Neural Crest Cell Development Research Articles

Dynamic regulation and requirement for ribosomal RNA transcription during mammalian development

Ribosomal RNA (rRNA) transcription by RNA polymerase I (Pol I) is a critical rate-limiting step in ribosome biogenesis, which is essential for cell survival. Despite its global function, disruptions in ribosome biogenesis cause tissue-specific birth defects called ribosomopathies, which frequently affect craniofacial development. Here, we describe a cellular and molecular mechanism underlying the susceptibility of craniofacial development to disruptions in Pol I transcription. We show that Pol I subunits are highly expressed in the neuroepithelium and neural crest cells (NCCs), which generate most of the craniofacial skeleton. High expression of Pol I subunits sustains elevated rRNA transcription in NCC progenitors, which supports their high tissue-specific levels of protein translation, but also makes NCCs particularly sensitive to rRNA synthesis defects. Consistent with this model, NCC-specific deletion of Pol I subunits Polr1a, Polr1c, and associated factor Tcof1 in mice cell-autonomously diminishes rRNA synthesis, which leads to p53 protein accumulation, resulting in NCC apoptosis and craniofacial anomalies. Furthermore, compound mutations in Pol I subunits and associated factors specifically exacerbate the craniofacial anomalies characteristic of the ribosomopathies Treacher Collins syndrome and Acrofacial Dysostosis-Cincinnati type. Mechanistically, we demonstrate that diminished rRNA synthesis causes an imbalance between rRNA and ribosomal proteins. This leads to increased binding of ribosomal proteins Rpl5 and Rpl11 to Mdm2 and concomitantly diminished binding between Mdm2 and p53. Altogether, our results demonstrate a dynamic spatiotemporal requirement for rRNA transcription during mammalian cranial NCC development and corresponding tissue-specific threshold sensitivities to disruptions in rRNA transcription in the pathogenesis of congenital craniofacial disorders.

The cell surface hyaluronidase TMEM2 plays an essential role in mouse neural crest cell development and survival.

Hyaluronan (HA) is a major extracellular matrix component whose tissue levels are dynamically regulated during embryonic development. Although the synthesis of HA has been shown to exert a substantial influence on embryonic morphogenesis, the functional importance of the catabolic aspect of HA turnover is poorly understood. Here, we demonstrate that the transmembrane hyaluronidase TMEM2 plays an essential role in neural crest development and the morphogenesis of neural crest derivatives, as evidenced by the presence of severe craniofacial abnormalities in Wnt1-Cre-mediated Tmem2 knockout (Tmem2CKO) mice. Neural crest cells (NCCs) are a migratory population of cells that gives rise to diverse cell lineages, including the craniofacial complex, the peripheral nervous system, and part of the heart. Analysis of Tmem2 expression during NCC formation and migration reveals that Tmem2 is expressed at the site of NCC delamination and in emigrating Sox9-positive NCCs. In Tmem2CKO embryos, the number of NCCs emigrating from the neural tube is greatly reduced. Furthermore, linage tracing reveals that the number of NCCs traversing the ventral migration pathway and the number of post-migratory neural crest derivatives are both significantly reduced in a Tmem2CKO background. In vitro studies using Tmem2-depleted mouse O9-1 neural crest cells demonstrate that Tmem2 expression is essential for the ability of these cells to form focal adhesions on and to migrate into HA-containing substrates. Additionally, we show that Tmem2-deficient NCCs exhibit increased apoptotic cell death in NCC-derived tissues, an observation that is corroborated by in vitro experiments using O9-1 cells. Collectively, our data demonstrate that TMEM2-mediated HA degradation plays an essential role in normal neural crest development. This study reveals the hitherto unrecognized functional importance of HA degradation in embryonic development and highlights the pivotal role of Tmem2 in the developmental process.

Aberrant expression of LINC00346 regulates cell migration and proliferation via competitively binding to miRNA-148a-3p/Dnmt1 in Hirschsprung's disease.

