Formation Of Neural Crest Cells Research Articles

Pharmacological Inhibition of the Spliceosome SF3b Complex by Pladienolide-B Elicits Craniofacial Developmental Defects in Mouse and Zebrafish.

Mutations in genes encoding spliceosome components result in craniofacial structural defects in humans, referred to as spliceosomopathies. The SF3b complex is a crucial unit of the spliceosome, but model organisms generated through genetic modification of the complex do not perfectly mimic the phenotype of spliceosomopathies. Since the phenotypes are suggested to be determined by the extent of spliceosome dysfunction, an alternative experimental system that can seamlessly control SF3b function is needed. To establish another experimental system for model organisms elucidating relationship between spliceosome function and human diseases, we administered Pladienolide-B (PB), a SF3b complex inhibitor, to mouse and zebrafish embryos and assessed resulting phenotypes. PB-treated mouse embryos exhibited neural tube defect and exencephaly, accompanied by apoptosis and reduced cell proliferation in the neural tube, but normal structure in the midface and jaw. PB administration to heterozygous knockout mice of Sf3b4, a gene coding for a SF3b component, influenced the formation of cranial neural crest cells (CNCCs). Despite challenges in continuous PB administration and a high death rate in mice, PB was stably administered to zebrafish embryos, resulting in prolonged survival. Brain, cranial nerve, retina, midface, and jaw development were affected, mimicking spliceosomopathy phenotypes. Additionally, alterations in cell proliferation, cell death, and migration of CNCCs were detected. We demonstrated that zebrafish treated with PB exhibited phenotypes similar to those observed in human spliceosomopathies. This experimental system may serve as a valuable research tool for understanding spliceosome function and human diseases.

Clinical and Genetic Correlation in Neurocristopathies: Bridging a Precision Medicine Gap.

Neurocristopathies (NCPs) encompass a spectrum of disorders arising from issues during the formation and migration of neural crest cells (NCCs). NCCs undergo epithelial-mesenchymal transition (EMT) and upon key developmental gene deregulation, fetuses and neonates are prone to exhibit diverse manifestations depending on the affected area. These conditions are generally rare and often have a genetic basis, with many following Mendelian inheritance patterns, thus making them perfect candidates for precision medicine. Examples include cranial NCPs, like Goldenhar syndrome and Axenfeld-Rieger syndrome; cardiac-vagal NCPs, such as DiGeorge syndrome; truncal NCPs, like congenital central hypoventilation syndrome and Waardenburg syndrome; and enteric NCPs, such as Hirschsprung disease. Additionally, NCCs' migratory and differentiating nature makes their derivatives prone to tumors, with various cancer types categorized based on their NCC origin. Representative examples include schwannomas and pheochromocytomas. This review summarizes current knowledge of diseases arising from defects in NCCs' specification and highlights the potential of precision medicine to remedy a clinical phenotype by targeting the genotype, particularly important given that those affected are primarily infants and young children.

Effect of valproic acid on the formation and migration of cranial neural crest cells at the early developmental stages in rat embryos.

Cranial neural crest cells (NCCs) are critical for craniofacial development. The administration of valproic acid (VPA) to pregnant females causes craniofacial malformations in offspring. However, the in vivo influence of VPA on mammalian cranial NCCs remains unclear. In this study, we aimed to elucidate the developmental stage-specific effect of VPA on cranial NCCs through the administration of a single dose of VPA to pregnant rat females immediately prior to the formation of the cranial neural crest (NC). We performed whole-mount immunohistochemistry or in situ hybridization to examine localization changes of gene transcripts associated with the epithelial-mesenchymal transition of the cranial NC (i.e., cranial NCC formation) and cranial NCC migration. The results showed that Hoxa2 mRNA was abnormally detected and Sox9 mRNA expression was decreased in the midbrain-rhombomere (R) 1/2 NC, which forms cranial NCCs that migrate to the frontonasal mass (FNM) and branchial arch (BA) 1, through VPA administration, thus reducing the formation of SNAI2-positive NCCs. Hoxa2-positive NCCs were detected normally in BA2 and abnormally in FNM and BA1, which are normally Hox-free, implying VPA-induced abnormal cranial NCC migration. In vitro verification experiments using the whole embryo culture system revealed that midbrain-R4 NCC migration was abnormal. These results indicate that VPA reduces the formation/delamination of the midbrain-R1/2 NCCs in a developmental stage-specific manner and subsequently causes the abnormal migration of R4 NCCs, which suggests that the abnormal formation and migration of cranial NCCs contribute to the inhibition of axonal elongation in the trigeminal nerve and a reduction in head size.

