Human Birth Defects Research Articles

Self-assembling human heart organoids for the modeling of cardiac development and congenital heart disease

Congenital heart defects constitute the most common human birth defect, however understanding of how these disorders originate is limited by our ability to model the human heart accurately in vitro. Here we report a method to generate developmentally relevant human heart organoids by self-assembly using human pluripotent stem cells. Our procedure is fully defined, efficient, reproducible, and compatible with high-content approaches. Organoids are generated through a three-step Wnt signaling modulation strategy using chemical inhibitors and growth factors. Heart organoids are comparable to age-matched human fetal cardiac tissues at the transcriptomic, structural, and cellular level. They develop sophisticated internal chambers with well-organized multi-lineage cardiac cell types, recapitulate heart field formation and atrioventricular specification, develop a complex vasculature, and exhibit robust functional activity. We also show that our organoid platform can recreate complex metabolic disorders associated with congenital heart defects, as demonstrated by an in vitro model of pregestational diabetes-induced congenital heart defects.

Modeling human congenital disorders with neural crest developmental defects using patient-derived induced pluripotent stem cells.

The neural crest is said to be the fourth germ layer in addition to the ectoderm, mesoderm and endoderm because of its ability to differentiate into a variety of cells that contribute to the various tissues of the vertebrate body. Neural crest cells (NCCs) can be divided into three functional groups: cranial NCCs, cardiac NCCs and trunk NCCs. Defects related to NCCs can contribute to a broad spectrum of syndromes known as neurocristopathies. Studies on the neural crest have been carried out using animal models such as Xenopus, chicks, and mice. However, the precise control of human NCC development has not been elucidated in detail due to species differences. Using induced pluripotent stem cell (iPSC) technology, we developed an in vitro disease model of neurocristopathy by inducing the differentiation of patient-derived iPSCs into NCCs and/or neural crest derivatives. It is now possible to address complicated questions regarding the pathogenetic mechanisms of neurocristopathies by characterizing cellular biological features and transcriptomes and by transplanting patient-derived NCCs in vivo. Here, we provide some examples that elucidate the pathophysiology of neurocristopathies using disease modeling via iPSCs.

Identification of Single Nucleotide Polymorphism on Bone Morphogenetic Protein 2 Gene in Non-Syndromic Cleft Lip/ Palate Patient

Cleft lip/palate (CL/P) is one of the most common birth defects in humans. Haploinsufficiency in genesBone Morphogenetic Protein (BMP) 2 is thought to play an important role in the incidence of CL/P. Thisstudy aimed to identify changes in the nucleotide (Single Nucleotide Polymorphism/SNP) BMP 2 rs235768A>T gene in CL/P patient in Indonesia. Seventy samples of DNA that were successfully amplified andrestricted consisted of patient and control samples with the three of which were used for sequencing. Basedon the analysis using restriction enzymes and Finch TV and Bioedit software programs, this study identifieda change from nucleotide A to nucleotide T which is a mutation missense (Serine-Arginine/TCA-TCT).Based on the results of the Fisher’s exact test, there was difference in genotype frequency between the CL/Pgroup and the control. Meanwhile, there was no difference in allele frequencies between the two groups. Theallele frequency T has a higher value than the frequency of the allele A.

Misregulation of cell adhesion molecules in the Ciona neural tube closure mutant bugeye