Hirschsprung's disease (HSCR) is a common birth defect caused by dysplasia of neural crest cells in the gut. Long noncoding RNAs (lncRNAs) play an important role in cellular processes, including development and disease. Despite the known engagement of LINC00346 in several human diseases, its biological function in HSCR remains unknown. The relative expression levels of LINC00346, miR-148a-3p and Dnmt1 in HSCR colon tissues were detected by quantitative real-time PCR. Western blot assays were conducted to investigate the Dnmt1 protein expression level. Knockdown of LINC00346 and overexpression of miR-148a-3p in SH-SY5Y and SK-N-BE(2) cell lines was conducted. Cell proliferation and migration were detected by cell counting Kit-8 assays, 5-ethynyl-2'-deoxyuridine assays and transwell assays. Cell apoptosis was verified by flow cytometric analysis. Furthermore, the competing endogenous RNA (ceRNA) activity of LINC00346 on miR-148a-5p was investigated via bioinformatics analysis and luciferase reporter assays. Downregulation of LINC00346 and Dnmt1 was detected in HSCR tissues. Knockdown of LINC00346 and overexpression of miR-148a-3p in SK-N-BE(2) and SH-SY5Y cells inhibited cell migration and proliferation and promoted apoptosis. Moreover, the miR-148a-3p inhibitor rescued the downregulation of Dnmt1 in LINC00346 knockdown cell lines, which was evidence of the ceRNA regulatory mechanism of Dnmt1 by LINC00346. LINC00346 was downregulated in HSCR colon tissues and acted as a ceRNA to regulate the expression of Dnmt1 in vitro. Together, these findings indicate that LINC00346 could affect the occurrence of HSCR by participating in the development of enteric neural crest cells.

Aberrant high expression of the TET1 gene in Hirschsprung's disease.

The pathogenesis of Hirschsprung's disease (HSCR) remains unclear but might involve genes participating in neural crest development. Gene methylation controls the expression of many genes and is involved in the development and migration of neural crest cells, but the involvement of demethylation in HSCR is unknown. This study aimed to investigate the expression of ten-eleven translocation methylcytosine dioxygenase 1 (TET1) (a demethylation protein) in patients with HSCR. This is a retrospective study of surgical specimens from paediatric patients with and without HSCR (e.g., intussusception and incarcerated hernia) obtained from 07/2015 to 08/2017. TET1 expression was determined by qRT-PCR, western blotting, and immunohistochemistry. The levels of 5-hydroxymethylcytosine were determined by the dot blot assay. The specimens of 35 patients with HSCR and 25 controls were collected. The median TET1 mRNA expression values were 1.028 [HSCR-stenotic (S)], 0.908 [HSCR-dilated (D)], and 0.467 (control) (HSCR-S vs. control: P=0.002; HSCR-D vs. control: P=0.008; HSCR-S vs. HSCR-D: P=0.44). TET1 protein levels followed a similar pattern. The intensity of immunostaining identified higher expression of TET1 in HSCR colon tissues compared with control tissues. The 5-hmC levels in HSCR stenotic segment samples were significantly higher than those in controls. The expression of TET1 is higher in paediatric patients with HSCR than in controls. DNA demethylation initiated by TET1 may be related to HSCR, which demonstrates that TET1 may play a role in the development of HSCR.

Preparation and Morphological Analysis of Chick Cranial Neural Crest Cell Cultures.

During vertebrate development, neural crest cells (NCCs) migrate extensively and differentiate into various cell types that contribute to structures like the craniofacial skeleton and the peripheral nervous system. While it is critical to understand NCC migration in the context of a 3D embryo, isolating migratory cells in 2D culture facilitates visualization and functional characterization, complementing embryonic studies. The present protocol demonstrates a method for isolating chick cranial neural folds to generate primary NCC cultures. Migratory NCCs emerge from neural fold explants plated onto a fibronectin-coated substrate. This results in dispersed, adherent NCC populations that can be assessed by staining and quantitative morphological analyses. This simplified culture approach is highly adaptable and can be combined with other techniques. For example, NCC emigration and migratory behaviors can be evaluated by time-lapse imaging or functionally queried by including inhibitors or experimental manipulations of gene expression (e.g., DNA, morpholino, or CRISPR electroporation). Because of its versatility, this method provides a powerful system for investigating cranial NCC development.

