Differentiation Of Neural Crest Cells Research Articles

Developmental toxicity of dibutyl phthalate and citrate ester plasticizers in Xenopus laevis embryos

Citrate esters have been considered as alternatives to phthalate plasticizers. Being considered to have low toxicity in mammals, their toxicological information for aquatic animals remains poorly understood. We examined the developmental toxicity of citrate esters including tributyl O-acetylcitrate (ATBC), triethyl 2-acetylcitrate (ATEC), and trihexyl O-acetylcitrate (ATHC) together with dibutyl phthalate (DBP) based on the frog embryo teratogenesis assay-Xenopus (FETAX). ATBC has the lowest 96 h LC50 and 96 h EC50 values. In RT-qPCR, the ratio of bax and bcl-2 mRNA was significantly increased by DBP, but not by ATBC, ATEC and ATHC. DNA fragmentation was obvious in DBP-treated tadpoles, but not in those treated with ATBC and ATEC, whereas ATHC caused necrotic DNA degradation. Lipid hydroperoxide levels in tadpoles were significantly increased by DBP and ATHC, but not by ATBC and ATEC, suggesting that induction of oxidative stress by DBP and ATHC in embryos. In tadpoles with head abnormalities, basihyal bone, ceratohyal bone and Meckel's cartilage were frequently missed together with reduction in branchial gill bones. Col2a1 mRNA in the head of tadpoles was significantly decreased by low concentration of DBP, ATHC, and high concentration of ATEC. In stage 25 embryos FoxN3 mRNA, a master regulator for differentiation of neural crest cells to chondrocytes in head, was significantly decreased by DBP and ATHC, but not by ATBC and ATEC. In conclusion, ATEC was recommended as the alternative to phthalate plasticizer having the lowest developmental toxicity in amphibian embryos.

Migration and differentiation of enteric neural crest cells in the embryonic gut explant cultured ex vivo

Hirschsprung's disease (HSCR) is a common congenital gut motility disorder characterized by the reduction or absence of enteric neurons in the distal region of the colon. The Dominant megacolon (Dom) mutant mouse, which carries a spontaneous mutation in Sox10, exhibits HSCR‐like phenotypes, and hence has been used as an animal model of HSCR. It has been known that in Sox10Dom/+ mouse embryos, the migration and differentiation of neural crest cells (NCCs), which are progenitors of enteric neurons and glial cells, are abnormal, leading to hypoganglionosis or aganglionosis in the hindgut. In Sox10Dom/Dom homozygous embryos, the entire gut tube is aganglionic and the embryo dies before E13.5. However, it is still unknown whether hypoganglionic and aganglionic phenotypes exhibited by Dom mice is a result of abnormalities in NCCs only (cell‐autonomous effects), alterations of gut microenvironment through which NCCs migrate (niche effects), or both. Previously, an organotypic gut explant system has already been established, which allows ex vivo co‐culture of donor NCCs of different genotypes with a recipient embryonic gut segment isolated from embryos of the same or other genotypes. In the present study, E13.5 Dom mouse hindgut explants were used as recipient gut segments for transplantation of donor NCCs. Five days after cell transplantation, the hindgut explant cultured ex vivo appeared morphologically and histologically normal with rhythmic contractions and relaxations, indicating that the development of the hindgut explant ex vivo was similar to that in vivo. To obtain donor enteric neural crest cells (ENCCs), midgut segments which were genetically labeled with GFP were dissected from E11.5 mouse embryos and cultured on a fibronectin‐coated surface. ENCCs emigrated from the midgut segments were collected and transplanted to the hindgut explant, which was then cultured ex vivo for 5 days. We found that transplanted ENCCs survived, migrated, and differentiated into TuJ1‐immunoreative enteric neurons in normoganglionic and hypoganglionic segments of the hindgut explant. When transplanted to aganglionic segments of the hindgut, ENCCs were still able to differentiate into TuJ1‐immunoreactive neurons, but they were not able to migrate as far as those transplanted cells in the normoganglionic and hypoganglionic segments, and most of them stayed near the transplantation site without forming a normal neuronal network. Our results indicated that endogenous enteric neurons were required for the normal migration and integration of transplanted ENCCs within the hindgut explant.Support or Funding InformationThe work was supported by a grant from the Research Grants Council of the Hong Kong Special Administrative Region, China (Project No.: CUHK 14175017).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Hair Follicle‐derived Neural Crest Stem Cells (HF‐NCSCs) as a Cell Source of Enteric Neurons

