Xenopus Embryogenesis Research Articles

Actin depolymerizing factor destrin governs cell migration in neural development during Xenopus embryogenesis

The actin-based cytoskeleton is considered a fundamental driving force for cell differentiation and development. Destrin (dstn), a member of the actin depolymerizing factor family, regulates actin dynamics by treadmilling actin filaments and increasing globular actin pools. However, the specific developmental roles of dstn have yet to be fully elucidated. Here, we investigated the physiological functions of dstn during early embryonic development using Xenopus laevis as an experimental model organism. dstn is expressed in anterior neural tissue and neural plate during Xenopus embryogenesis. Depleting dstn promoted morphants with short body axes and small heads. Moreover, dstn inhibition extended the neural plate region, impairing cell migration and distribution during neurulation. In addition to the neural plate, dstn knockdown perturbed neural crest cell migration. Our data suggest new insights for understanding the roles of actin dynamics in embryonic neural development, simultaneously presenting a new challenge for studying the complex networks governing cell migration involving actin dynamics.

Ccl19 and ccl21 affect cell movements and differentiation in early Xenopus development.

We characterized Xenopus laevis C-C motif chemokine ligand 19.L (ccl19.L) and C-C motif chemokine ligand 21.L (ccl21.L) during early Xenopus embryogenesis. The temporal and spatial expression patterns of ccl19.L and ccl21.L tended to show an inverse correlation, except that the expression level was higher in the dorsal side at the gastrula stage. For example, even at the dorsal sector of the gastrulae, ccl19.L was expressed in the axial region and ccl21.L was expressed in the paraxial region. Dorsal overexpression of ccl19.L and ccl21.L and knockdown of Ccl19.L and Ccl21.L inhibited gastrulation, but their functions were different in cell behaviors during morphogenesis. Observation of Keller sandwich explants revealed that overexpression of both ccl19.L and ccl21.L and knockdown of Ccl21.L inhibited the convergent extension movements, while knockdown of Ccl19.L did not. ccl19.L-overexpressing explants attracted cells at a distance and ccl21.L-overexpressing explants attracted neighboring cells. Ventral overexpression of ccl19.L and ccl21.L induced secondary axis-like structures and chrd.1 expression at the ventral side. Upregulation of chrd.1 was induced by ligand mRNAs through ccr7.S. Knockdown of Ccl19.L and Ccl21.L inhibited gastrulation and downregulated chrd.1 expression at the dorsal side. The collective findings indicate that ccl19.L and ccl21.L might play important roles in morphogenesis and dorsal-ventral patterning during early embryogenesis in Xenopus.

The role of Wnt signaling in Xenopus neural induction.

Development of the central nervous system in amphibians has called attention from scientists for over a century. Interested in the matter of embryonic inductions, Hans Spemann and Hilde Mangold found out that the dorsal blastopore lip of the salamander's embryo has organizer properties. Such an ectopic graft could induce structures in the host embryo, including a neural tube overlying the notochord of a perfect secondary body axis. A couple of decades later, the frog Xenopus laevis emerged as an excellent embryological experimental model and seminal concepts involving embryonic inductions began to be revealed. The so-called primary induction is, in fact, a composition of signaling and inductive events that are triggered as soon as fertilization takes place. In this regard, since early 1990s an intricate network of signaling pathways has been built. The Wnt pathway, which began to be uncovered in cancer biology studies, is crucial during the establishment of two signaling centers in Xenopus embryogenesis: Nieuwkoop center and the blastula chordin noggin expression center (BCNE). Here we will discuss the historical events that led to the discovery of those centers, as well as the molecular mechanisms by which they operate. This chapter highlights the cooperation of both signaling centers with potential to be further explored in the future. We aim to address the essential morphological transformation during gastrulation and neurulation as well as the role of Wnt signaling in patterning the organizer and the neural plate.