Abstract 1670: A human pluripotent stem cell derived differentiation model for the study of normal and transformed sympatho-adrenal progenitors

Abstract Background: Neuroblastoma (NB) is a clinically and genetically heterogeneous embryonal cancer thought to arise due to a disruption of normal sympatho-adrenergic progenitors (SAPs) development. Defining the unknown molecular pathways of normal SAP differentiation is essential to improve our limited understanding of NB pathogenesis, and devise more effective and safer therapies. Currently, accurate human model systems for normal and aberrant SAP development are lacking. Using human pluripotent stem cells (hPSCs), we optimised and characterised an in vitro differentiation model to generate SAPs to dissect normal SAP development and NB oncogenesis. Aims: Using hPSCs, we optimised and validated an in vitro differentiation model to investigate normal and aberrant SAP development. Methods/materials: Utilizing a differentiation protocol developed by the Studer laboratory at Memorial Sloan Kettering Cancer Center, we performed in vitro differentiations of hPSCs into SAPs. Throughout a 40-day differentiation process, we harvested cells for bulk RNA-sequencing to analyse time-course expression profiles of specific neurodevelopmental markers. Next, utilizing 10XGenomics, we further mapped molecular phenotypes at the single-cell level. Additionally, we combined our data with the publicly available human SAP single cell-data set by the Adamayko laboratory to identify our cells of interest and pinpoint the distinct cell populations. Results: During differentiation, we could confirm the development of truncal neural crest cells (NCCs) based on the expression of SOX10 and truncal HOX genes, including B7 and B9. Further differentiation of truncal NCCs into SAPs confirmed SOX10 downregulation and upregulation of neurodevelopmental transcription factors such as ASCL1, PHOX2B and STMN2. By comparing our in vitro results with in vivo data generated from human embryos, we discovered that we could generate the in vivo SAP developmental subpopulations. We further characterised all the subpopulations using sets of tissue-specific makers (i.e., PLP1, CHGA, and ISL1). These results confirm that our model can accurately generate SAPs that are comparable to their in vivo counterparts. Conclusion: We have developed a human in vitro differentiation model to generate SAPs resembling their in vivo counterparts. This model allows for mechanistic studies that are unfeasible using in vivo systems, with human cells that faithfully recapitulate the NB cell of origin. We are now using our model to explore NB transformation in vitro using genetically modified and patient-specific hPSCs. Our system will provide critical insights into early NB oncogenesis and will serve as a platform for pre-clinical studies in the context of NB. Citation Format: Stephane Van Haver, Yujie Fan, Wouter Van Loocke, Katleen De Preter, Alex Kentsis, Lorenz Studer, Stephen Roberts, Frank Speleman. A human pluripotent stem cell derived differentiation model for the study of normal and transformed sympatho-adrenal progenitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1670.

The Vitamin A Paradox: Too Much or Too Little Impacts Development and Disease

Vitamin A is essential for proper human embryonic development particularly with respect to brain, craniofacial, limb, and organ, patterning and morphogenesis. Later, in adulthood, vitamin A plays important roles in fertility, immune function, maintenance of vision, and skeletal homeostasis. Surprisingly, deficiency or excess of vitamin A and its metabolic derivative, retinoic acid, can elicit similar effects. Therefore, strict feedback and other mechanisms must exist to regulate levels within a narrow physiological range. We discovered retinol dehydrogenase 10 (Rdh10) as a novel critical regulator of vitamin A metabolism and retinoic acid (RA) synthesis during embryogenesis. Rdh10 oxidizes all trans retinol (vitamin A) to retinal, which is then oxidized by retinaldehyde dehydogenases to retinoic acid. Rdh10 loss‐of‐function mouse embryos exhibit craniofacial anomalies such as choanal atresia, facial clefts, and abnormal branchiomeric muscle development, cardiac malformations including choanal atresia, and gastrointestinal defects such as Hirschsprung disease. Underpinning each phenotype were defects in the development of neural crest cells, a migratory progenitor cell population essential for normal craniofacial and organ morphogenesis. We determined that Rdh10 and retinoic acid signaling are precisely required between embryonic day (E) 7.5‐9.5 in both an intrinsic/autocrine and extrinsic/paracrine manner for proper neural crest cell development. Thus, Rdh10 forms a key nodal point in the regulation of vitamin A metabolism and retinoic acid synthesis and is essential for regulating basic developmental and homeostasis signaling pathways. Furthermore, Rdh10 and vitamin A signaling are both genetic and non‐genetic risk factors in the etiology and pathogenesis of congenital birth defects.