Neural tube closure (NTC) is a complex multi-step morphogenetic process that transforms the flat neural plate found on the surface of the post-gastrulation embryo into the hollow and subsurface central nervous system (CNS). Errors in this process underlie some of the most prevalent human birth defects, and occur in about 1 out of every 1000 births. Previously, we discovered a mutant in the basal chordate Ciona savignyi (named bugeye) that revealed a novel role for a T-Type Calcium Channel (Cav3) in this process. Moreover, the requirement for CAV3s in Xenopus NTC suggests a conserved function among the chordates. Loss of CAV3 leads to defects restricted to anterior NTC, with the brain apparently fully developed, but protruding from the head. Here we report first on a new Cav3 mutant in the related species C. robusta. RNAseq analysis of both C. robusta and C. savignyi bugeye mutants reveals misregulation of a number of transcripts including ones that are involved in cell-cell recognition and adhesion. Two in particular, Selectin and Fibronectin leucine-rich repeat transmembrane, which are aberrantly upregulated in the mutant, are expressed in the closing neural tube, and when disrupted by CRISPR gene editing lead to the open brain phenotype displayed in bugeye mutants. We speculate that these molecules play a transient role in tissue separation and adhesion during NTC and failure to downregulate them leads to an open neural tube.

Engineered mucoperiosteal scaffold for cleft palate regeneration towards the non-immunogenic transplantation

Cleft lip and palate (CL/P) is the most prevalent craniofacial birth defect in humans. None of the surgical procedures currently used for CL/P repair lead to definitive correction of hard palate bone interruption. Advances in tissue engineering and regenerative medicine aim to develop new strategies to restore palatal bone interruption by using tissue or organ-decellularized bioscaffolds seeded with host cells. Aim of this study was to set up a new natural scaffold deriving from a decellularized porcine mucoperiosteum, engineered by an innovative micro-perforation procedure based on Quantum Molecular Resonance (QMR) and then subjected to in vitro recellularization with human bone marrow-derived mesenchymal stem cells (hBM-MSCs). Our results demonstrated the efficiency of decellularization treatment gaining a natural, non-immunogenic scaffold with preserved collagen microenvironment that displays a favorable support to hMSC engraftment, spreading and differentiation. Ultrastructural analysis showed that the micro-perforation procedure preserved the collagen mesh, increasing the osteoinductive potential for mesenchymal precursor cells. In conclusion, we developed a novel tissue engineering protocol to obtain a non-immunogenic mucoperiosteal scaffold suitable for allogenic transplantation and CL/P repair. The innovative micro-perforation procedure improving hMSC osteogenic differentiation potentially impacts for enhanced palatal bone regeneration leading to future clinical applications in humans.

The Mandibular and Hyoid Arches-From Molecular Patterning to Shaping Bone and Cartilage.

The mandibular and hyoid arches collectively make up the facial skeleton, also known as the viscerocranium. Although all three germ layers come together to assemble the pharyngeal arches, the majority of tissue within viscerocranial skeletal components differentiates from the neural crest. Since nearly one third of all birth defects in humans affect the craniofacial region, it is important to understand how signalling pathways and transcription factors govern the embryogenesis and skeletogenesis of the viscerocranium. This review focuses on mouse and zebrafish models of craniofacial development. We highlight gene regulatory networks directing the patterning and osteochondrogenesis of the mandibular and hyoid arches that are actually conserved among all gnathostomes. The first part of this review describes the anatomy and development of mandibular and hyoid arches in both species. The second part analyses cell signalling and transcription factors that ensure the specificity of individual structures along the anatomical axes. The third part discusses the genes and molecules that control the formation of bone and cartilage within mandibular and hyoid arches and how dysregulation of molecular signalling influences the development of skeletal components of the viscerocranium. In conclusion, we notice that mandibular malformations in humans and mice often co-occur with hyoid malformations and pinpoint the similar molecular machinery controlling the development of mandibular and hyoid arches.

Cellular Viability of Partial Heart Transplant Grafts in Cold Storage.