Zbtb21 is required for the anterior-posterior patterning of neural tissue in the early Xenopus embryo

The vertebrate body is organized along the dorsal-ventral (DV) and anterior-posterior (AP) axes by the BMP and Wnt pathways, respectively. We previously reported that Xenopus Zbtb14 promotes dorsalization (neural induction) of ectoderm by inhibiting BMP signaling and also posteriorizes neural tissue by activating Wnt signaling, thereby coordinating the patterning of the DV and AP axes during early development. Although it has been reported that human ZBTB21 binds to ZBTB14 and is involved in gene expression in cultured mammalian cells, the function of Zbtb21 in early embryonic development remains unknown. Here, we show that Xenopus Zbtb21 plays an essential role in AP axis formation in the early Xenopus embryo. zbtb21 and zbtb14 are co-expressed in the dorsal region of embryos during gastrulation. Simultaneous overexpression of Zbtb21 and Zbtb14 in ectodermal explants enhances the neural-inducing activity of Zbtb14. Moreover, knockdown experiments showed that Zbtb21 is required for the formation of posterior neural tissue and the suppression of anterior neural development. Collectively, these results suggest that in cooperation with Zbtb14, Zbtb21 has a crucial function in AP patterning during early Xenopus embryogenesis.

Uncoupling the BMP receptor antagonist function from the WNT agonist function of R-spondin 2 using the inhibitory peptide dendrimer RWd

Signaling by bone morphogenetic proteins (BMPs) plays pivotal roles in embryogenesis, adult tissue homeostasis, and disease. Recent studies revealed that the well-established WNT agonist R-spondin 2 (RSPO2) is also a BMP receptor (BMP receptor type 1A) antagonist, with roles in early Xenopus embryogenesis and human acute myeloid leukemia (AML). To uncouple the BMP antagonist function from the WNT agonist function and to promote development of AML therapeutics, here we identified a 10-mer peptide (RW) derived from the thrombospondin 1 domain of RSPO2, which specifically prevents binding between RSPO2 and BMP receptor type 1A without altering WNT signaling. We also show that a corresponding RW dendrimer (RWd) exhibiting improved half-life relieves inhibition of BMP receptor signaling by RSPO2 in human AML cells, reduces cell growth, and induces differentiation. Moreover, microinjection of RWd in Xenopus embryos ventralizes the dorsoventral embryonic patterning by upregulating BMP signaling without affecting WNT signaling. Our study corroborates the function of RSPO2 as a BMP receptor antagonist and provides a proof of concept for pharmacologically uncoupling BMP antagonist from WNT agonist functions of RSPO2 using the inhibitor peptide RWd with enhanced target selectivity and limited side effects.

Galloway-Mowat syndrome: New insights from bioinformatics and expression during Xenopus embryogenesis

Galloway-Mowat syndrome (GAMOS) is a rare developmental disease. Patients suffer from congenital brain anomalies combined with renal abnormalities often resulting in an early-onset steroid-resistant nephrotic syndrome.The etiology of GAMOS has a heterogeneous genetic contribution. Mutations in more than 10 different genes have been reported in GAMOS patients. Among these are mutations in four genes encoding members of the human KEOPS (kinase, endopeptidase and other proteins of small size) complex, including OSGEP, TP53RK, TPRKB and LAGE3. Until now, these components have been functionally mainly investigated in bacteria, eukarya and archaea and in humans in the context of the discovery of its role in GAMOS, but the KEOPS complex members’ expression and function during embryogenesis in vertebrates is still unknown.In this study, in silico analysis showed that both gene localization and the protein sequences of the three core KEOPS complex members Osgep, Tp53rk and Tprkb are highly conserved across different species including Xenopus laevis. In addition, we examined the spatio-temporal expression pattern of osgep, tp53rk and tprkb using RT-PCR and whole mount in situ hybridization approaches during early Xenopus development. We observed that all three genes were expressed during early embryogenesis and enriched in tissues and organs affected in GAMOS. More precisely, KEOPS complex genes are expressed in the pronephros, but also in neural tissue such as the developing brain, eye and cranial cartilage.These findings suggest that the KEOPS complex plays an important role during vertebrate embryonic development.

A systemic cell cycle block impacts stage-specific histone modification profiles during Xenopus embryogenesis.