Elucidating the Role of N‐cadherin during Trigeminal Gangliogenesis

Cadherin‐based adhesion is critical to the formation of multicellular tissues, including the cranial sensory ganglia of the peripheral nervous system. Initial assembly of the cranial trigeminal ganglion, which houses the cell bodies for the trigeminal nerve, relies on the coalescence of N‐cadherin (N‐cad)‐expressing placode cell‐derived neurons and Cadherin‐7‐expressing neural crest cells. The trigeminal ganglion has a role that is primarily sensory, transmitting information on pain, touch, and temperature from different facial regions back to the central nervous system. Proper survival and differentiation of trigeminal neurons relies, in part, upon binding of neurotrophic and other growth factors secreted from target tissues to their cognate receptors found on peripheral axons. These complexes are then trafficked to neuronal cell bodies, resulting in the activation of downstream signaling pathways. Prior studies have shown that N‐cad internalization and trafficking are required for proper neuronal outgrowth. Moreover, N‐cad knockdown in chick trigeminal placode cells leads to early defects in trigeminal ganglion assembly by increasing placodal neuron dispersal. Later functions for N‐cad in chick trigeminal gangliogenesis, however, remain obscure. We hypothesize that N‐cad controls trigeminal ganglion development through the regulation of multiple signaling pathways. To address this, we performed morpholino‐mediated knockdown of N‐cad in chick trigeminal placode cells followed by immunohistochemistry and biochemistry to evaluate trigeminal gangliogenesis. Our preliminary data show that N‐cad depletion has a sustained negative impact on the trigeminal ganglion through cell and non‐cell autonomous effects, resulting in decreased ganglion size, aberrant nerve outgrowth and branching, and reduced levels of Cadherin‐7, neurotrophins and their receptors, and Smad family members. Future studies will aim to elucidate the mechanisms by which N‐cad controls trigeminal ganglion development via these molecules and pathways, including experiments designed to examine protein trafficking in trigeminal neurons. Taken together, our findings reveal potential adhesion‐dependent and ‐independent functions for N‐cad in the trigeminal ganglion, which will aid in the understanding of its tissue‐specific roles. This knowledge will shed light on the etiology of disorders characterized by abnormal neural crest cell and placode cell development and those affecting the peripheral nervous system.

Novel roles for rRNA and ribosome biogenesis associated factors in neural crest cell colonization of the gut and in the pathogenesis of Hirschsprung disease

The enteric nervous system regulates the functions of the gastrointestinal system, which include food transportation, digestion, nutrient and liquid absorption and expulsion of waste. The enteric nervous system is comprised of neurons and glia that are primarily derived from a multipotent, migratory population of cells called neural crest cells. Defects in the formation, migration and or differentiation of neural crest cells can lead to incomplete colonization of the gastrointestinal tract and perturbed ENS development resulting in Hirschsprung disease. Hirschsprung disease is a congenital disorder characterized by the absence of ganglia in variable regions of the gastrointestinal tract and ~50% of the known cases of Hirschsprung disease have an unknown genetic diagnosis. This makes it imperative to identify other genes and biological processes that contribute to the etiology and pathogenesis of Hirschsprung disease. We have identified three new genes, Tcof1, Polr1a and Polr1c, and the process of rDNA transcription which is a rate‐limiting step in ribosome biogenesis, as novel regulators of neural crest cell development, enteric nervous system formation and the etiology of Hirschsprung disease. Loss of function mutation of Tcof1, Polr1a and Polr1c in neural crest cells results in total intestinal aganglionosis, the most severe form of Hirschsprung disease in mice. Mechanistically, downregulation of Tcof1, Polr1a and Polr1c results in reduced rRNA synthesis, which leads to increased p53‐dependent NCC apoptosis, and insufficient NCC to colonize the gut. This led us to hypothesize that inhibition of p53 could rescue the Hirschsprung phenotype in the mutants, and we are currently testing this and other and other approaches as potential preventative therapies.