The enteric nervous system (ENS), which regulates most of the gastrointestinal functions, is derived from neural crest cells during early embryonic development. Abnormal migration and differentiation of neural crest cells may cause defective formation of the ENS, thus leading to enteric aganglionosis (absence of ganglia consisting of neurons and glial cells) in the distal colon. In the present study, we aimed to regenerate enteric neurons by transplanting hair follicle‐derived neural crest stem cells (HF‐NCSCs) to the aganglionic distal colon. We first isolated hair follicle bulges from the vibrissa whisker pad of mice, and cultured the bulges for 3 to 4 days. After HF‐NCSCs migrated onto the culture surface, the bulges were removed, and HF‐NCSCs were cultured in an expansion medium for another week. HF‐NCSCs were found to be able to differentiate into myocytes, neurons and glial cells in different differentiation media. Ten days after they were transplanted to the hindgut explants isolated from E13.5 mouse embryos, the transplanted HF‐NCSCs spread along the gut explant cultured ex vivo and differentiated into neurons. Immunostaining of the serial cross sections indicated most of the transplanted cells were located in the myenteric plexus between two muscle layers. HF‐NCSCs were also transplanted to the descending colon of an adult mouse in vivo after laparotomy. One week later, the cells spread along the colon and differentiate into Tuj1 and PGP9.5 immunoreactive cells. Ten days after transplantation in vivo, subtype inhibitory neurons immunoreactive to nNOS were found among the transplanted cells, and a small number of transplanted cells differentiated into excitatory neurons immunoreactive to ChAT. Additionally, almost all the transplanted cells were found in the myenteric plexus which is also the normal location of endogenous enteric neurons between the longitudinal muscle and circular muscle layers 6 days after transplantation. These results showed that (1) enough HF‐NCSCs were obtained for cell transplantation from a small number of skin hair follicles, and (2) HF‐NCSCs from hair follicles could serve as a cell source for stem cell‐based therapy. This work also gives insights into the feasibility of using autologous cell transplantation with a less invasive hair follicle isolation method on patients with ENS defects in the colon.Support or Funding InformationThe work was supported by a grant from the Research Grants Council of the Hong Kong Special Administrative Region, China (Project No.: 14175017).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Birth Defects of the Craniofacial Skeleton and the Potential for Prevention

Craniofacial anomalies account for approximately one‐third of all birth defects, and common anomalies such as cleft palate, craniosynostosis, and mandibulofacial dysostosis can occur in isolation or as part of one of nearly 700 distinct syndromes. Most of the bone, cartilage and connective tissue of the head and face is derived from a stem cell and progenitor cell population called neural crest cells. Congenital craniofacial anomalies are therefore typically associated with defects in neural crest cell development. Understanding the etiology and pathogenesis of craniofacial birth defects is dependent upon knowledge of the mechanisms that govern neural crest cell formation, migration, survival and differentiation. Recently, we discovered that ribosome biogenesis, the process of making ribosomes which are essential for protein translation in all cells, plays an essential role in neural crest cell proliferation and survival during craniofacial development. Ribosome biogenesis commences with transcription of rDNA by RNA Polymerases I and III, and this event is considered a rate‐limiting step in ribosome biogenesis. Mutations in genes that encode subunits of RNA Polymerase I, or its associated factors, lead to deficiencies in ribosome biogenesis, which results in neural crest cell apoptosis, and consequently congenital craniofacial disorders such as Treacher Collins syndrome, and Acrofacial Dysostosis ‐ Cincinnati type. Inhibition of neural crest cell death is capable of ameliorating and in some cases completely preventing craniofacial malformations in mouse and zebrafish models of these syndromes. Through investigating fundamental mechanisms that regulate neural crest cell biology, our work is facilitating the development of therapeutic approaches to prevent the pathogenesis of congenital craniofacial birth defects.Support or Funding InformationStowers Institute for Medical ResearchThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

TFII-I and AP2α Co-Occupy the Promoters of Key Regulatory Genes Associated with Craniofacial Development.