Congenital heart defects are the most common types of birth defects in humans. Children with congenital heart defects frequently require heart valve replacement with an implant. Unfortunately, conventional heart valve implants do not grow. Therefore, these children are committed to serial re-operations for successively larger implant exchanges. Partial heart transplantation is a new and innovative approach to deliver growing heart valve implants. However, the transplant biology of partial heart transplant grafts remains unexplored. This is a critical barrier for clinical translation. Therefore, we investigated the cellular viability of partial heart transplants in cold storage. Histology and immunohistochemistry revealed no morphological differences in heart valves after 6, 24, or 48 h of cold storage. Moreover, immunohistochemistry showed that the marker for apoptosis activated caspase 3 and the marker for cell division Ki67 remained unchanged after 48 h of cold storage. Finally, quantification of fluorescing resorufin showed no statistically significant decrease in cellular metabolic activity in heart valves after 48 h of cold storage. We conclude that partial heart transplants remain viable after 48 h of cold storage. These findings represent the first step toward translating partial heart transplantation from the bench to the bedside because they have direct clinical implications for the procurement logistics of this new type of transplant.

CT morphology of anomalous systemic arterial supply to normal lung in dogs.

Anomalous systemic arterial supply to the normal lung (ASANL) is a rare congenital anomaly in humans, in which the systemic arteries supply the basal segments of the lower lobe. It has a normal bronchial connection, but lacks a normal pulmonary artery. This anomaly has not been previously reported in the veterinary literature. The objectives of this retrospective descriptive study were to characterize the CT findings and clinical features of ASANL , and to determine the breed predisposition in a population of referral canine cases. Thoracic CT images, in which the caudal lung lobes were fully inflated and the pulmonary artery could be traced to the periphery, were reviewed. A total of 1,950 dogs were enrolled, and the aberrant vasculature equivalent to ASANL in humans was detected in 48 dogs. Shetland Sheepdogs (7/48, odds ratio [OR]=8.0, P<0.00001), Miniature Dachshunds (19/48, OR=3.9, P<0.00001), and Labrador Retrievers (6/48, OR=4.5, P=0.0009) were over-represented. The affected lung lobes were the right caudal lobe (24/48, 50%), the left caudal lobe (21/48, 43.8%), and bilateral caudal lobes (3/48, 6.3%). The aberrant vessels originated from the left gastric artery (14/48), descending thoracic aorta (8/48), celiac artery (6/48), and splenic artery (1/48). In the remaining 19 cases, the origin of the aberrant vessels could not be determined. Although the clinical significance of ASANL in dogs remains unclear, surgeons should be aware of this finding prior to lobectomy of the caudal lung lobes to avoid intraoperative systemic arterial bleeding.

Pleiotropy method reveals genetic overlap between orofacial clefts at multiple novel loci from GWAS of multi-ethnic trios.

Based on epidemiologic and embryologic patterns, nonsyndromic orofacial clefts– the most common craniofacial birth defects in humans– are commonly categorized into cleft lip with or without cleft palate (CL/P) and cleft palate alone (CP), which are traditionally considered to be etiologically distinct. However, some evidence of shared genetic risk in IRF6, GRHL3 and ARHGAP29 regions exists; only FOXE1 has been recognized as significantly associated with both CL/P and CP in genome-wide association studies (GWAS). We used a new statistical approach, PLACO (pleiotropic analysis under composite null), on a combined multi-ethnic GWAS of 2,771 CL/P and 611 CP case-parent trios. At the genome-wide significance threshold of 5 × 10−8, PLACO identified 1 locus in 1q32.2 (IRF6) that appears to increase risk for one OFC subgroup but decrease risk for the other. At a suggestive significance threshold of 10−6, we found 5 more loci with compelling candidate genes having opposite effects on CL/P and CP: 1p36.13 (PAX7), 3q29 (DLG1), 4p13 (LIMCH1), 4q21.1 (SHROOM3) and 17q22 (NOG). Additionally, we replicated the recognized shared locus 9q22.33 (FOXE1), and identified 2 loci in 19p13.12 (RAB8A) and 20q12 (MAFB) that appear to influence risk of both CL/P and CP in the same direction. We found locus-specific effects may vary by racial/ethnic group at these regions of genetic overlap, and failed to find evidence of sex-specific differences. We confirmed shared etiology of the two OFC subtypes comprising CL/P, and additionally found suggestive evidence of differences in their pathogenesis at 2 loci of genetic overlap. Our novel findings include 6 new loci of genetic overlap between CL/P and CP; 3 new loci between pairwise OFC subtypes; and 4 loci not previously implicated in OFCs. Our in-silico validation showed PLACO is robust to subtype-specific effects, and can achieve massive power gains over existing approaches for identifying genetic overlap between disease subtypes. In summary, we found suggestive evidence for new genetic regions and confirmed some recognized OFC genes either exerting shared risk or with opposite effects on risk to OFC subtypes.