Forming an embryo from a zygote poses an apparent conflict for epigenetic regulation. On the one hand, the de novo induction of cell fate identities requires the establishment and subsequent maintenance of epigenetic information to harness developmental gene expression. On the other hand, the embryo depends on cell proliferation, and every round of DNA replication dilutes preexisting histone modifications by incorporation of new unmodified histones into chromatin. Here, we investigated the possible relationship between the propagation of epigenetic information and the developmental cell proliferation during Xenopus embryogenesis. We systemically inhibited cell proliferation during the G1/S transition in gastrula embryos and followed their development until the tadpole stage. Comparing wild-type and cell cycle-arrested embryos, we show that the inhibition of cell proliferation is principally compatible with embryo survival and cellular differentiation. In parallel, we quantified by mass spectrometry the abundance of a large set of histone modification states, which reflects the developmental maturation of the embryonic epigenome. The arrested embryos developed abnormal stage-specific histone modification profiles (HMPs), in which transcriptionally repressive histone marks were overrepresented. Embryos released from the cell cycle block during neurulation reverted toward normality on morphological, molecular, and epigenetic levels. These results suggest that the cell cycle block by HUA alters stage-specific HMPs. We propose that this influence is strong enough to control developmental decisions, specifically in cell populations that switch between resting and proliferating states such as stem cells.

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.

TMEM79/MATTRIN defines a pathway for Frizzled regulation and is required for Xenopus embryogenesis.

Wnt signaling through the Frizzled (FZD) family of serpentine receptors is essential for embryogenesis and homeostasis, and stringent control of the FZD protein level is critical for stem cell regulation. Through CRISPR/Cas9 genome-wide screening in human cells, we identified TMEM79/MATTRIN, an orphan multi-span transmembrane protein, as a specific inhibitor of Wnt/FZD signaling. TMEM79 interacts with FZD during biogenesis and promotes FZD degradation independent of ZNRF3/RNF43 ubiquitin ligases (R-spondin receptors). TMEM79 interacts with ubiquitin-specific protease 8 (USP8), whose activating mutations underlie human tumorigenesis. TMEM79 specifically inhibits USP8 deubiquitination of FZD, thereby governing USP8 substrate specificity and promoting FZD degradation. Tmem79 and Usp8 genes have a pre-bilaterian origin, and Tmem79 inhibition of Usp8 and Wnt signaling is required for anterior neural development and gastrulation in Xenopus embryos. TMEM79 is a predisposition gene for Atopic dermatitis, suggesting deregulation of Wnt/FZD signaling a possible cause for this most common yet enigmatic inflammatory skin disease.

Dach1 regulates neural crest migration during embryonic development

Dachshund 1(Dach1) is a key component of the retinal determination gene network that plays significant roles in cell fate regulation. The vertebrate homolog of Drosophila dachshund has gained considerable importance as an essential regulator of development, but its functions during embryonic development remain elusive. We investigated the functional significance of dach1 during Xenopus embryogenesis using loss-of-function studies. Reverse transcription-polymerase chain reaction demonstrated the maternal nature of dach1, showing enhanced expression at the neurula stage of development, and morpholino oligonucleotide injection of dach1 induced phenotypic anomalies of microcephaly and reduced body length. Animal cap assays followed by whole-mount in-situ hybridization indicated the perturbed expression of neural and neural crest (NC) markers. Our data suggest the prerequisite functions of dach1 in NC migration during Xenopus embryogenesis. However, the developmental pathways regulated by dach1 during embryogenesis require further elucidation.

The secreted BMP antagonist ERFE is required for the development of a functional circulatory system in Xenopus

The hormone Erythroferrone (ERFE) is a member of the C1q/TNF-related protein family that regulates iron homeostasis through the suppression of hamp. In a gain of function screen in Xenopus embryos, we identified ERFE as a potent secondary axis-inducing agent. Experiments in Xenopus embryos and ectodermal explants revealed that ERFE functions as a selective inhibitor of the BMP pathway and the conserved C1q domain is not required for this activity. Inhibition occurs at the extracelluar level, through the interaction of ERFE with the BMP ligand. During early Xenopus embryogenesis, erfe is first expressed in the ventral blood islands where initial erythropoiesis occurs and later in circulating blood cells. ERFE knockdown does not alter the expression of etv.2, aplnr and flt1 in tailbud stage embryos indicating endothelial cell specification is independent of ERFE. However, in tadpole embryos, defects of the vascular network and primitive blood circulation are observed as well as edema formation. RNAseq analysis of ERFE morphant embryos also revealed the inhibition of gja4 indicating disruption of dorsal aorta formation.