Global ubiquitinome profiling identifies NEDD4 as a regulator of Profilin 1 and actin remodelling in neural crest cells

The ubiquitin ligase NEDD4 promotes neural crest cell (NCC) survival and stem-cell like properties to regulate craniofacial and peripheral nervous system development. However, how ubiquitination and NEDD4 control NCC development remains unknown. Here we combine quantitative analysis of the proteome, transcriptome and ubiquitinome to identify key developmental signalling pathways that are regulated by NEDD4. We report 276 NEDD4 targets in NCCs and show that loss of NEDD4 leads to a pronounced global reduction in specific ubiquitin lysine linkages. We further show that NEDD4 contributes to the regulation of the NCC actin cytoskeleton by controlling ubiquitination and turnover of Profilin 1 to modulate filamentous actin polymerization. Taken together, our data provide insights into how NEDD4-mediated ubiquitination coordinates key regulatory processes during NCC development.

16p12.1 Deletion Orthologs are Expressed in Motile Neural Crest Cells and are Important for Regulating Craniofacial Development in Xenopus laevis.

Copy number variants (CNVs) associated with neurodevelopmental disorders are characterized by extensive phenotypic heterogeneity. In particular, one CNV was identified in a subset of children clinically diagnosed with intellectual disabilities (ID) that results in a hemizygous deletion of multiple genes at chromosome 16p12.1. In addition to ID, individuals with this deletion display a variety of symptoms including microcephaly, seizures, cardiac defects, and growth retardation. Moreover, patients also manifest severe craniofacial abnormalities, such as micrognathia, cartilage malformation of the ears and nose, and facial asymmetries; however, the function of the genes within the 16p12.1 region have not been studied in the context of vertebrate craniofacial development. The craniofacial tissues affected in patients with this deletion all derive from the same embryonic precursor, the cranial neural crest, leading to the hypothesis that one or more of the 16p12.1 genes may be involved in regulating neural crest cell (NCC)-related processes. To examine this, we characterized the developmental role of the 16p12.1-affected gene orthologs, polr3e, mosmo, uqcrc2, and cdr2, during craniofacial morphogenesis in the vertebrate model system, Xenopus laevis. While the currently-known cellular functions of these genes are diverse, we find that they share similar expression patterns along the neural tube, pharyngeal arches, and later craniofacial structures. As these genes show co-expression in the pharyngeal arches where NCCs reside, we sought to elucidate the effect of individual gene depletion on craniofacial development and NCC migration. We find that reduction of several 16p12.1 genes significantly disrupts craniofacial and cartilage formation, pharyngeal arch migration, as well as NCC specification and motility. Thus, we have determined that some of these genes play an essential role during vertebrate craniofacial patterning by regulating specific processes during NCC development, which may be an underlying mechanism contributing to the craniofacial defects associated with the 16p12.1 deletion.

Research progress on neural crest cells and neurocristopathies and its pathogenesis.

Neural crest cells (NCCs) are multipotent progenitor cells unique to vertebrates, and they have the ability to differentiate into a variety of cells, such as chondrocytes, neurons, and melanocytes. The formation, migration, and differentiation of NCCs are tightly regulated, and the disruption of NCC development results in abnormal embryo development. Neurocristopathies (NCPs) refer to a group of diseases that develop in response to abnormal development of NCCs. NCPs are of various types and exhibit complex phenotypes, which can affect many parts of the human body, such as the craniofacial structure, heart, intestine, and skin. NCPs negatively impact the physical function and mental health of the affected patients. NCPs account for one third of the defects in children with birth defects. Genetic factors are the main risk factors for NCPs, but environmental factors and abnormal gene-environment interactions can also lead to the development of NCPs. In this review, we introduce NCCs, NCPs, and their pathogenesis, so as to provide a reference point for a systematic understanding of NCPs and NCC development, and to provide scientific support for understanding the etiology of NCPs and their effective prevention and control.

Elevated Hoxb5b Expands Vagal Neural Crest Pool and Blocks Enteric Neuronal Development in Zebrafish.