Exposure to ethanol leads to midfacial hypoplasia in a zebrafish model of FASD via indirect interactions with the Shh pathway

BackgroundGene-environment interactions are likely to underlie most human birth defects. The most common known environmental contributor to birth defects is prenatal alcohol exposure. Fetal alcohol spectrum disorders (FASD) describe the full range of defects that result from prenatal alcohol exposure. Gene-ethanol interactions underlie susceptibility to FASD, but we lack a mechanistic understanding of these interactions. Here, we leverage the genetic tractability of zebrafish to address this problem.ResultsWe first show that vangl2, a member of the Wnt/planar cell polarity (Wnt/PCP) pathway that mediates convergent extension movements, strongly interacts with ethanol during late blastula and early gastrula stages. Embryos mutant or heterozygous for vangl2 are sensitized to ethanol-induced midfacial hypoplasia. We performed single-embryo RNA-seq during early embryonic stages to assess individual variation in the transcriptional response to ethanol and determine the mechanism of the vangl2-ethanol interaction. To identify the pathway(s) that are disrupted by ethanol, we used these global changes in gene expression to identify small molecules that mimic the effects of ethanol via the Library of Integrated Network-based Cellular Signatures (LINCS L1000) dataset. Surprisingly, this dataset predicted that the Sonic Hedgehog (Shh) pathway inhibitor, cyclopamine, would mimic the effects of ethanol, despite ethanol not altering the expression levels of direct targets of Shh signaling. Indeed, we found that ethanol and cyclopamine strongly, but indirectly, interact to disrupt midfacial development. Ethanol also interacts with another Wnt/PCP pathway member, gpc4, and a chemical inhibitor of the Wnt/PCP pathway, blebbistatin, phenocopies the effect of ethanol. By characterizing membrane protrusions, we demonstrate that ethanol synergistically interacts with the loss of vangl2 to disrupt cell polarity required for convergent extension movements.ConclusionsOur results show that the midfacial defects in ethanol-exposed vangl2 mutants are likely due to an indirect interaction between ethanol and the Shh pathway. Vangl2 functions as part of a signaling pathway that regulates coordinated cell movements during midfacial development. Ethanol exposure alters the position of a critical source of Shh signaling that separates the developing eye field into bilateral eyes, allowing the expansion of the midface. Collectively, our results shed light on the mechanism by which the most common teratogen can disrupt development.

Maternal iron deficiency perturbs embryonic cardiovascular development in mice

Congenital heart disease (CHD) is the most common class of human birth defects, with a prevalence of 0.9% of births. However, two-thirds of cases have an unknown cause, and many of these are thought to be caused by in utero exposure to environmental teratogens. Here we identify a potential teratogen causing CHD in mice: maternal iron deficiency (ID). We show that maternal ID in mice causes severe cardiovascular defects in the offspring. These defects likely arise from increased retinoic acid signalling in ID embryos. The defects can be prevented by iron administration in early pregnancy. It has also been proposed that teratogen exposure may potentiate the effects of genetic predisposition to CHD through gene–environment interaction. Here we show that maternal ID increases the severity of heart and craniofacial defects in a mouse model of Down syndrome. It will be important to understand if the effects of maternal ID seen here in mice may have clinical implications for women.

