological differences between the elaborate vertebrate brain and its simpler invertebrate counterpart remain poorly understood. Recently, we hypothesized that the emergence of the most anterior unit of the vertebrate brain, the telencephalon, could be related to the appearance in vertebrates' ancestors of a unique homeobox gene, Anf/Hesx1(further Anf), which is absent from all invertebrates and regulates the earliest steps of telencephalon development in vertebrates. However, the failure of Anf to be detected in one of the most basal extant vertebrate species, the lamprey, seriously compromises this hypothesis. Here, we report the cloning of Anf in three lamprey species and demonstrate that this gene is indeed expressed in embryos in the same pattern as in other vertebrates and executes the same functions by inhibiting the expression of the anterior general regulator Otx2 in favour of the telencephalic regulator FoxG1. These results are consistent with the hypothesis that the Anf homeobox gene may have been important in the evolution of the telencephalon.

Noggin4 is a long-range inhibitor of Wnt8 signalling that regulates head development in Xenopus laevis.

Noggin4 is a Noggin family secreted protein whose molecular and physiological functions remain unknown. In this study, we demonstrate that in contrast to other Noggins, Xenopus laevis Noggin4 cannot antagonise BMP signalling; instead, it specifically binds to Wnt8 and inhibits the Wnt/ß -catenin pathway. Live imaging demonstrated that Noggin4 diffusivity in embryonic tissues significantly exceeded that of other Noggins. Using the Fluorescence Recovery After Photobleaching (FRAP) assay and mathematical modelling, we directly estimated the affinity of Noggin4 for Wnt8 in living embryos and determined that Noggin4 fine-tune the Wnt8 posterior-to-anterior gradient. Our results suggest a role for Noggin4 as a unique, freely diffusing, long-range inhibitor of canonical Wnt signalling, thus explaining its ability to promote head development.

The cytoskeletal protein Zyxin inhibits Shh signaling during the CNS patterning in Xenopus laevis through interaction with the transcription factor Gli1.

Zyxin is a cytoskeletal protein that controls cell movements by regulating actin filaments assembly, but it can also modulate gene expression owing to its interactions with the proteins involved in signaling cascades. Therefore, identification of proteins that interact with Zyxin in embryonic cells is a promising way to unravel mechanisms responsible for coupling of two major components of embryogenesis: morphogenetic movements and cell differentiation. Now we show that in Xenopus laevis embryos Zyxin can bind to and suppress activity of the primary effector of Sonic hedgehog (Shh) signaling cascade, the transcription factor Gli1. By using loss- and gain-of-function approaches, we demonstrate that Zyxin is essential for reduction of Shh signaling within the dorsal part of the neural tube of X. laevis embryo. Thus, our finding discloses a novel function of Zyxin in fine tuning of the central neural system patterning which is based on the ventral-to-dorsal gradient of Shh signaling.

Noggin4 expression during chick embryonic development.

We describe here the expression pattern of Noggin4 during the early development of the chick embryo (Gallus gallus). The expression of this gene starts with the onset of gastrulation (stage HH4), in two bilateral bands along the primitive streak, with a local maximum around Hensen's node. By the end of gastrulation, Noggin4 transcripts are distributed diffusely throughout the epiblast, with the highest concentration in the head ectoderm. Interestingly, the expression of Noggin4 during the first half of gastrulation demonstrates a clear left-right asymmetry in Hensen's node, being much more intensive in its right anterior portion. During neurulation, Noggin4 is expressed mainly in the neuroectoderm, with the most intensive expression in the head and lateral neural folds. In mesoderm derivatives, expression is seen in somites but not in the notochord. In general, primarily ectodermal and diffusive expression of Noggin4 in chick embryo, with a maximum in the anterior neurectoderm, resembles that of its ortholog in Xenopus, which indicates a conservative function of this gene in evolution.

Novel functions of Noggin proteins: inhibition of Activin/Nodal and Wnt signaling.

The secreted protein Noggin1 is an embryonic inducer that can sequester TGFß cytokines of the BMP family with extremely high affinity. Owing to this function, ectopic Noggin1 can induce formation of the headless secondary body axis in Xenopus embryos. Here, we show that Noggin1 and its homolog Noggin2 can also bind, albeit less effectively, to ActivinB, Nodal/Xnrs and XWnt8, inactivation of which, together with BMP, is essential for the head induction. In support of this, we show that both Noggin proteins, if ectopically produced in sufficient concentrations in Xenopus embryo, can induce a secondary head, including the forebrain. During normal development, however, Noggin1 mRNA is translated in the presumptive forebrain with low efficiency, which provides the sufficient protein concentration for only its BMP-antagonizing function. By contrast, Noggin2, which is produced in cells of the anterior margin of the neural plate at a higher concentration, also protects the developing forebrain from inhibition by ActivinB and XWnt8 signaling. Thus, besides revealing of novel functions of Noggin proteins, our findings demonstrate that specification of the forebrain requires isolation of its cells from BMP, Activin/Nodal and Wnt signaling not only during gastrulation but also at post-gastrulation stages.