Neural crest cells (NCCs) are a migratory, transient, and multipotent stem cell population essential to vertebrate embryonic development, contributing to numerous cell lineages in the adult organism. While great strides have been made in elucidating molecular and cellular events that drive NCC specification, comprehensive knowledge of the genetic factors that orchestrate NCC developmental programs is still far from complete. We discovered that elevated Hoxb5b levels promoted an expansion of zebrafish NCCs, which persisted throughout multiple stages of development. Correspondingly, elevated Hoxb5b also specifically expanded expression domains of the vagal NCC markers foxd3 and phox2bb. Increases in NCCs were most apparent after pulsed ectopic Hoxb5b expression at early developmental stages, rather than later during differentiation stages, as determined using a novel transgenic zebrafish line. The increase in vagal NCCs early in development led to supernumerary Phox2b+ enteric neural progenitors, while leaving many other NCC-derived tissues without an overt phenotype. Surprisingly, these NCC-derived enteric progenitors failed to expand properly into sufficient quantities of enterically fated neurons and stalled in the gut tissue. These results suggest that while Hoxb5b participates in vagal NCC development as a driver of progenitor expansion, the supernumerary, ectopically localized NCC fail to initiate expansion programs in timely fashion in the gut. All together, these data point to a model in which Hoxb5b regulates NCCs both in a tissue specific and temporally restricted manner.

Spatiotemporal expression of NRAS and occurrence of giant congenital melanocytic nevi.

The mechanism underlying giant congenital melanocytic nevus (GCMN) formation is not fully understood. According to recent research, NRAS gene mutation is the main driving factor in GCMN. Melanocytic precursor cells proliferate during the embryonic stage after acquiring NRAS mutations. However, why GCMN undergoes intense proliferation in the embryonic stage and then stops postnatally remains unknown. The current theory for this phenomenon is that the GCMN undergoes oncogene-induced senescence. However, there is not enough evidence to indicate that senescence induces growth arrest in GCMN. In this study, we hypothesized that the expression level of the NRAS gene changes dynamically during the development and differentiation of neural crest cells into melanocytes and that the NRAS expression level determines whether the cell proliferates or becomes quiescent.

PACSIN proteins in vivo: Roles in development and physiology.

Protein kinase C and casein kinase substrate in neurons (PACSINs), or syndapins (synaptic dynamin‐associated proteins), are a family of proteins involved in the regulation of cell cytoskeleton, intracellular trafficking and signalling. Over the last twenty years, PACSINs have been mostly studied in the in vitro and ex vivo settings, and only in the last decade reports on their function in vivo have emerged. We first summarize the identification, structure and cellular functions of PACSINs, and then focus on the relevance of PACSINs in vivo. During development in various model organisms, PACSINs participate in diverse processes, such as neural crest cell development, gastrulation, laterality development and neuromuscular junction formation. In mouse, PACSIN2 regulates angiogenesis during retinal development and in human, PACSIN2 associates with monosomy and embryonic implantation. In adulthood, PACSIN1 has been extensively studied in the brain and shown to regulate neuromorphogenesis, receptor trafficking and synaptic plasticity. Several genetic studies suggest a role for PACSIN1 in the development of schizophrenia, which is also supported by the phenotype of mice depleted of PACSIN1. PACSIN2 plays an essential role in the maintenance of intestinal homeostasis and participates in kidney repair processes after injury. PACSIN3 is abundant in muscle tissue and necessary for caveolar biogenesis to create membrane reservoirs, thus controlling muscle function, and has been linked to certain genetic muscular disorders. The above examples illustrate the importance of PACSINs in diverse physiological or tissue repair processes in various organs, and associations to diseases when their functions are disturbed.

Up-regulation of microRNA-34a mediates ethanol-induced impairment of neural crest cell migration in vitro and in zebrafish embryos through modulating epithelial-mesenchymal transition by targeting Snail1

Prenatal ethanol exposure can impair neural crest cell (NCC) development, including NCC survival, differentiation and migration, contributing to the craniofacial dysmorphology in Fetal Alcohol Spectrum Disorders (FASD). Epithelial-mesenchymal transition (EMT) plays an important role in regulating the migration of NCCs. The objective of this study is to determine whether ethanol exposure can suppress NCC migration through inhibiting EMT and whether microRNA-34a (miR-34a) is involved in the ethanol-induced impairment of EMT in NCCs. We found that exposure to 100 mM ethanol significantly inhibited the migration of NCCs. qRT-PCR and Western Blot analysis revealed that exposure to ethanol robustly reduced the mRNA and protein expression of Snail1, a critical transcriptional factor that has a pivotal role in the regulation of EMT. Ethanol exposure also significantly increased the mRNA expression of the Snail1 target gene E-cadherin1 and inhibited EMT in NCCs. We also found that exposure to ethanol significantly elevated the expression of miR-34a that targets Snail1 in NCCs. In addition, down-regulation of miR-34a prevented ethanol-induced repression of Snail1 and diminished ethanol-induced upregulation of Snail1 target gene E-cadherin1 in NCCs. Inhibition of miR-34a restored EMT and prevented ethanol-induced inhibition of NCC migration in vitro and in zebrafish embryos in vivo. These results demonstrate that ethanol-induced upregulation of miR-34a contributes to the impairment of NCC migration through suppressing EMT by targeting Snail1.