Correlation between TGF-β2/3 promoter DNA methylation and Smad signaling during palatal fusion induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a persistent organic pollutant that is strongly associated with a number of human diseases and birth defects, including cleft palate. Transforming growth factor (TGF) plays a significant role during mammalian palatogenesis. However, the epigenetic mechanism of transforming growth factors in the process of TCDD-induced cleft palate is unclear. The purpose of this research was to investigate the relationship and potential mechanism between TGF-β2/3 promoter DNA methylation and Smad signaling during TCDD-induced cleft palate. Pregnant C57BL/6N mice were exposed to 64 µg/kg TCDD on gestational day 10 (GD10) to establish the cleft palate model and palatal tissues of embryos were collected on GD13, GD14, and GD15 for subsequent experiments. TGF-β2/3 mRNA expression, TGF-β2/3 promoter methylation, and Smad signaling molecules expression were assessed in the palate of the two groups. The results showed that the incidence of cleft palate was 94.7% in the TCDD-treated group whereas no cleft palate was found in the control group. TCDD-treated group altered specific CpG sites of TGF-β2/3 promoter methylation. Compared to the control group, the proliferation of mouse embryonic palate mesenchymal stromal cells (MEPM), the expressions of TGF-β2/3, p-Smad2, and Smad4 were all reduced, while the expression of Smad7 was significantly increased in the atAR group. Smad signaling was downregulated by TCDD. Therefore, we suggest that TGF-β2/3 promoter methylation and Smad signaling may be involved in TCDD-induced cleft palate formation in fetal mice.

Mechanical heterogeneity along single cell-cell junctions is driven by lateral clustering of cadherins during vertebrate axis elongation.

Morphogenesis is governed by the interplay of molecular signals and mechanical forces across multiple length scales. The last decade has seen tremendous advances in our understanding of the dynamics of protein localization and turnover at subcellular length scales, and at the other end of the spectrum, of mechanics at tissue-level length scales. Integrating the two remains a challenge, however, because we lack a detailed understanding of the subcellular patterns of mechanical properties of cells within tissues. Here, in the context of the elongating body axis of Xenopus embryos, we combine tools from cell biology and physics to demonstrate that individual cell-cell junctions display finely-patterned local mechanical heterogeneity along their length. We show that such local mechanical patterning is essential for the cell movements of convergent extension and is imparted by locally patterned clustering of a classical cadherin. Finally, the patterning of cadherins and thus local mechanics along cell-cell junctions are controlled by Planar Cell Polarity signaling, a key genetic module for CE that is mutated in diverse human birth defects.

Diabetes, Oxidative Stress, and DNA Damage Modulate Cranial Neural Crest Cell Development and the Phenotype Variability of Craniofacial Disorders.

Craniofacial malformations are among the most common birth defects in humans and they often have significant detrimental functional, aesthetic, and social consequences. To date, more than 700 distinct craniofacial disorders have been described. However, the genetic, environmental, and developmental origins of most of these conditions remain to be determined. This gap in our knowledge is hampered in part by the tremendous phenotypic diversity evident in craniofacial syndromes but is also due to our limited understanding of the signals and mechanisms governing normal craniofacial development and variation. The principles of Mendelian inheritance have uncovered the etiology of relatively few complex craniofacial traits and consequently, the variability of craniofacial syndromes and phenotypes both within families and between families is often attributed to variable gene expression and incomplete penetrance. However, it is becoming increasingly apparent that phenotypic variation is often the result of combinatorial genetic and non-genetic factors. Major non-genetic factors include environmental effectors such as pregestational maternal diabetes, which is well-known to increase the risk of craniofacial birth defects. The hyperglycemia characteristic of diabetes causes oxidative stress which in turn can result in genotoxic stress, DNA damage, metabolic alterations, and subsequently perturbed embryogenesis. In this review we explore the importance of gene-environment associations involving diabetes, oxidative stress, and DNA damage during cranial neural crest cell development, which may underpin the phenotypic variability observed in specific craniofacial syndromes.