The aim of this study is to define the candidate target genes for TFII-I and AP2α regulation in neural crest progenitor cells. The GTF2I and GTF2IRD1 genes encoding the TFII-I family of transcription factors are prime candidates for the Williams-Beuren syndrome, a complex multisystem disorder characterized by craniofacial, skeletal, and neurocognitive deficiencies. AP2α, a product of the TFAP2A gene, is a master regulator of neural crest cell lineage. Mutations in TFAP2A cause branchio-oculo-facial syndrome characterized by dysmorphic facial features and orofacial clefts. In this study, we examined the genome-wide promoter occupancy of TFII-I and AP2α in neural crest progenitor cells derived from in vitro-differentiated human embryonic stem cells. Our study revealed that TFII-I and AP2α co-occupy a selective set of genes that control the specification of neural crest cells. The data suggest that TFII-I and AP2α may coordinately control the expression of genes encoding chromatin-modifying proteins, epigenetic enzymes, transcription factors, and signaling proteins.

Cut loose and run: The complex role of ADAM proteases during neural crest cell development.

ADAM metalloproteases have been shown to play critical roles during development. In this review, we will describe functional evidence that implicates ADAM proteins during the genesis, migration and differentiation of neural crest cells. We will restrict our analysis to the transmembrane ADAMs as other reviews have addressed the role of extracellular metalloproteases (Christian et al. [2013] Critical Reviews in Biochemistry and Molecular Biology 48:544-560). This review will describe advances that have been obtained mainly through the use of two vertebrate model systems, the frog, and avian embryos. The role of the principal substrates of ADAMs, the cadherins, has been extensively described in other reviews, most recently in (Cousin [1997] Mechanisms of Development 148:79-88; Taneyhill and Schiffmacher [2017] Genesis, 55). The function of ADAMs in the migration of other cell types, including the immune system, wound healing and cancer has been described previously in (Dreymueller et al. [2017] Mediators of Inflammation 2017: 9621724). Our goal is to illustrate both the importance of ADAMs in controlling neural crest behavior and how neural crest cells have helped us understand the molecular interactions, substrates, and functions of ADAM proteins in vivo.

FOXD3/FOXD4 is required for the development of hindgut in the rat model of anorectal malformation.

Congenital anorectal malformation is the most common digestive tract malformation in newborns. It has been reported that FOXD3/FOXD4, a forkhead transcription factor, regulates the generation, migration, and differentiation of neural crest cells. However, whether FOXD3/FOXD4 takes part in anorectal malformation remains unclear. In the present study, we used ethylene thiourea to induce the animal models of anorectal malformation in rat embryos and to interrogate the role of FOXD3/FOXD4 in anorectal malformation pathogenesis. Hindgut samples of the animal models were collected at E15, E17, E19, and E21 days of age. The expression of FOXD3/FOXD4 was detected by immunohistochemistry, western blot, and quantitative real-time fluorescence PCR. By immunohistochemical staining, FOXD3/FOXD4 was observed in epithelial cells of the rectum and the anus both in normal and rat embryos with anorectal malformation. Expression level analysis by western blot indicated that FOXD3/FOXD4 expression increased in ethylene thiourea-induced anorectal malformation groups. mRNA expression as determined by quantitative real-time fluorescence PCR analysis was consistent with the western blot results. Tentative conclusions were drawn that FOXD3/FOXD4 is expressed in the hindgut in rat embryos and is upregulated in anorectal malformation. FOXD3/FOXD4 is required for the development of the hindgut, and its aberrant expression may be an important factor leading to the incidence of anorectal malformation. Impact statement Congenital anorectal malformation (ARM) is the most common digestive tract malformation in newborns. The pathophysiological ground remains unclear. In this study, we used animal models of ARM for the first time to interrogate the role of FOXD3/FOXD4 in ARM pathogenesis. The animal models of ARM were successfully induced by ethylene thiourea (ETU) in rat embryos providing a strong basis for pathogenesis study of this disease. Expression analysis of FOXD3/FOXD4 was carried out in these models, and the results shape a deeper understanding of FOXD3/FOXD4 being required for the normal development of the hindgut. The aberrant expression of FOXD3/FOXD4 may be an important factor leading to ARM incidence.