The LIM-domain protein Zyxin binds the homeodomain factor Xanf1/Hesx1 and modulates its activity in the anterior neural plate of Xenopus laevis embryo.

The question of how subdivision of embryo into cell territories acquiring different fates is coordinated with morphogenetic movements shaping the embryonic body still remains poorly resolved. In the present report, we demonstrate that a key regulator of anterior neural plate patterning, the homeodomain transcriptional repressor Xanf1/Hesx1, can bind to the LIM-domain protein Zyxin, which is known to regulate cell morphogenetic movements via influence on actin cytoskeleton dynamics. Using a set of deletion mutants, we found that the Engrailed-type repressor domain of Xanf1 and LIM2-domain of Zyxin are primarily responsible for interaction of these proteins. We also demonstrate that Zyxin overexpression in Xenopus embryos elicits effects similar to those observed in embryos with downregulated Xanf1. In contrast, when the repressor-fused variant of Zyxin is expressed, the forebrain enlargements typical for embryos overexpressing Xanf1 develop. These results are consistent with a possible role of Zyxin as a negative modulator of Xanf1 transcriptional repressing activity.

Multiple noggins in vertebrate genome: cloning and expression of noggin2 and noggin4 in Xenopus laevis.

Noggin is a neural inducer secreted by cells of the Spemann organizer. A single noggin gene was identified until very recently in all tested vertebrates. The only exception was zebrafish, in which two close homologs of noggin, named noggin1 and noggin3, and one gene more diverged from them, noggin2, were cloned. Nevertheless, finding of three zebrafish noggins was attributed exclusively to specific genomic duplications in the fish evolutionary branch. However, very recently it was shown that Xenopus tropicalis have additional noggin homolog, called noggin2 [Fletcher, R.B., Watson, A.L., Harland, R.M. (2004). Expression of Xenopus tropicalis noggin1 and noggin2 in early development: two noggin genes in a tetrapod. Gene Expr. Patterns 5, 225-230], which indicates at least two independent noggin genes in vertebrate phylum. Now we report identification of two novel noggin homologs in each of so evolutionary distant species as Xenopus laevis, chicken and fugu. One of these noggins is ortholog of the X. tropicalis and zebrafish noggin2, whereas another, named noggin4, was not known previously. In the X. laevis embryos, the expression of noggin2 very resembles that of its counterpart in X. tropicalis: it begins with neurulation at the anterior margin of the neural plate and, afterward, continues mainly in the forebrain and dorsal hindbrain. At the same time, noggin4 is expressed starting from the beginning of gastrulation, throughout the ectoderm, with a local expression maximum in the prospective anterior neurectoderm. Later, it is widely expressed on the dorsal side of embryo, including neural tube, eyes, otic vesicles, cranial placodes, branchial arches, and somites. The data presented here demonstrate that the vertebrate phylum contains at least three distinct noggin genes.

The homeodomain-containing transcription factor X-nkx-5.1 inhibits expression of the homeobox gene Xanf-1 during the Xenopus laevis forebrain development.

Expression of the homeobox gene Xanf-1 starts within the presumptive forebrain primordium of the Xenopus embryo at the midgastrula stage and is inhibited by the late neurula. Such stage-specific inhibition is essential for the normal development as the experimental prolongation of the Xanf-1 expression elicits severe brain abnormalities. To identify transcriptional regulators that are responsible for the Xanf-1 inhibition, we have used the yeast one-hybrid system and identified a novel Xenopus homeobox gene X-nkx-5.1 that belongs to a family of Nkx-5.1 transcription factors. In terms of gene expression, X-nkx-5.1 shares many common features with its orthologs in other species, including expression in the embryonic brain and in the ciliated cells of the otic and lateral line placodes. However, we have also observed several features specific for X-nkx-5.1, such as expression in precursors of the epidermal ciliated cells that may indicate a possible common evolutionary origin of all ciliated cells derived from the embryonic ectoderm. Another specific feature is that the X-nkx-5.1 expression in the anterior neural plate starts early, within the area overlapping the Xanf-1 expression territory at the midneurula stage, and it correlates with the beginning of the Xanf-1 inhibition. Using various loss and gain-of-function techniques, including microinjections of antisense morpholino oligonucleotides and mRNA encoding for the X-nkx-5.1 and its dominant repressor and activator versions, we have shown that X-nkx-5.1 can indeed play a role of stage-specific inhibitor of Xanf-1 in the anterior neural plate during the Xenopus development.