Toward Decoding Bioelectric Events in Xenopus Embryogenesis: New Methodology for Tracking Interplay Between Calcium and Resting Potentials In Vivo

Although chemical signaling during embryogenesis is readily addressed by a plethora of available techniques, the developmental functions of ionic signaling are still poorly understood. It is increasingly realized that bioelectric events in nonneural cells are critical for pattern regulation, but their study has been hampered by difficulties in monitoring and manipulating them in vivo. Recent developments in visualizing electrical signaling dynamics in the field of neuroscience have facilitated functional experiments that reveal instructive developmental bioelectric signals. However, there is a pressing need for additional tools to explore time-dependent ionic signaling to understand complex endogenous dynamics. Here, we present methodological advances, including 4D imaging and data analysis, for improved tracking of calcium flux in the Xenopus laevis embryo, lowering the barrier for in vivo physiology work in this important model system. Using these techniques, we investigated the relationship between bioelectric ion channel activity and calcium, finding that cell hyperpolarization and depolarization both induce persistent static elevation of cytoplasmic calcium levels that fade over developmental time. These calcium changes correlate with increased cell mobility in early embryos and abnormal craniofacial morphology in later embryos. We thus highlight membrane potential modulation as a tractable tool for modulation of signaling cascades that rely on calcium as a transduction mechanism. The methods we describe facilitate the study of important novel aspects of developmental physiology, are extendable to numerous classes of existing and forthcoming fluorescent physiological reporters, and establish highly accessible, inexpensive protocols for their investigation.

Cdc2-like kinase 2 (Clk2) promotes early neural development in Xenopus embryos.

Neural induction and patterning in vertebrates are regulated during early development by several morphogens, such as bone morphogenetic proteins (BMPs) and fibroblast growth factors (FGFs). Ventral ectoderm differentiates into epidermis in response to BMPs, whereas BMP signaling is tightly inhibited in the dorsal ectoderm which develops into neural tissues. Here, we show that Cdc2-like kinase 2 (Clk2) promotes early neural development and inhibits epidermis differentiation in Xenopus embryos. clk2 is specifically expressed in neural tissues along the anterior-posterior axis during early Xenopus embryogenesis. When overexpressed in ectodermal explants, Clk2 induces the expression of both anterior and posterior neural marker genes. In agreement with this observation, overexpression of Clk2 in whole embryos expands the neural plate at the expense of epidermal ectoderm. Interestingly, the neural-inducing activity of Clk2 is increased following BMP inhibition and activation of the FGF signaling pathway in ectodermal explants. Clk2 also downregulates the level of p-Smad1/5/8 in cooperation with BMP inhibition, in addition to increasing the level of activated MAPK together with FGF. These results suggest that Clk2 plays a role in early neural development of Xenopus possibly via modulation of morphogen signals such as the BMP and FGF pathways.

Morpholinos Do Not Elicit an Innate Immune Response during Early Xenopus Embryogenesis

It has recently been reported that a common side effect of translation-blocking morpholino antisense oligonucleotides is the induction of a set of innate immune response genes in Xenopus embryos and that splicing-blocking morpholinos lead to unexpected off-target mis-splicing events. Here, we present ananalysis of all publicly available Xenopus RNA sequencing (RNA-seq) data in a reexamination of the effects of translation-blocking morpholinos on the innate immune response. Our analysis does notsupport the authors' general conclusion, which wasbased on a limited number of RNA-seq datasets. Moreover, the strong induction of an immune response appears to be specific to the tbxt/tbxt2 morpholinos. The more comprehensive study presented here indicates that using morpholinos for targeted gene knockdowns remains of considerable value for the rapid identification of gene function.

Membrane Microdomains as Platform to Study Membrane-Associated Events During Oogenesis, Meiotic Maturation, and Fertilization in Xenopus laevis.