Feeder-free differentiation of cells exhibiting characteristics of corneal endothelium from human induced pluripotent stem cells.

ABSTRACTThe purpose of this study was to devise a strategy for the derivation of corneal endothelial cells (CEnCs) from adult fibroblast-derived induced pluripotent stem cells (iPSCs). IPSCs were generated from an adult human with normal ocular history via expression of OCT4, SOX2, KLF4 and c-MYC. Neural crest cells (NCCs) were differentiated from iPSCs via addition of CHIR99021 and SB4315542. NCCs were driven toward a CEnC fate via addition of B27, PDGF-BB and DKK-2 to CEnC media. Differentiation of NCCs and CEnCs was evaluated via rt-PCR, morphological and immunocytochemical analysis. At 17 days post-NCC induction, there were notable changes in cell morphology and upregulation of the neural crest lineage transcripts PAX3, SOX9, TFAP2A, SOX10 and p75NTR and the proteins p75/NGFR and SOX10. Exposure of NCCs to B27, PDGF-BB and DKK-2 induced a shift in morphology from a spindle-shaped neural phenotype to a tightly-packed hexagonal appearance and increased expression of the transcripts ATP1A1, COL8A1, COL8A2, AQP1 and CDH2 and the proteins ZO-1, N-Cad, AQP-1 and Na+/K+ATPase. Replacement of NCC media with CEnC media on day 3, 5 or 8 reduced the differentiation time needed to yield CEnCs. IPSC-derived CEnCs could be used for evaluation of cornea endothelial disease pathophysiology and for testing of novel therapeutics.

Establishment of a murine culture system for modeling the temporal progression of cranial and trunk neural crest cell differentiation.

ABSTRACTThe neural crest (NC) is a transient population of embryonic progenitors that are implicated in a diverse range of congenital birth defects and pediatric syndromes. The broad spectrum of NC-related disorders can be attributed to the wide variety of differentiated cell types arising from the NC. In vitro models of NC development provide a powerful platform for testing the relative contributions of intrinsic and extrinsic factors mediating NC differentiation under normal and pathogenic conditions. Although differentiation is a dynamic process that unfolds over time, currently, there is no well-defined chronology that characterizes the in vitro progression of NC differentiation towards specific cell fates. In this study, we have optimized culture conditions for expansion of primary murine NC cells that give rise to both ectodermal and mesoectodermal derivatives, even after multiple passages. Significantly, we have delineated highly reproducible timelines that include distinct intermediate stages for lineage-specific NC differentiation in vitro. In addition, isolating both cranial and trunk NC cells from the same embryos enabled us to make direct comparisons between the two cell populations over the course of differentiation. Our results define characteristic changes in cell morphology and behavior that track the temporal progression of NC cells as they differentiate along the neuronal, glial and chondrogenic lineages in vitro. These benchmarks constitute a chronological baseline for assessing how genetic or environmental disruptions may facilitate or impede NC differentiation. Introducing a temporal dimension substantially increases the power of this platform for screening drugs or chemicals for developmental toxicity or therapeutic potential. This article has an associated First Person interview with the first author of the paper.

Pten regulates neural crest proliferation and differentiation during mouse craniofacial development.