Gene Environment Interactions in the Etiology of Neural Tube Defects.

Human structural congenital malformations are the leading cause of infant mortality in the United States. Estimates from the United States Center for Disease Control and Prevention (CDC) determine that close to 3% of all United States newborns present with birth defects; the worldwide estimate approaches 6% of infants presenting with congenital anomalies. The scientific community has recognized for decades that the majority of birth defects have undetermined etiologies, although we propose that environmental agents interacting with inherited susceptibility genes are the major contributing factors. Neural tube defects (NTDs) are among the most prevalent human birth defects and as such, these malformations will be the primary focus of this review. NTDs result from failures in embryonic central nervous system development and are classified by their anatomical locations. Defects in the posterior portion of the neural tube are referred to as meningomyeloceles (spina bifida), while the more anterior defects are differentiated as anencephaly, encephalocele, or iniencephaly. Craniorachischisis involves a failure of the neural folds to elevate and thus disrupt the entire length of the neural tube. Worldwide NTDs have a prevalence of approximately 18.6 per 10,000 live births. It is widely believed that genetic factors are responsible for some 70% of NTDs, while the intrauterine environment tips the balance toward neurulation failure in at risk individuals. Despite aggressive educational campaigns to inform the public about folic acid supplementation and the benefits of providing mandatory folic acid food fortification in the United States, NTDs still affect up to 2,300 United States births annually and some 166,000 spina bifida patients currently live in the United States, more than half of whom are now adults. Within the context of this review, we will consider the role of maternal nutritional status (deficiency states involving B vitamins and one carbon analytes) and the potential modifiers of NTD risk beyond folic acid. There are several well-established human teratogens that contribute to the population burden of NTDs, including: industrial waste and pollutants [e.g., arsenic, pesticides, and polycyclic aromatic hydrocarbons (PAHs)], pharmaceuticals (e.g., anti-epileptic medications), and maternal hyperthermia during the first trimester. Animal models for these teratogens are described with attention focused on valproic acid (VPA; Depakote). Genetic interrogation of model systems involving VPA will be used as a model approach to discerning susceptibility factors that define the gene-environment interactions contributing to the etiology of NTDs.

Single‐Cell Level Identification Of Cephalic Epithelial Transcriptomic Signatures To Elucidate The Pathogenesis Of Cleft Lip/Palate

Craniofacial development results from a tightly regulated choreography involving patterning, morphogenesis, and growth of multiple embryonic cell types. Clefts of the lip and/or palate are maiming disruptions of facial shape and represent the most common human craniofacial birth defect. During mammalian facial development, the maxillary prominence extends medially to contact the two nasal prominences (medial and lateral) at a three-way anatomical seam named lambdoidal junction (λ), wherein the three prominences coalesce. However, if there is an impaired fusion of the facial prominences orofacial clefting ensues. We have previously reported an essential role for the cephalic epithelium at the λ to enable normal fusion of the murine facial prominences. Using the mouse as a model, we have now generated single-cell RNAseq (scRNAseq) transcriptional profiles that identified the various murine epithelial subpopulations at the λ. I am currently studying the spatial and temporal characteristics of the epithelial subpopulations using microdissection the facial prominences prior/after the fusion process combined with bioimaging approaches. In parallel, we have also conducted all analyses also directly comparing wild-type and available genetic mutant mouse lines with orofacial clefting (PBX1/2 compound mutants). These analyses have identified key transcriptomic changes in mutant mice embryos providing a deeper understanding of the molecular mechanisms underlying the pathogenesis of orofacial clefting. Altogether, these approaches are helping to elucidate the spatiotemporal dynamics of the cell subpopulations comprising the λ epithelium in vivo, and the perturbed landscapes that cause disfiguring anomalies to the anatomical features of the mammalian face.