Innervation of the pancreas in development and disease.

The autonomic nervous system innervates the pancreas by sympathetic, parasympathetic and sensory branches during early organogenesis, starting with neural crest cell invasion and formation of an intrinsic neuronal network. Several studies have demonstrated that signals from pancreatic neural crest cells direct pancreatic endocrinogenesis. Likewise, autonomic neurons have been shown to regulate pancreatic islet formation, and have also been implicated in type I diabetes. Here, we provide an overview of recent progress in mapping pancreatic innervation and understanding the interactions between pancreatic neurons, epithelial morphogenesis and cell differentiation. Finally, we discuss pancreas innervation as a factor in the development of diabetes.

The Core Splicing Factors EFTUD2, SNRPB and TXNL4A Are Essential for Neural Crest and Craniofacial Development.

Mandibulofacial dysostosis (MFD) is a human congenital disorder characterized by hypoplastic neural-crest-derived craniofacial bones often associated with outer and middle ear defects. There is growing evidence that mutations in components of the spliceosome are a major cause for MFD. Genetic variants affecting the function of several core splicing factors, namely SF3B4, SF3B2, EFTUD2, SNRPB and TXNL4A, are responsible for MFD in five related but distinct syndromes known as Nager and Rodriguez syndromes (NRS), craniofacial microsomia (CFM), mandibulofacial dysostosis with microcephaly (MFDM), cerebro-costo-mandibular syndrome (CCMS) and Burn–McKeown syndrome (BMKS), respectively. Animal models of NRS and MFDM indicate that MFD results from an early depletion of neural crest progenitors through a mechanism that involves apoptosis. Here we characterize the knockdown phenotype of Eftud2, Snrpb and Txnl4a in Xenopus embryos at different stages of neural crest and craniofacial development. Our results point to defects in cranial neural crest cell formation as the likely culprit for MFD associated with EFTUD2, SNRPB and TXNL4A haploinsufficiency, and suggest a commonality in the etiology of these craniofacial spliceosomopathies.

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.

Inability to switch from ARID1A-BAF to ARID1B-BAF impairs exit from pluripotency and commitment towards neural crest formation in ARID1B-related neurodevelopmental disorders

Subunit switches in the BAF chromatin remodeler are essential during development. ARID1B and its paralog ARID1A encode for mutually exclusive BAF subunits. De novo ARID1B haploinsufficient mutations cause neurodevelopmental disorders, including Coffin-Siris syndrome, which is characterized by neurological and craniofacial features. Here, we leveraged ARID1B+/− Coffin-Siris patient-derived iPSCs and modeled cranial neural crest cell (CNCC) formation. We discovered that ARID1B is active only during the first stage of this process, coinciding with neuroectoderm specification, where it is part of a lineage-specific BAF configuration (ARID1B-BAF). ARID1B-BAF regulates exit from pluripotency and lineage commitment by attenuating thousands of enhancers and genes of the NANOG and SOX2 networks. In iPSCs, these enhancers are maintained active by ARID1A-containing BAF. At the onset of differentiation, cells transition from ARID1A- to ARID1B-BAF, eliciting attenuation of the NANOG/SOX2 networks and triggering pluripotency exit. Coffin-Siris patient cells fail to perform the ARID1A/ARID1B switch, and maintain ARID1A-BAF at the pluripotency enhancers throughout all stages of CNCC formation. This leads to persistent NANOG/SOX2 activity which impairs CNCC formation. Despite showing the typical neural crest signature (TFAP2A/SOX9-positive), ARID1B-haploinsufficient CNCCs are also aberrantly NANOG-positive. These findings suggest a connection between ARID1B mutations, neuroectoderm specification and a pathogenic mechanism for Coffin-Siris syndrome.

SUMOylation Potentiates ZIC Protein Activity to Influence Murine Neural Crest Cell Specification.

The mechanisms of neural crest cell induction and specification are highly conserved among vertebrate model organisms, but how similar these mechanisms are in mammalian neural crest cell formation remains open to question. The zinc finger of the cerebellum 1 (ZIC1) transcription factor is considered a core component of the vertebrate gene regulatory network that specifies neural crest fate at the neural plate border. In mouse embryos, however, Zic1 mutation does not cause neural crest defects. Instead, we and others have shown that murine Zic2 and Zic5 mutate to give a neural crest phenotype. Here, we extend this knowledge by demonstrating that murine Zic3 is also required for, and co-operates with, Zic2 and Zic5 during mammalian neural crest specification. At the murine neural plate border (a region of high canonical WNT activity) ZIC2, ZIC3, and ZIC5 function as transcription factors to jointly activate the Foxd3 specifier gene. This function is promoted by SUMOylation of the ZIC proteins at a conserved lysine immediately N-terminal of the ZIC zinc finger domain. In contrast, in the lateral regions of the neurectoderm (a region of low canonical WNT activity) basal ZIC proteins act as co-repressors of WNT/TCF-mediated transcription. Our work provides a mechanism by which mammalian neural crest specification is restricted to the neural plate border. Furthermore, given that WNT signaling and SUMOylation are also features of non-mammalian neural crest specification, it suggests that mammalian neural crest induction shares broad conservation, but altered molecular detail, with chicken, zebrafish, and Xenopus neural crest induction.