The phosphatase and tensin homolog deleted on chromosome TEN (Pten) is implicated in a broad range of developmental events and diseases. However, its role in neural crest and craniofacial development has not been well illustrated. Using genetically engineered mouse models, we showed that inactivating Pten specifically in neural crest cells causes malformation of craniofacial structures. Pten conditional knockout mice exhibit perinatal lethality with overgrowth of craniofacial structures. At the cellular level, Pten deficiency increases cell proliferation rate and enhances osteoblast differentiation. Our data further revealed that inactivating Pten elevates PI3K/Akt signaling activity in neural crest derivatives, and confirmed that attenuation of PI3K/Akt activity led to decreased neural crest cell proliferation and differentiation both in vitro and in vivo. Our study revealed that Pten is essential for craniofacial morphogenesis in mice. Inactivating Pten in neural crest cells increases proliferation rate and promotes their differentiation toward osteoblasts. Our data further indicate that Pten acts via modulating PI3K/Akt activity during these processes. Developmental Dynamics 247:304-314, 2018. © 2017 Wiley Periodicals, Inc.

Development of interstitial cells of Cajal in the human digestive tract as the result of reciprocal induction of mesenchymal and neural crest cells.

Neural crest cells (NCC) can migrate into different parts of the body and express their strong inductive potential. In addition, they are multipotent and are able to differentiate into various cell types with diverse functions. In the primitive gut, NCC induce differentiation of muscular structures and interstitial cells of Cajal (ICC), and they themselves differentiate into the elements of the enteric nervous system (ENS), neurons and glial cells. ICC develop by way of mesenchymal cell differentiation in the outer parts of the primitive gut wall around the myenteric plexus (MP) ganglia, with the exception of colon, where they appear simultaneously also at the submucosal border of the circular muscular layer around the submucosal plexus (SMP) ganglia. However, in a complex process of reciprocal induction of NCC and local mesenchyma, c‐kit positive precursors are the first to differentiate, representing probably the common precursors of ICC and smooth muscle cells (SMC). C‐kit positive precursors could represent a key impact factor regarding the final differentiation of NCC into neurons and glial cells with neurons subsequently excreting stem cell factor (SCF) and other signalling molecules. Under the impact of SCF, a portion of c‐kit positive precursors lying immediately around the ganglia differentiate into ICC, while the rest differentiate into SMC.

Structural and functional differences in PHOX2B frameshift mutations underlie isolated or syndromic congenital central hypoventilation syndrome.

Heterozygous mutations in the PHOX2B gene are causative of congenital central hypoventilation syndrome (CCHS), a neurocristopathy characterized by defective autonomic control of breathing due to the impaired differentiation of neural crest cells. Among PHOX2B mutations, polyalanine (polyAla) expansions are almost exclusively associated with isolated CCHS, whereas frameshift variants, although less frequent, are often more severe than polyAla expansions and identified in syndromic CCHS. This article provides a complete review of all the frameshift mutations identified in cases of isolated and syndromic CCHS reported in the literature as well as those identified by us and not yet published. These were considered in terms of both their structure, whether the underlying indels induced frameshifts of either 1 or 2 steps (“frame 2” and “frame 3” mutations respectively), and clinical associations. Furthermore, we evaluated the structural and functional effects of one “frame 3” mutation identified in a patient with isolated CCHS, and one “frame 2” mutation identified in a patient with syndromic CCHS, also affected with Hirschsprung's disease and neuroblastoma. The data thus obtained confirm that the type of translational frame affects the severity of the transcriptional dysfunction and the predisposition to isolated or syndromic CCHS.

Trunk neural crest origin of dermal denticles in a cartilaginous fish

Cartilaginous fishes (e.g., sharks and skates) possess a postcranial dermal skeleton consisting of tooth-like "denticles" embedded within their skin. As with teeth, the principal skeletal tissue of dermal denticles is dentine. In the head, cranial neural crest cells give rise to the dentine-producing cells (odontoblasts) of teeth. However, trunk neural crest cells are generally regarded as nonskeletogenic, and so the embryonic origin of trunk denticle odontoblasts remains unresolved. Here, we use expression of FoxD3 to pinpoint the specification and emigration of trunk neural crest cells in embryos of a cartilaginous fish, the little skate (Leucoraja erinacea). Using cell lineage tracing, we further demonstrate that trunk neural crest cells do, in fact, give rise to odontoblasts of trunk dermal denticles. These findings expand the repertoire of vertebrate trunk neural crest cell fates during normal development, highlight the likely primitive skeletogenic potential of this cell population, and point to a neural crest origin of dentine throughout the ancestral vertebrate dermal skeleton.

