Suckling, Feeding, and Swallowing: Behaviors, Circuits, and Targets for Neurodevelopmental Pathology.

All mammals must suckle and swallow at birth, and subsequently chew and swallow solid foods, for optimal growth and health. These initially innate behaviors depend critically upon coordinated development of the mouth, tongue, pharynx, and larynx as well as the cranial nerves that control these structures. Disrupted suckling, feeding, and swallowing from birth onward-perinatal dysphagia-is often associated with several neurodevelopmental disorders that subsequently alter complex behaviors. Apparently, a broad range of neurodevelopmental pathologic mechanisms also target oropharyngeal and cranial nerve differentiation. These aberrant mechanisms, including altered patterning, progenitor specification, and neurite growth, prefigure dysphagia and may then compromise circuits for additional behavioral capacities. Thus, perinatal dysphagia may be an early indicator of disrupted genetic and developmental programs that compromise neural circuits and yield a broad range of behavioral deficits in neurodevelopmental disorders.

Identification and functional analysis of rare HECTD1 missense variants in human neural tube defects.

Neural tube defects (NTDs) are severe malformations of the central nervous system that arise from failure of neural tube closure. HECTD1 is an E3 ubiquitin ligase required for cranial neural tube closure in mouse models. NTDs in the Hectd1 mutant mouse model are due to the failure of cranial mesenchyme morphogenesis during neural fold elevation. Our earlier research has linked increased extracellular heat shock protein 90 (eHSP90) secretion to aberrant cranial mesenchyme morphogenesis in the Hectd1 model. Furthermore, overexpression of HECTD1 suppresses stress-induced eHSP90 secretion in cell lines. In this study, we report the identification of five rare HECTD1 missense sequence variants in NTD cases. The variants were found through targeted next-generation sequencing in a Chinese cohort of 352 NTD cases and 224 ethnically matched controls. We present data showing that HECTD1 is a highly conserved gene, extremely intolerant to loss-of-function mutations and missense changes. To evaluate the functional consequences of NTD-associated missense variants, functional assays in HEK293T cells were performed to examine protein expression and the ability of HECTD1 sequence variants to suppress eHSP90 secretion. One NTD-associated variant (A1084T) had significantly reduced expression in HEK293T cells. All five NTD-associated variants (p.M392V, p.T801I, p.I906V, p.A1084T, and p.P1835L) reduced regulation of eHSP90 secretion by HECTD1, while a putative benign variant (p.P2474L) did not. These findings are the first association of HECTD1 sequence variation with NTDs in humans.

ALDH1A2-related disorder: A new genetic syndrome due to alteration of the retinoic acid pathway.

Aldehyde Dehydrogenase 1, Family Member A2 (ALDH1A2) is essential for the synthesis of retinoic acid from vitamin A. Studies in model organisms demonstrate a critical role for ALDH1A2 in embryonic development, yet few pathogenic variants are linked to congenital anomalies in humans. We present three siblings with multiple congenital anomaly syndrome linked to biallelic sequence variants in ALDH1A2. The major congenital malformations affecting these children include tetralogy of Fallot, absent thymus, diaphragmatic eventration, and talipes equinovarus. Upper airway anomalies, hypocalcemia, and dysmorphic features are newly reported in this manuscript. In vitro functional validation of variants indicated that substitutions reduced the expression of the enzyme. Our clinical and functional data adds to a recent report of biallelic ALDH1A2 pathogenic variants in two families with a similar constellation of congenital malformations. These findings provide further evidence for an autosomal recessive ALDH1A2-deficient recognizable malformation syndrome involving the diaphragm, cardiac and musculoskeletal systems.

Mouse Models of Neural Tube Defects.

During embryonic development, the central nervous system forms as the neural plate and then rolls into a tube in a complex morphogenetic process known as neurulation. Neural tube defects (NTDs) occur when neurulation fails and are among the most common structural birth defects in humans. The frequency of NTDs varies greatly anywhere from 0.5 to 10 in 1000 live births, depending on the genetic background of the population, as well as a variety of environmental factors. The prognosis varies depending on the size and placement of the lesion and ranges from death to severe or moderate disability, and some NTDs are asymptomatic. This chapter reviews how mouse models have contributed to the elucidation of the genetic, molecular, and cellular basis of neural tube closure, as well as to our understanding of the causes and prevention of this devastating birth defect.

Reduced maternal vitamin A status increases the incidence of normal aortic arch variants.

While common in the general population, the developmental origins of "normal" anatomic variants of the aortic arch remain unknown. Aortic arch development begins with the establishment of the second heart field (SHF) that contributes to the pharyngeal arch arteries (PAAs). The PAAs remodel during subsequent development to form the mature aortic arch and arch vessels. Retinoic acid signaling involving the biologically active metabolite of vitamin A, plays a key role in multiple steps of this process. Recent work from our laboratory indicates that the E3 ubiquitin ligase Hectd1 is required for full activation of retinoic acid signaling during cardiac development. Furthermore, our study suggested that mild alterations in retinoic acid signaling combined with reduced gene dosage of Hectd1, results in a benign aortic arch variant where the transverse aortic arch is shortened between the brachiocephalic and left common carotid arteries. These abnormalities are preceded by hypoplasia of the fourth PAA. To further explore this interaction, we investigate whether reduced maternal dietary vitamin A intake can similarly influence aortic arch development. Our findings indicate that the incidence of hypoplastic fourth PAAs, as well as the incidence of shortened transverse arch are increased with reduced maternal vitamin A intake during pregnancy. These studies provide new insights as to the developmental origins of these benign aortic arch variants.