Studies on the egg plasma membrane-associated tyrosine kinase Src have shed light on the identity of the molecular machinery that is responsible for gamete interaction and possibly fusion in African clawed frog Xenopus laevis. Here we describe our protocol for identifying and analyzing molecular and cellular machinery that contributes to a variety of biological processes in the course of oogenesis, oocyte maturation, egg fertilization, and early embryogenesis in Xenopus. Our current special interest is to evaluate the hypothesis that the oocyte/egg membrane microdomain (MD)-associated uroplakin III-Src system is responsible for mediating sperm-egg membrane interaction/fusion signal to the oocyte/egg cytoplasm to initiate embryonic and zygotic development in this species. Therefore, this chapter contains a brief introduction to biology of oocytes and eggs in Xenopus and addresses the following questions: (1) What is oocyte/egg MD? (2) Why do we study oocyte/egg MD? (3) How to manipulate oocyte/egg MD? (4) What has been achieved by oocyte/egg MD studies? (5) What are the next steps in oocyte/egg MD studies?

Axis elongation during Xenopus tail-bud stage is regulated by GABA expressed in the anterior-to-mid neural tube.

The receptors of gamma-aminobutyric acid (GABA), which is a well-known neurotransmitter, are expressed in the anterior-to-mid neural tube at an early stage of Xenopus development, but there has been no report on the role of GABA in the presumptive central nervous system. Therefore, we tried to reveal the function of GABA for Xenopus early embryogenesis. We first confirmed that the region expressing a gene encoding glutamate decarboxylase 1 (gad1), which is an enzyme that catalyzes the decarboxylation of L-glutamate to GABA, overlapped with that of several genes encoding GABA receptors (gabr) in the neural tube. Metabolome analysis of culture medium of dorsal tail-bud explants containing the neural tube region of tail-bud stage embryos also revealed that GABA was expressed at this stage. Then, we examined the treatment of pentylenetetrazole (PTZ) and picrotoxin (PTX), which are known as inhibitors of GABA receptors (GABA-R), on the early stages of Xenopus embryogenesis, and found that axis elongation in the tail-bud was inhibited by both treatments, and these phenotypic effects were rescued by co-treatment with GABA. Moreover, our spatial- and temporal-specific inhibitor treatments revealed that the gabr- and gad1-overlapped region, which presents at the anterior-to-mid neural tube during the tail-bud stages, was much more sensitive to PTZ and thus caused severe inhibition of axis elongation. Taken together, our results indicate that the small ligand molecule GABA functions as a regulator to induce the axis elongation event in the tail-bud during early embryogenesis via direct stimulation of the neural tube and indirect stimulation of the surrounding area.

Lysine demethylase 3a in craniofacial and neural development during Xenopus embryogenesis.

Epigenetic modifier lysine demethylase 3a(Kdm3a) specifically demethylates mono‑ and di‑methylated ninth lysine of histone3 and belongs to the Jumonji domain‑containing group of demethylases. Kdm3a serves roles during various biological and pathophysiological processes, including spermatogenesis and metabolism, determination of sex, androgen receptor‑mediated transcription and embryonic carcinoma cell differentiation. In the present study, physiological functions of Kdm3a were evaluated during embryogenesis of Xenopus laevis. Spatiotemporal expression pattern indicated that kdm3a exhibited its expression from early embryonic stages until tadpole stage, however considerable increase of kdm3a expression was observed during the neurula stage of Xenopus development. Depleting kdm3a using kdm3a antisense morpholino oligonucleotides induced anomalies, including head deformities, small‑sized eyes and abnormal pigmentation. Whole‑mount insitu hybridization results demonstrated that kdm3a knockdown was associated with defects in neural crest migration. Further, quantitative polymerase chain reaction revealed abnormal expression of neural markers in kdm3a morphants. RNA sequencing of kdm3a morphants indicated that kdm3a was implicated in mesoderm formation, cell adhesion and metabolic processes of embryonic development. In conclusion, the results of the present study indicated that Kdm3a may serve a role in neural development during Xenopus embryogenesis and may be targeted for treatment of developmental disorders. Further investigation is required to elucidate the molecular mechanism underlying the regulation of neural development by Kdm3a.

