Shinya Matsukawa

Transcriptional regulator PRDM12 is essential for human pain perception

Pain perception has evolved as a warning mechanism to alert organisms to tissue damage and dangerous environments. In humans, however, undesirable, excessive or chronic pain is a common and major societal burden for which available medical treatments are currently suboptimal. New therapeutic options have recently been derived from studies of individuals with congenital insensitivity to pain (CIP). Here we identified 10 different homozygous mutations in PRDM12 (encoding PRDI-BF1 and RIZ homology domain-containing protein 12) in subjects with CIP from 11 families. Prdm proteins are a family of epigenetic regulators that control neural specification and neurogenesis. We determined that Prdm12 is expressed in nociceptors and their progenitors and participates in the development of sensory neurons in Xenopus embryos. Moreover, CIP-associated mutants abrogate the histone-modifying potential associated with wild-type Prdm12. Prdm12 emerges as a key factor in the orchestration of sensory neurogenesis and may hold promise as a target for new pain therapeutics.

Rapamycin treatment causes developmental delay, pigmentation defects, and gastrointestinal malformation on Xenopus embryogenesis

Rapamycin is a drug working as an inhibitor of the TOR (target of rapamycin) signaling pathway and influences various life phenomena such as cell growth, proliferation, and life span extension in eukaryote. However, the extent to which rapamycin controls early developmental events of amphibians remains to be understood. Here we report an examination of rapamycin effects during Xenopus early development, followed by a confirmation of suppression of TOR downstream kinase S6K by rapamycin treatment. First, we found that developmental speed was declined in dose-dependent manner of rapamycin. Second, black pigment spots located at dorsal and lateral skin in tadpoles were reduced by rapamycin treatment. Moreover, in tadpole stages severe gastrointestinal malformations were observed in rapamycin-treated embryos. Taken together with these results, we conclude that treatment of the drug rapamycin causes enormous influences on early developmental period.

The requirement of histone modification by PRDM12 and Kdm4a for the development of pre-placodal ectoderm and neural crest in Xenopus

In vertebrates, pre-placodal ectoderm and neural crest development requires morphogen gradients and several transcriptional factors, while the involvement of histone modification remains unclear. Here, we report that histone-modifying factors play crucial roles in the development of pre-placodal ectoderm and neural crest in Xenopus. During the early neurula stage, PRDM12 was expressed in the lateral pre-placodal ectoderm and repressed the expression of neural crest specifier genes via methylation of histone H3K9. ChIP-qPCR analyses indicated that PRDM12 promoted the occupancy of the trimethylated histone H3K9 (H3K9me3) on the Foxd3, Slug, and Sox8 promoters. Injection of the PRDM12 MO inhibited the expression of presumptive trigeminal placode markers and decreased the occupancy of H3K9me3 on the Foxd3 promoter. Histone demethylase Kdm4a also inhibited the expression of presumptive trigeminal placode markers in a similar manner to PRDM12 MO and could compensate for the effects of PRDM12. ChIP-qPCR analyses revealed that promotion of the occupancy of H3K9me3 on the Foxd3, Slug, and Sox8 promoters was inhibited by Kdm4a overexpression. Taken together, these data indicate that histone modification was essential for pre-placodal ectoderm and neural crest development.

Sirtuin inhibitor Ex-527 causes neural tube defects, ventral edema formations, and gastrointestinal malformations in Xenopus laevis embryos

Chemical reagent Ex‐527 is widely used as a major inhibitor of Sirtuin enzymes, which are a family of highly conserved protein deacetylases and have been linked with caloric restriction and aging by modulating energy metabolism, genomic stability, and stress resistance. However, the extent to which Ex‐527 controls early developmental events of vertebrate embryos remains to be understood. Here, we report an examination of Ex‐527 effects during Xenopus early development, followed by a confirmation of expressions of xSirt1 and xSirt2 in embryonic stages and enhancement of acetylation by Ex‐527. First, we found that reductions in size of neural plate at neurula stages were induced by Ex‐527 treatment. Second, tadpoles with short body length and large edematous swellings in the ventral side were frequently observed. Moreover, Ex‐527‐treated embryos showed severe gastrointestinal malformations in late tadpole stages. Taken together with these results, we conclude that the Sirtuin family start functioning at early embryonic stages and is required for various developmental events.