Anisodamine affects the pigmentation, mineral density, craniofacial area, and eye development in zebrafish embryos.

Anisodamine is one of the major components of the tropine alkaloid family and is widely used in the treatment of pain, motion sickness, pupil dilatation, and detoxification of organophosphorus poisoning. As a muscarinic receptor antagonist, the low toxicity and moderate drug effect of anisodamine often result in high doses for clinical use, making it important to fully investigate its toxicity. In this study, zebrafish embryos were exposed to 1.3-, 2.6-, and 5.2-mM anisodamine for 7 days to study the toxic effects of drug exposure on pigmentation, mineral density, craniofacial area, and eye development. The results showed that exposure to anisodamine at 1.3 mM resulted in cranial malformations and abnormal pigmentation in zebrafish embryos; 2.6- and 5.2-mM anisodamine resulted in significant eye development defects and reduced bone density in zebrafish embryos. The associated toxicities were correlated with functional development of neural crest cells through gene expression (col1a2, ddb1, dicer1, mab21l1, mab21l2, sox10, tyrp1b, and mitfa) in the dose of 5.2-mM exposed group. In conclusion, this study provides new evidence of the developmental toxicity of high doses of anisodamine in aqueous solutions to organisms and provides a warning for the safe use of this drug.

The double-edged sword role of TGF-β signaling pathway between intrauterine inflammation and cranial neural crest development.

Intrauterine infection would harm a developing embryo/fetus, thereby increasing the risk of developmental malformation. But, whether or not the infection-induced inflammation affects neural crest development still remains obscure. In this study, we employed meta-analysis to demonstrate the potential correlation between infection-induced inflammation and craniofacial anomalies, which was usually derived from the problems in neural crest cell development. The correlation was further verified by inflammatory cytokine release and the activation of nuclear factor kappa-light-chain enhancer of activated B cells signaling in lipopolysaccharide-treated HH10 chicken embryos. In such an inflammatory condition, AP-2α- and Pax7-labeled pre-migratory and migratory neural crest cells in HH10 chicken embryos were significantly less than the ones in control. The bioinformatics analysis of RNA-seq data demonstrated that the principal differential gene expression occurred in transforming growth factor-beta (TGF-β) signaling pathway, which was confirmed by the subsequent experimental results of quantitative PCR and immunofluorescent staining. Under this inflammatory circumstance, whole-mount in situ hybridization, immunofluorescence, and quantitative PCR showed the gene expression changes of key EMT-related transcription factors including upregulated Msx1, downregulated Slug, and FoxD3, as well as adhesion molecules and extracellular matrix protein including upregulated Cadherrin6B, E-cadherin, N-cadherin, and Laminin at the dorsal portion of neural tube of HH10 chicken embryos. Meanwhile, the bioinformatics analysis of RNA-seq data also manifested the differential gene expressions relevant to cell proliferation, which was confirmed by proliferating cell nuclear antigen Western blot data and co-immunofluorescence staining of human natural killer-1 and phosphorylated histone H3. In brief, this study revealed for the first time that the double-edged sword role of TGF-β signaling pathway between intrauterine inflammation (protective role) and cranial neural crest development (harmful role).

Contribution of sox9b to pigment cell formation in medaka fish.