Gldc is essential for embryonic development and kidney organogenesis

Glycine is a simple amino acid that is vital to the proper functioning of the body, but its roles during ontogeny are not well understood. Glycine levels are precisely regulated through the glycine cleavage system (GCS), a complex molecular machine that produces one-carbon units for later metabolism and folate production. Elevated glycine levels due to congenital mutations in GCS components, such as glycine dehydrogenase (gldc), cause devastating human birth defects and the rare disease nonketotic hyperglycinemia (NKH). The exact pathological implications of glycine accumulation are unknown, but NKH patients suffer from pleiotropic symptoms including seizures, apnea, lethargy, severe mental retardation, and early death. Animal models utilizing mice and zebrafish have shown increased ventriculomegaly and motor dysfunction in gldc deficient animals, but have not analyzed organogenesis. Here, we have utilized zebrafish as a genetic platform to explore the roles of gldc during early development. Whole mount in situ hybridization revealed that gldc transcripts were highly expressed in multiple tissues, including the central nervous system and embryonic kidney, recapitulating both mouse and human expression studies. gldc deficiency caused hydrocephalus and pericardial edema, suggesting defects in brain and renal ontogeny. Elevated apoptosis was detected in locations within the brain, kidney, and cloaca. Furthermore, formation of nephron cell populations in the kidney was disrupted in gldc deficient embryos, where the proximal straight tubule was increased at the expense of the distal tubule. Morphological and expression studies revealed severe alterations in brain patterning and morphogenesis as well. Taken together, these studies indicate that gldc has essential roles during early development and have revealed novel functions for pattern formation and cell survival in tissues such as the brain and kidney. Our evidence further emphasizes the zebrafish as a faithful model to examine NKH. Ongoing work with the zebrafish model is poised to expand our medical understanding about the developmental defects consequent to NKH and can be used to perform chemical screens to identify potential therapeutic interventions.

Defining the Role of MEMO1 in Craniodental Mineralization

Defects in the mineralization of craniofacial tissues are an underlying pathology in a variety of human congenital disorders. Our understanding of these developmental processes is confounded by the complex intertwinement of different tissue types (e.g. ectoderm, neural crest and mesoderm) and mineralization methods (e.g. endochondral and intramembranous ossification). Understanding the unique cellular and molecular mechanisms driving the development and mineralization of craniodental tissues (e.g. bone and teeth) is critical to explain, prevent, and treat associated genetic disorders. Our lab has identified the protein Mediator of Cell Motility 1 (MEMO1)—initially found to associate with breast cancer metastasis—is critical for bone and enamel mineralization. However, MEMO1’s biological and molecular function in these processes is unknown.We are using in vivo mouse-based and in vitro cell culture-based models to address this knowledge gap. With our in vivo models, we have utilized immunofluorescence, scanning electron microscopy, µCT, single cell RNA-sequencing, and basic histological examination to define the craniodental defects upon loss of Memo1 in the ectoderm or oral epithelium. With our in vitro models, we have utilized an oral epithelial cell line, along with CRISPR/Cas9 genome editing, Western Blot analysis, bulk RNA-sequencing and immunofluorescence to gain insight into the cellular and molecular function of MEMO1 within oral epithelial cells. Analyses of the in vivo Memo1 ectoderm or oral epithelial mutants, relative to controls, identifies an important role for MEMO1 in enamel development. Conditional mutants display chalky enamel associated with a significant decrease in enamel density (p-value < 0.03). Compromised enamel in conditional mutants is further supported by alterations in gene and protein expression within ameloblasts and may include defects in the cytoskeleton. Supporting this observation, our oral epithelial in vitro Memo1 loss-of-function model displays changes in cytoskeletal dynamics, including increased cellular contractility.These findings provide novel insight into how MEMO1 contributes to mineralization, potentially deploying its breast cancer metastatic function (i.e., cytoskeletal regulation), within a novel biological setting—amelogenesis. The oral epithelial specific role of MEMO1 expands our understanding of craniodental mineralization and MEMO1’s molecular control of these processes. Furthermore, this provides novel insight into genetic diseases of mineralization, including those impacting the craniodental complex. Additional investigation into our Memo1 loss-of-function models will progress both the specific and general knowledge of tissue mineralization—providing novel opportunities to exploit this protein, or its interacting partners, in a clinical or therapeutic setting.