KDEL tagging: a method for generating dominant-negative inhibitors of the secretion of TGF-beta superfamily proteins

Most endoplasmic reticulum (ER)-retained proteins contain a carboxy-terminal signal sequence called the ER retention signal motif such as the Lys-Asp-Glu-Leu (KDEL) motif. Using this molecular mechanism, we developed a new dominant-negative assay, designated the KDEL-tag trap assay, to negatively regulate secretion of disulfide bond-dependent protein dimers, as typified by TGF-beta superfamily proteins. First, we tested this method on the Nodal protein Xnr5, which is a well-studied mesoderm inducer in vertebrates. Tagging of Xnr5 protein with KDEL at the carboxy-terminus effectively blocked the secretion of Xnr5, resulting in complete inhibition of mesoderm induction in Xenopus embryogenesis. Second, we examined the usefulness of the KDEL-tag trap assay on BMPs, which are well-known negative regulators of neural induction and ventralizing factors during early development, and demonstrated that the functions of the BMP family proteins BMP4 and ADMP were blocked by the KDEL-tag trap assay. Moreover, the technical feasibility of the KDEL-tag trap assay was confirmed in a cell culture system using mouse osteoblasts. Taken together, these results suggest that the KDEL-tag trap assay can be adapted to inhibit a variety of plasma membrane or secreted proteins of a multimeric nature.

Specific induction of cranial placode cells from Xenopus ectoderm by modulating the levels of BMP, Wnt, and FGF signaling.

The neural–epidermal boundary tissues include the neural crest and preplacodal ectoderm (PPE) as primordial constituents. The PPE region is essential for the development of various sensory and endocrine organs, such as the anterior lobe of the pituitary, olfactory epithelium, lens, trigeminal ganglion, and otic vesicles. During gastrulation, a neural region is induced in ectodermal cells that interacts with mesendodermal tissue and responds to several secreted factors. Among them, inhibition of bone morphogenetic protein (BMP) in the presumptive neuroectoderm is essential for the induction of neural regions, and formation of a Wnt and fibroblast growth factor (FGF) signaling gradient along the midline determines anterior–posterior patterning. In this study, we attempted to specifically induce PPE cells from undifferentiated Xenopus cells by regulating BMP, Wnt, and FGF signaling. We showed that the proper level of BMP inhibition with an injection of truncated BMP receptor or treatment with a chemical antagonist triggered the expression of PPE genes. In addition, by varying the amount of injected chordin, we optimized specific expression of the PPE genes. PPE gene expression is increased by adding an appropriate dose of an FGF receptor antagonist. Furthermore, co‐injection with either wnt8 or the Wnt inhibitor dkk‐1 altered the expression levels of several region‐specific genes according to the injected dose. We specifically induced PPE cell differentiation in animal cap cells from early‐stage Xenopus embryos by modulating BMP, Wnt, and FGF signaling. This is not the first research on placode induction, but our simple method could potentially be applied to mammalian stem cell systems. genesis 53:652–659, 2015.

Xnr3 affects brain patterning via cell migration in the neural-epidermal tissue boundary during early Xenopus embryogenesis

Neural induction and anteroposterior neural patterning occur simultaneously during Xenopus gastrulation by the inhibition of BMP and Wnt signaling, respectively. However, other processes might be necessary for determining the neural-epidermal boundary. Xenopus nodal-related-3 (Xnr3) is expressed in dorsal blastula and plays a role in neural formation. In this study, we analyzed how Xnr3 affects neural patterning to identify novel mechanisms of neural-epidermal-boundary determination. In situ hybridization revealed that ventro-animal injection with Xnr3 shifted the lateral krox20 expression domain anteriorly and reduced Otx2 expression. The mature region of Xnr3 is necessary for these effects to occur, and the pro-region accelerated them. Phalloidin labeling revealed that cells around the neural-epidermal boundary lost their slender shape following Xnr3 injection. Moreover, we analyzed the cell migration of ectodermal cells and found specific Xnr3-induced effects at the neural-epidermal boundary. These findings together suggested that Xnr3 affects anterior ectoderm migration around the neural-epidermal boundary to induce a specific neural pattern abnormality. Change of the shape of surrounding ectodermal cells and the specific migratory pattern might therefore reflect the novel mechanism of neural-epidermal boundary.

mNanog Possesses Dorsal Mesoderm-Inducing Ability by Modulating Both BMP and Activin/Nodal Signaling in Xenopus Ectodermal Cells

Background In Xenopus early embryogenesis, various genes are involved with mesoderm formation. In particular, dorsal mesoderm contains the organizer region and induces neural tissues through the inhibition of bone morphogenetic protein (BMP) signaling. In our initial study to identify novel genes necessary for maintaining the undifferentiated state, we unexpectedly revealed mesoderm-inducing activity for mNanog in Xenopus. Methodology/Principal Findings The present series of experiments investigated the effect of mNanog gene expression on Xenopus embryo. Ectopic expression of mNanog induced dorsal mesoderm gene activity, secondary axis formation, and weakly upregulated Activin/nodal signaling. The injection of mNanog also effectively inhibited the target genes of BMP signaling, while Xvent2 injection downregulated the dorsal mesoderm gene expression induced by mNanog injection. Conclusions/Significance These results suggested that mNanog expression induces dorsal mesoderm by regulating both Activin/nodal signaling and BMP signaling in Xenopus. This finding highlights the possibly novel function for mNanog in stimulating the endogenous gene network in Xenopus mesoderm formation.