Current concepts on ocular vascular abnormalities in the phakomatoses

ABSTRACT Background: Neurofibromatosis, Von Hippel Lindau disease, and tuberous sclerosis complex are classified under the term phakomatoses. They are characterized by ocular vascular abnormalities such as vascular tortuosity, corkscrew retinal vessel configuration, moyamoya-like aspect, microaneurysms, hemangioblastomas, and focal sheathing of retinal arteries, possibly due to abnormal formation, migration, and differentiation of neural crest cells. These alterations can be the first sign or the hallmark of disease and can be related to vasoproliferative tumors. Purpose: Novel imaging technologies in ophthalmology, such as near-infrared reflectances and spectral domain optical coherence tomography, have improved our knowledge in the diagnosis of these pathologies. Previously undetected macular vascular alterations have been reported in phakomatoses using optical coherence tomography angiography. This review will summarize the ophthalmic vascular abnormalities and novel imaging methods in the phakomatoses. Conclusion: Active research is being led into the ophthalmic management of these conditions and their complications, and owing to elevated vascular endothelial growth factor production from hemangioblastoma, hamartoma, and retinal vascular proliferative tumors, increasing interest in this line of therapy has been conducted although research is still ongoing in this area.

Sorting Sox: Diverse Roles for Sox Transcription Factors During Neural Crest and Craniofacial Development.

Sox transcription factors play many diverse roles during development, including regulating stem cell states, directing differentiation, and influencing the local chromatin landscape. Of the twenty vertebrate Sox factors, several play critical roles in the development the neural crest, a key vertebrate innovation, and the subsequent formation of neural crest-derived structures, including the craniofacial complex. Herein, we review the specific roles for individual Sox factors during neural crest cell formation and discuss how some factors may have been essential for the evolution of the neural crest. Additionally, we describe how Sox factors direct neural crest cell differentiation into diverse lineages such as melanocytes, glia, and cartilage and detail their involvement in the development of specific craniofacial structures. Finally, we highlight several SOXopathies associated with craniofacial phenotypes.

Investigating the Effects of Histone H3.3 Point Mutations in Neural Crest Cells and Craniofacial Development

Craniofacial development is an intricate process that requires the meticulous and coordinated expression of many genes. Histone proteins which are fundamental to both the structural integrity and organization of DNA into nucleosomes and consequently, chromatin play an important role in coordinated gene expression. Furthermore, the modification of histone tails is now known to be an essential mechanism for coordinated expression of a large number of genes. Recently, a point mutation in the gene h3f3a which encodes the histone variant Histone H3.3, was identified in a forward genetic screen in zebrafish. H3.3A was found to be important for neural crest cell induction and generation of cranial neural crest cell derived head bones. The authors proposed that incorporation of histone H3.3 was required for expression of genes important for cranial neural crest cell specification, whereas remodelling of this histone tail may be important for later stages in neural crest cell differentiation. However, it remains to be confirmed if disrupted modification of histone tails is also important in neural crest cells. We postulate that modification of the H3.3 tail is required for coordinated expression of genes important for neural crest migration and/or differentiation. To test this hypothesis, we used CRISPR/Cas9 to generate a mouse line with a conditional missense mutation in H3f3a. The mutation generated was previously shown to result in an abnormal increase in methylation of K27 and a decrease in methylation of K36 of the histone H3.3 tail. In this poster, we will present preliminary data using the Wnt‐1 Cre2 mouse line to drive expression of this dominant negative mutation in neural crest cells. Alcian Blue Staining will be used to analyze cartilage in E14.5 embryos and alizarin red will be used to stain bones formed in heads of E17.5 embryos. Bones will be analyzed in control and mutant embryos. Our result will enable us to uncover if K27 or K36 methylation is important in neural crest cell formation, migration and/or differentiation. This work will begin to unravel the role of epigenetics in neural crest cell formation.Support or Funding Information

The histone methyltransferase KMT2D, mutated in Kabuki syndrome patients, is required for neural crest cell formation and migration.