Physiological effects of KDM5C on neural crest migration and eye formation during vertebrate development

BackgroundLysine-specific histone demethylase 5C (KDM5C) belongs to the jumonji family of demethylases and is specific for the di- and tri-demethylation of lysine 4 residues on histone 3 (H3K4 me2/3). KDM5C is expressed in the brain and skeletal muscles of humans and is associated with various biologically significant processes. KDM5C is known to be associated with X-linked mental retardation and is also involved in the development of cancer. However, the developmental significance of KDM5C has not been explored yet. In the present study, we investigated the physiological roles of KDM5C during Xenopus laevis embryonic development.ResultsLoss-of-function analysis using kdm5c antisense morpholino oligonucleotides indicated that kdm5c knockdown led to small-sized heads, reduced cartilage size, and malformed eyes (i.e., small-sized and deformed eyes). Molecular analyses of KDM5C functional roles using whole-mount in situ hybridization, β-galactosidase staining, and reverse transcription-polymerase chain reaction revealed that loss of kdm5c resulted in reduced expression levels of neural crest specifiers and genes involved in eye development. Furthermore, transcriptome analysis indicated the significance of KDM5C in morphogenesis and organogenesis.ConclusionOur findings indicated that KDM5C is associated with embryonic development and provided additional information regarding the complex and dynamic gene network that regulates neural crest formation and eye development. This study emphasizes the functional significance of KDM5C in Xenopus embryogenesis; however, further analysis is needed to explore the interactions of KDM5C with specific developmental genes.

Ventx1.1 as a Direct Repressor of Early Neural Gene zic3 in Xenopus laevis.

From Xenopus embryo studies, the BMP4/Smad1-targeted gene circuit is a key signaling pathway for specifying the cell fate between the ectoderm and neuro-ectoderm as well as the ventral and dorsal mesoderm. In this context, several BMP4/Smad1 target transcriptional factors have been identified as repressors of the neuro-ectoderm. However, none of these direct target transcription factors in this pathway, including GATA1b, Msx1 and Ventx1.1 have yet been proven as direct repressors of early neuro-ectodermal gene expression. In order to demonstrate that Ventx1.1 is a direct repressor of neuro-ectoderm genes, a genome-wide Xenopus ChIP-Seq of Ventx1.1 was performed. In this study, we demonstrated that Ventx1.1 bound to the Ventx1.1 response cis-acting element 1 and 2 (VRE1 and VRE2) on the promoter for zic3, which is a key early neuro-ectoderm gene, and this Ventx1.1 binding led to repression of zic3 transcription. Site-directed mutagenesis of VRE1 and VRE2 within zic3 promoter completely abolished the repression caused by Ventx1.1. In addition, we found both the positive and negative regulation of zic3 promoter activity by FoxD5b and Xcad2, respectively, and that these occur through the VREs and via modulation of Ventx1.1 levels. Taken together, the results demonstrate that the BMP4/Smad1 target gene, Ventx1.1, is a direct repressor of neuro-ectodermal gene zic3 during early Xenopus embryogenesis.

Serine Threonine Kinase Receptor-Associated Protein Deficiency Impairs Mouse Embryonic Stem Cells Lineage Commitment Through CYP26A1-Mediated Retinoic Acid Homeostasis.

Retinoic acid (RA) signaling is essential for the differentiation of embryonic stem cells (ESCs) and vertebrate development. RA biosynthesis and metabolism are controlled by a series of enzymes, but the molecular regulators of these enzymes remain largely obscure. In this study, we investigated the functional role of the WD-domain protein STRAP (serine threonine kinase receptor-associated protein) in the pluripotency and lineage commitment of murine ESCs. We generated Strap knockout (KO) mouse ESCs and subjected them to spontaneous differentiation. We observed that, despite the unchanged characteristics of ESCs, Strap KO ESCs exhibited defects for lineage differentiation. Signature gene expression analyses revealed that Strap deletion attenuated intracellular RA signaling in embryoid bodies (EBs), and exogenous RA significantly rescued this deficiency. Moreover, loss of Strap selectively induced Cyp26A1 expression in mouse EBs, suggesting a potential role of STRAP in RA signaling. Mechanistically, we identified putative Krüppel-like factor 9 (KLF9) binding motifs to be critical in the enhancement of non-canonical RA-induced transactivation of Cyp26A1. Increased KLF9 expression in the absence of STRAP is partially responsible for Cyp26A1 induction. Interestingly, STRAP knockdown in Xenopus embryos influenced anterior-posterior neural patterning and impaired the body axis and eye development during early Xenopus embryogenesis. Taken together, our study reveals an intrinsic role for STRAP in the regulation of RA signaling and provides new molecular insights for ESC fate determination. Stem Cells 2018;36:1368-1379.