A Novel Bone Substitute Based on Recombinant Type I Collagen for Reconstruction of Alveolar Cleft.

This study aimed to examine the optimal cross-link density of recombinant peptide (RCP) particles, based on human collagen type I, for bone reconstruction in human alveolar cleft. Low- (group 1), medium- (group 2), and high- (group 3) cross-linked RCP particles were prepared by altering the duration of the heat-dependent dehydration reaction. Rat palatine fissures (n = 45), analogous to human congenital bone defects, were examined to evaluate the potential of bone formation by the three different RCP particles. Microcomputed tomography images were obtained to measure bone volume and bone mineral density at 4, 8, 12, and 16 weeks post grafting. Specimens were obtained for histological analysis at 16 weeks after grafting. Additionally, alkaline phosphatase and tartrate acid phosphatase staining were performed to visualize the presence of osteoblasts and osteoclasts. At 16 weeks, bone volume, bone mineral density, and new bone area measurements in group 2 were significantly higher than in any other group. In addition, the number of osteoblasts and osteoclasts on the new bone surface in group 2 was significantly higher than in any other group. Our results demonstrated that medium cross-linking was more suitable for bone formation—and could be useful in human alveolar cleft repairs as well.

Онтологія варіантів будови та вад розвитку черепа. Частина I

Annually severe congenital abnormalities occur in 1 of 33 newborns or about 125.000 live births in the United States and are the leading cause of infant mortality. Craniofacial anomalies account for about one-third of all abnormalities. The prevalence of human congenital malformations is an important characteristic of public health. According to the World Health Organization, the birth rate of children with cleft lip and palate is on average 1:750 newborns, which is 20-30% of all human malformations and 86% of maxillofacial malformations. They contribute significantly to infant morbidity and disability as well as millions of dollars in health care costs each year. Development of the human skull begins about 23-26 days after fertilization, when a multipotent population of cranial neural crest cells migrates from the dorsal part of the neuraxis to the embryonic head region, giving rise to the frontal bone and interparietal part of the occipital bone, while the rest of the cranial arch comes mainly from the mesoderm. A malformation is a deviation from the normal development of an organ or tissue. Among the causes are chromosomal abnormalities, single gene defects, teratogenic or multi-factor agents, that is, a combination of genetic and external factors; the smallest number of cases are idiopathic. Exogenous causes of human congenital abnormalities include: physical (mechanical, thermal, radiation); chemical (hypoxia, malnutrition, hormonal discordance, teratogenic poisons); biological (viruses, bacteria and their toxins, protozoa); mental factors. Endogenous causes are heredity (changes in hereditary structures, mutations); biological inadequacy of germ cells (endocrine disorders, “over-ripening” of germ cells); parents’ age. Congenital craniofacial abnormalities are caused by improper growth and/or development of the brain and facial parts of the skull and soft tissue. Craniofacial abnormalities affecting the skull include macrocephaly, microcephaly and unilateral deformities. Sometimes cranial sutures fuse too early and craniosynostosis occurs, which is accompanied by various cranial deformities. This leads to abnormal cranial development, which can cause dysmorphia, and in the most severe cases catastrophically affect brain development. Conclusion. Based on the analysis of the literature sources, an attempt has been made to summarize the existing information and classifications of congenital cranial abnormalities, as well as the interpretation of various anomalies of the brain and facial skull in alphabetic order. The most common stigmas and malformations of the skull bones are briefly characterized