Rho-associated kinase is a therapeutic target in neuroblastoma

Neuroblastoma is a peripheral neural system tumor that originates from the neural crest and is the most common and deadly tumor of infancy. Here we show that neuroblastoma harbors frequent mutations of genes controlling the Rac/Rho signaling cascade important for proper migration and differentiation of neural crest cells during neuritogenesis. RhoA is activated in tumors from neuroblastoma patients, and elevated expression of Rho-associated kinase (ROCK)2 is associated with poor patient survival. Pharmacological or genetic inhibition of ROCK1 and 2, key molecules in Rho signaling, resulted in neuroblastoma cell differentiation and inhibition of neuroblastoma cell growth, migration, and invasion. Molecularly, ROCK inhibition induced glycogen synthase kinase 3β-dependent phosphorylation and degradation of MYCN protein. Small-molecule inhibition of ROCK suppressed MYCN-driven neuroblastoma growth in TH-MYCN homozygous transgenic mice and MYCN gene-amplified neuroblastoma xenograft growth in nude mice. Interference with Rho/Rac signaling might offer therapeutic perspectives for high-risk neuroblastoma.

Functional analysis of the zebrafish ortholog of HMGCS1 reveals independent functions for cholesterol and isoprenoids in craniofacial development.

There are 8 different human syndromes caused by mutations in the cholesterol synthesis pathway. A subset of these disorders such as Smith-Lemli-Opitz disorder, are associated with facial dysmorphia. However, the molecular and cellular mechanisms underlying such facial deficits are not fully understood, primarily because of the diverse functions associated with the cholesterol synthesis pathway. Recent evidence has demonstrated that mutation of the zebrafish ortholog of HMGCR results in orofacial clefts. Here we sought to expand upon these data, by deciphering the cholesterol dependent functions of the cholesterol synthesis pathway from the cholesterol independent functions. Moreover, we utilized loss of function analysis and pharmacological inhibition to determine the extent of sonic hedgehog (Shh) signaling in animals with aberrant cholesterol and/or isoprenoid synthesis. Our analysis confirmed that mutation of hmgcs1, which encodes the first enzyme in the cholesterol synthesis pathway, results in craniofacial abnormalities via defects in cranial neural crest cell differentiation. Furthermore targeted pharmacological inhibition of the cholesterol synthesis pathway revealed a novel function for isoprenoid synthesis during vertebrate craniofacial development. Mutation of hmgcs1 had no effect on Shh signaling at 2 and 3 days post fertilization (dpf), but did result in a decrease in the expression of gli1, a known Shh target gene, at 4 dpf, after morphological deficits in craniofacial development and chondrocyte differentiation were observed in hmgcs1 mutants. These data raise the possibility that deficiencies in cholesterol modulate chondrocyte differentiation by a combination of Shh independent and Shh dependent mechanisms. Moreover, our results describe a novel function for isoprenoids in facial development and collectively suggest that cholesterol regulates craniofacial development through versatile mechanisms.