SoxE-type transcription factors, Sox10 and Sox9, are key regulators of the development of neural crest cells. Sox10 specifies pigment cell, glial, and neuronal lineages, whereas Sox9 is reportedly closely associated with skeletogenic lineages in the head, but its involvement in pigment cell formation has not been investigated genetically. Thus, it is not fully understood whether or how distinctly these genes as well as their paralogs in teleosts are subfunctionalized. We have previously shown using the medaka fish Oryzias latipes that pigment cell formation is severely affected by the loss of sox10a, yet unaffected by the loss of sox10b. Here we aimed to determine whether Sox9 is involved in the specification of pigment cell lineage. The sox9b homozygous mutation did not affect pigment cell formation, despite lethality at the early larval stages. By using sox10a, sox10b, and sox9b mutations, compound mutants were established for the sox9b and sox10 genes and pigment cell phenotypes were analyzed. Simultaneous loss of sox9b and sox10a resulted in the complete absence of melanophores and xanthophores from hatchlings and severely defective iridophore formation, as has been previously shown for sox10a-/- ; sox10b-/- double mutants, indicating that Sox9b as well as Sox10b functions redundantly with Sox10a in pigment cell development. Notably, leucophores were present in sox9b-/- ; sox10a-/- and sox10a-/- ; sox10b-/- double mutants, but their numbers were significantly reduced in the sox9b-/- ; sox10a-/- mutants. These findings highlight that Sox9b is involved in pigment cell formation, and plays a more critical role in leucophore development than Sox10b.

Different myocardial microRNA expression patterns are observed in pressure overload- and volume overload-induced chronic heart failure

Abstract Introduction MicroRNAs (miRNA) are short, single-stranded non-coding RNA molecules that regulate gene expression on the post-transcriptional level. Dysregulation of distinct miRNAs has been found to contribute to the development of chronic heart failure (CHF). However, whether distinct types of CHF are associated with different miRNA expression patterns is debated. Aim To characterize left ventricular (LV) miRNA expression in rat models of pressure overload (PO) versus volume overload (VO)-induced CHF. Methods Transverse aortic constriction (TAC) was performed to evoke PO-induced CHF. Aortocaval fistula (ACF) was created to establish VO-induced CHF. Age-matched sham-operated rats served as controls for TAC (ShamT) and ACF (ShamA), respectively. Pressure-volume analysis, echocardiography, histology and quantitative real-time PCR were carried out to assess alterations of the LV. Global miRNA expression profiling was performed using Nanostring technology. Bioinformatics analysis of differentially expressed miRNAs was also carried out to predict relevant miRNA-target interactions. Results Reduced LV systolic function (ejection fraction: 38±5 vs. 65±2% TAC vs. ShamT, 55±3 vs. 67±3%, ACF vs. ShamA, P<0.01) as well as elevated myocardial B-type natriuretic peptide and increased β-to-α myosin heavy chain gene expression confirmed the development of pathological remodeling and CHF in both models. Nevertheless, characteristic differences could be observed in LV morphology and ultrastructure. Accordingly, the TAC model was associated with robustly increased wall thickness, concentric LV hypertrophy and marked fibrotic remodeling. On the contrary, LV dilatation, eccentric LV hypertrophy and moderate fibrosis were the main morphometric findings in the ACF model. A group of miRNA (rno-miR-130a, 132, 199a-5p, 21, 210, 27b, 326) showed similar alterations in both phenotypes of CHF. However, other miRNAs demonstrated unique (specific to TAC: rno-miR-148b-3p, 150, 199a-3p, 203, 23b, 27b, let-7e; specific to ACF: rno-miR-140, 142-3p, 17-5p, 195, 20a, 204, 214, 27a, 29b, 322, 365, 425, 450a, let-7i) LV expressional changes in distinct phenotypes of CHF. In silico bioinformatics analysis revealed that the altered miRNA expression pattern predominantly controls the cardiac neural crest cell development, the inositol-phosphate pathway and the expression of microtubules-binding proteins. In contrast, alterations in the expression of genes responsible for redox state and epithelial-mesenchymal transition were modified only in the ACF group. Despite of the different signaling cascades, expression of Arhgap12 (Rho GTPase activating protein 12) was predicted to be strongly inhibited in both CHF models. Conclusions PO and VO-induced CHF are associated with unique miRNA expression patterns, which drive different signaling pathways. miRNA-controlled downregulation of Arhgap12 might represent a common feature in both phenotypes of CHF. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): ÚNKP-20-4-II-SE-20/New National Excellence Program of the Ministry of Human CapacitiesNVKP_16-1–2016-0017 (“National Heart Program”) National Research, Development and Innovation Fund of Hungary