Kabuki syndrome is an autosomal dominant developmental disorder with high similarities to CHARGE syndrome. It is characterized by a typical facial gestalt in combination with short stature, intellectual disability, skeletal findings and additional features like cardiac and urogenital malformations, cleft palate, hearing loss and ophthalmological anomalies. The major cause of Kabuki syndrome are mutations in KMT2D, a gene encoding a histone H3 lysine 4 (H3K4) methyltransferase belonging to the group of chromatin modifiers. Here we provide evidence that Kabuki syndrome is a neurocrestopathy, by showing that Kmt2d loss-of-function inhibits specific steps of neural crest (NC) development. Using the Xenopus model system, we find that Kmt2d loss-of-function recapitulates major features of Kabuki syndrome including severe craniofacial malformations. A detailed marker analysis revealed defects in NC formation as well as migration. Transplantation experiments confirm that Kmt2d function is required in NC cells. Furthermore, analyzing in vivo and in vitro NC migration behavior demonstrates that Kmt2d is necessary for cell dispersion but not protrusion formation of migrating NC cells. Importantly, Kmt2d knockdown correlates with a decrease in H3K4 monomethylation and H3K27 acetylation supporting a role of Kmt2d in the transcriptional activation of target genes. Consistently, using a candidate approach, we find that Kmt2d loss-of-function inhibits Xenopus Sema3F expression, and overexpression of Sema3F can partially rescue Kmt2d loss-of-function defects. Taken together, our data reveal novel functions of Kmt2d in multiple steps of NC development and support the hypothesis that major features of Kabuki syndrome are caused by defects in NC development.

Exploring Shared Susceptibility between Two Neural Crest Cells Originating Conditions: Neuroblastoma and Congenital Heart Disease.

In the past years, genome wide association studies (GWAS) have provided evidence that inter-individual susceptibility to diverse pathological conditions can reveal a common genetic architecture. Through the analysis of congenital heart disease (CHD) and neuroblastoma (NB) GWAS data, we aimed to dissect the genetic susceptibility shared between these conditions, which are known to arise from neural crest cell (NCC) migration or development abnormalities, via identification and functional characterization of common regions of association. Two loci (2q35 and 3q25.32) harbor single nucleotide polymorphisms (SNPs) that are associated at a p-value < 10−3 with conotruncal malformations and ventricular septal defect respectively, as well as with NB. In addition, the lead SNP in 4p16.2 for atrial septal defect and the lead SNP in 3q25.32 for tetralogy of Fallot are less than 250 Kb distant from the lead SNPs for NB at the same genomic regions. Some of these shared susceptibility loci regulate the expression of relevant genes involved in NCC formation and developmental processes (such as BARD1, MSX1, and SHOX2) and are enriched in several epigenetic markers from NB and fetal heart cell lines. Although the clinical correlation between NB and CHD is unclear, our exploration of a possible common genetic basis between NB and a subset of cardiac malformations can help shed light on their shared embryological origin and pathogenetic mechanisms.

Sequence alteration in the enhancer contributes to the heterochronic Sox9 expression in marsupial cranial neural crest

Neonates of marsupial mammals are altricial at birth, because their gestation period is relatively short compared to placental mammals. Yet, as they need to travel to the teat from the birth canal, and suckle on the mother’s milk, forelimbs and jaws develop significantly early. Previous studies in opossum (Monodelphis domestica), an experimental marsupial model, have revealed that cranial neural crest cells are generated significantly early compared to those in placental mammals, such as mouse, leading to an early development of jaw primordia. We have previously found that Sox9, an important neural crest-specifier gene, is expressed in the future cranial neural crest of the opossum embryonic ectoderm significantly earlier than that in mouse or quail embryos. As Sox9 is essential for neural crest formation in various vertebrates, it seems likely that the heterochronic expression of Sox9 is critical for the early cranial neural crest formation in the marsupial embryos. In this study, we show a marsupial-specific sequence in the Sox9 neural crest enhancer E3. We also reveal that the mouse E3 enhancer is activated in the cranial neural crest cells of quail embryos, that the E3 enhancer with marsupial-specific sequence is activated earlier in the Pax7-expressing neural border prior to the onset of endogenous Sox9 expression, and that a misexpression of cMyb, which is also a transcriptional activator of Pax7, in the neural border can ectopically activate the “marsupialized” enhancer. Thus, we suggest that the modification of the E3 enhancer sequence in the marsupial ancestor would have promoted the early expression of Sox9 in the neural border, facilitating the early formation of the cranial neural crest cells and the subsequent heterochronic development of the jaw primordia.