Abstract 5832: Rho-associated kinase is a therapeutic target in neuroblastoma

Abstract Background: Neuroblastoma is a peripheral neural system tumor that originates from the neural crest and is the most common and deadly tumor of infancy. The non-canonical Wnt/planar cell polarity (PCP) signaling pathway regulates cytoskeletal organization, migration and maturation of neural crest cells during neuritogenesis. Here we show that neuroblastoma harbors frequent mutations of genes controlling the PCP signaling pathway and Rac/Rho signaling cascade important for proper migration and differentiation of neural crest cells during neuritogenesis. The majority of these mutations were detected in GTPase activating proteins and guanine nucleotide exchange factors genes, which may result in inhibition of Rac and activation of Rho and was associated with aggressive high-stage neuroblastomas. Methods: We performed whole-exome sequencing of neuroblastoma samples of all clinical subgroups to search for mutations and genetic aberrations. Cytotoxic activity of Rho-associated kinases (ROCK) inhibitors was studied in cell viability assays. Morphology and invasion were studied with microscopy. The molecular mechanisms were characterized using cell- and molecular biology techniques. In vivo studies (xenografts and the transgenic mouse model TH-MYCN) in mice were carried out to validate the therapeutic effects and toxicity. Results: Exome sequencing detected frequent mutations and gene aberrations in genes controlling the activity of PCP signaling. Analysis of gene signatures and immunohistochemistry showed that high expression of ROCK1 and ROCK2 correlated with poor patient survival. Several mediators of Rac/Rho were differentially expressed in cell lines and patient samples. Pharmacological or genetic inhibition of ROCK, a key molecule in Rac/Rho signaling resulted in differentiation, inhibition of neuroblastoma cell growth and migration and degradation of MYCN protein. Finally, ROCK inhibition reduced the growth of established neuroblastoma xenografts in nude mice and repressed tumor progression in a MYCN-driven mouse model of neuroblastoma (T-MYCN) Conclusions: These results provide evidence that inhibition of the non-canonical/PCP signaling cascade using ROCK inhibitors suppress the growth of neuroblastoma in preclinical models and suggest possibilities for improved treatment of high-risk neuroblastomas. Citation Format: Cecilia H. Dyberg, Susanne Fransson, Teodora Andonova, Baldur Sveinbjörnsson, Jessika Lännerholm-Palm, David Forsberg, Eric Herlenius, Tommy Martinsson, Per Kogner, John Inge Johnsen, Malin Wickström. Rho-associated kinase is a therapeutic target in neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5832. doi:10.1158/1538-7445.AM2017-5832

Multivalent binding of PWWP2A to H2A.Z regulates mitosis and neural crest differentiation.

Replacement of canonical histones with specialized histone variants promotes altering of chromatin structure and function. The essential histone variant H2A.Z affects various DNA-based processes via poorly understood mechanisms. Here, we determine the comprehensive interactome of H2A.Z and identify PWWP2A as a novel H2A.Z-nucleosome binder. PWWP2A is a functionally uncharacterized, vertebrate-specific protein that binds very tightly to chromatin through a concerted multivalent binding mode. Two internal protein regions mediate H2A.Z-specificity and nucleosome interaction, whereas the PWWP domain exhibits direct DNA binding. Genome-wide mapping reveals that PWWP2A binds selectively to H2A.Z-containing nucleosomes with strong preference for promoters of highly transcribed genes. In human cells, its depletion affects gene expression and impairs proliferation via a mitotic delay. While PWWP2A does not influence H2A.Z occupancy, the C-terminal tail of H2A.Z is one important mediator to recruit PWWP2A to chromatin. Knockdown of PWWP2A in Xenopus results in severe cranial facial defects, arising from neural crest cell differentiation and migration problems. Thus, PWWP2A is a novel H2A.Z-specific multivalent chromatin binder providing a surprising link between H2A.Z, chromosome segregation, and organ development.

Methyltransferase G9A Regulates Osteogenesis via Twist Gene Repression

Here we investigate the role of epigenetic factors in controlling the timing of cranial neural crest cell differentiation. The gene coding for histone H3 lysine 9 methyltransferase G9A was conditionally deleted in neural crest cells with Wnt1-Cre. The majority of homozygous-null animals survived to birth but thereafter failed to thrive. Phenotypic analysis of postnatal animals revealed that the mutants displayed incomplete ossification and 20% shorter jaws as compared to their wild-type littermates. At E13.5, patterns of expression of the osteogenic transcription factor RUNX2 and the mesenchymal transcription factor TWIST are similar in controls and mutants; both overlap in areas of future intramembranous bone formation. At E14.5, the nonosteogenic mesenchyme expressed TWIST, whereas the ossification center had strong RUNX2 and osteopontin expression. In the mutants, TWIST protein was present in the osteogenic mesenchyme, while osteopontin was not expressed until E15.5. In addition, in mutants, small regions of TWIST-positive osteogenic mesenchyme were visible until E15.5. The delay in ossification and reduction in size of the ossification centers were correlated with an earlier decrease in proliferation. We used micromass cultures of the face to investigate the direct effects of G9A inhibition on skeletal differentiation. Addition of a small molecule inhibitor for G9A, BIX-01294, to wild-type cells upregulated Twist genes similar to what was observed in vivo. The inhibitor also caused decreases in several osteogenic markers. Chromatin immunoprecipitation analysis of primary osteogenic mesenchyme from calvaria revealed that Twist1 and Twist2 regulatory regions contain the repressive H3K9me2 marks catalyzed by G9A, which are removed when BIX-01294 is added. Our results establish a role for G9A and H3K9me2 in the regulation of Twist genes and provide novel insights into the significance of epigenetic mechanisms in controlling temporal and tissue-specific gene expression during development.

What do we know about the structure of human thymic Hassall’s corpuscles? A histochemical, immunohistochemical, and electron microscopic study

Hassall’s corpuscles are the most prominent structures in the human thymus. However, relatively few analyses have been performed to determine their function and cellular origins during development. In this study, we evaluated the cellular microenvironment of human thymic Hassall’s corpuscles using histochemistry, immunohistochemistry, and transmission electron microscopy. We examined 95 human thymic tissue samples, which were perioperatively obtained from children undergoing cardiac surgery. To characterize the complex cellular microenvironment of human thymic corpuscles, we used a panel of 14 different antibodies to identify discrete cell types. We also utilized various histochemical methods (PAS reaction, alcian blue staining, alkaline phosphatase and acid phosphatase activity staining, von Kossa staining of calcified particles) and transmission electron microscopy to visualize these structures. Considerable variation in the sizes, shapes, and numbers of Hassall’s corpuscles was observed, even amongst children of the same age. Inside the largest Hassall’s corpuscles, cystic dilatation with an accumulation of cellular debris was found. These morphological observations might be associated with disruptions in the formation, migration, or differentiation of cardiac neural crest cells, which are essential for heart and thymus development. Immunohistochemical staining and electron microscopy revealed that Hassall’s corpuscles resemble other types of stratified squamous epithelia. Most Hassall’s corpuscles are heterocellular, consisting of thymic epithelial cells, macrophages, interdigitating dendritic cells, myoid cells, and, occasionally, mast cells and lymphocytes. To explore the potential functions of Hassall’s corpuscles, we found that the concentrations of B-lymphocytes and BCL2-positive lymphocytes suggested a role in regulation of lymphopoiesis. We also found that these structures do not originate from the perivascular epithelium as previously proposed, nor could we identify blood or lymph endothelial cells in close proximity. This leaves the origins of Hassall’s corpuscles an open question.

Inhibition of FGF signaling accelerates neural crest cell differentiation of human pluripotent stem cells

Neural crest (NC) is a transient population, arising during embryonic development and capable of differentiating into various somatic cells. The defects of neural crest development leads to neurocristopathy. Several signaling pathways were revealed their significance in NC cell specification. Fibroblast growth factor (FGF) is recognized as an important signaling during NC development, for instance Xenopus and avian; however, its contributions in human species are remained elusive. Here we used human pluripotent stem cells (hPSCs) to investigate the consequences of FGF inhibition during NC cell differentiation. The specific-FGF receptor inhibitor, SU5402, was used in this investigation. The inhibition of FGF did not found to affect the proliferation or death of hPSC-derived NC cells, but promoted hPSCs to commit NC cell fate. NC-specific genes, including PAX3, SLUG, and TWIST1, were highly upregulated, while hPSC genes, such as OCT4, and E-CAD, rapidly reduced upon FGF signaling blockage. Noteworthy, TFAP-2α, a marker of migratory NC cells, abundantly presented in SU5402-induced cells. This accelerated NC cell differentiation could be due to the activation of Notch signaling upon the blockage of ERK1/2 phosphorylation, since NICD was increased by SU5402. Altogether, this study proposed the contributions of FGF signaling in controlling human NC cell differentiation from hPSCs, the crosstalk between FGF and Notch, and might imply to the influences of FGF signaling in neurocristophatic diseases.