SOX10 as an immunohistochemical marker in cancer diagnostics

SOX10 is a transcription factor that plays an important role in the formation, specification and differentiation of neural crest cells. Recent studies indicate that this protein is expressed in tumors of the nervous system, salivary glands, breast, soft tissues and melanoma cells. Due to its higher sensitivity and specificity in comparison to other available markers, SOX10 protein is mainly used in the diagnosis of malignant peripheral nerve sheet tumor and primary and metastatic melanoma including desmoplastic type. SOX10 has a more readable immunohistochemical staining, which also facilitates histopathological interpretation. Several monoclonal and polyclonal antibodies for diagnostics are currently available. Unification of anti-SOX10 antibodies to increase specificity, eliminate variability and strength of immunohistochemical reactions will support its diagnostic usefulness.

EMT is the major target for okadaic acid-suppressed the development of neural crest cells in chick embryo

As a main marine phycotoxin, okadaic acid (OA) is mainly responsible for diarrheic shellfish poisoning (DSP), through specifically inhibiting phosphatase (PP1 and PP2A). It has been shown that isotope labelled-OA could cross the placental barrier in mice. However, it remains obscure how OA exposure could affect the formation of neural crest cells (NCCs), especially cranial NCCs in early embryo development. Here, we explored the effects of OA exposure on the generation of neural crest cells during embryonic development using the classic chick embryo model. We found that OA exposure at 100 nM (80.5 μg/L) could cause craniofacial bone defects in the developing chick embryo and delay the development of early chick embryos. Immunofluorescent staining of HNK-1, Pax7, and Ap-2α demonstrated that cranial NCC generation was inhibited by OA exposure. Double immunofluorescent staining with Ap-2α/PHIS3 or Pax7/c-Caspase3 manifested that both NCC proliferation and apoptosis were restrained by OA exposure. Furthermore, the expression of Msx1 and BMP4 were down-regulated in the developing chick embryonic neural tubes, which could contribute the inhibitive production of NCCs. We also discovered that expression of EMT-related adhesion molecules, such as Cadherin 6B (Cad6B) and E-cadherin, was altered following OA exposure. In sum, OA exposure negatively affected the development of embryonic neural crest cells, which in turn might result in cranial bone malformation.

Expression of Neural Crest Markers GLDC and ERRFI1 is Correlated with Melanoma Prognosis.

Regulation of particular genes during the formation of neural crest (NC) cells is also described during progression of malignant melanoma. In this context, it is of paramount importance to develop neural crest models allowing the identification of candidate genes, which could be used as biomarkers for melanoma prognosis. Here, we used a human induced Pluripotent Stem Cells (iPSC)-based approach to present novel NC-associated genes, expression of which was upregulated in melanoma. A list of 8 candidate genes, based on highest upregulation, was tested for prognostic value in a tissue microarray analysis containing samples from advanced melanoma (good versus bad prognosis) as well as from high-risk primary melanomas (early metastasizing versus non or late-metastasizing). CD271, GLDC, and ERRFI1 showed significantly higher expression in metastatic patients who died early than the ones who survived at least 30 months. In addition, GLDC and TWIST showed a significantly higher immunohistochemistry (IHC) score in primary melanomas from patients who developed metastases within 12 months versus those who did not develop metastases in 30 months. In conclusion, our iPSC-based study reveals a significant association of NC marker GLDC protein expression with melanoma prognosis.

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.

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.

CHARGE syndrome gastrointestinal involvement: from mouth to anus.

CHARGE syndrome is an autosomal dominant disorder that occurs as a result of a heterozygous loss-of-function mutation in the chromodomain helicase DNA-binding (CHD7) gene, which is important for neural crest cell formation. Gastrointestinal (GI) symptoms and feeding difficulties are highly prevalent but are often a neglected area of diagnosis, treatment, and research. Cranial nerve dysfunction, craniofacial abnormalities, and other physical manifestations of this syndrome lead to gut dysmotility, sensory impairment, and oral-motor function abnormalities. Over 90% of children need tube feeding early in their life and many experience weak sucking/chewing, gastroesophageal reflux disease (GERD), and aspiration. The mainstay of treatment thus far has consisted of feeding therapy, GERD medications, Nissen fundoplication, gastrostomy/jejunostomy, and food texture limitation. Owing to the multitude of severe medical issues associated with this genetic disorder, GI involvement is often overlooked. Here, we report on five patients with CHARGE syndrome who manifested a range of severe GI and feeding difficulties.