Supt20 is required for development of the axial skeleton.

Somitogenesis and subsequent axial skeletal development is regulated by the interaction of pathways that determine the periodicity of somite formation, rostrocaudal somite polarity and segment identity. Here we use a hypomorphic mutant mouse line to demonstrate that Supt20 (Suppressor of Ty20) is required for development of the axial skeleton. Supt20 hypomorphs display fusions of the ribs and vertebrae at lower thoracic levels along with anterior homeotic transformation of L1 to T14. These defects are preceded by reduction of the rostral somite and posterior shifts in Hox gene expression. While cycling of Notch target genes in the posterior presomitic mesoderm (PSM) appeared normal, expression of Lfng was reduced. In the anterior PSM, Mesp2 expression levels and cycling were unaffected; yet, expression of downstream targets such as Lfng, Ripply2, Mesp1 and Dll3 in the prospective rostral somite was reduced accompanied by expansion of caudal somite markers such as EphrinB2 and Hes7. Supt20 interacts with the Gcn5-containing SAGA histone acetylation complex. Gcn5 hypomorphic mutant embryos show similar defects in axial skeletal development preceded by posterior shift of Hoxc8 and Hoxc9 gene expression. We demonstrate that Gcn5 and Supt20 hypomorphs show similar defects in rostral-caudal somite patterning potentially suggesting shared mechanisms.

Hard to swallow: Developmental biological insights into pediatric dysphagia.

Pediatric dysphagia-feeding and swallowing difficulties that begin at birth, last throughout childhood, and continue into maturity--is one of the most common, least understood complications in children with developmental disorders. We argue that a major cause of pediatric dysphagia is altered hindbrain patterning during pre-natal development. Such changes can compromise craniofacial structures including oropharyngeal muscles and skeletal elements as well as motor and sensory circuits necessary for normal feeding and swallowing. Animal models of developmental disorders that include pediatric dysphagia in their phenotypic spectrum can provide mechanistic insight into pathogenesis of feeding and swallowing difficulties. A fairly common human genetic developmental disorder, DiGeorge/22q11.2 Deletion Syndrome (22q11DS) includes a substantial incidence of pediatric dysphagia in its phenotypic spectrum. Infant mice carrying a parallel deletion to 22q11DS patients have feeding and swallowing difficulties that approximate those seen in pediatric dysphagia. Altered hindbrain patterning, craniofacial malformations, and changes in cranial nerve growth prefigure these difficulties. Thus, in addition to craniofacial and pharyngeal anomalies that arise independently of altered neural development, pediatric dysphagia may result from disrupted hindbrain patterning and its impact on peripheral and central neural circuit development critical for feeding and swallowing. The mechanisms that disrupt hindbrain patterning and circuitry may provide a foundation to develop novel therapeutic approaches for improved clinical management of pediatric dysphagia.

Hectd1 is required for development of the junctional zone of the placenta.

The placenta plays a critical role in the growth and survival of the fetus. Here we demonstrate that the Homologous to the E6-AP Carboxyl Terminus (HECT) domain E3 ubiquitin ligase, Hectd1, is essential for development of the mouse placenta. Hectd1 is widely expressed during placentation with enrichment in trophoblast giant cells (TGCs) and other trophoblast-derived cell subtypes in the junctional and labyrinth zones of the placenta. Disruption of Hectd1 results in mid-gestation lethality and intrauterine growth restriction (IUGR). Variable defects in the gross structure of the mutant placenta are found including alterations in diameter, thickness and lamination. The number and nuclear size of TGCs is reduced. Examination of subtype specific markers reveals altered TGC development with decreased expression of Placental lactogen-1 and -2 (Pl1 and Pl2) and increased expression of Proliferin (Plf). Reduced numbers of spongiotrophoblasts and glycogen trophoblasts were also found at the junctional zone of the Hectd1 mutant placenta. Finally, there was an increase in immature uterine natural killer (uNK) cells in the maternal decidua of the Hectd1 mutant placenta. Proliferation and apoptosis are differentially altered in the layers of the placenta with an increase in both apoptosis and proliferation in the maternal decidua, a decrease in proliferation and increase in apoptosis in the labyrinth layer and both unchanged in the junctional zone. Together these data demonstrate that Hectd1 is required for development of multiple cell types within the junctional zone of the placenta.

HectD1 E3 ligase modifies adenomatous polyposis coli (APC) with polyubiquitin to promote the APC-axin interaction.

The adenomatous polyposis coli (APC) protein functions as a negative regulator of the Wnt signaling pathway. In this capacity, APC forms a "destruction complex" with Axin, CK1α, and GSK3ß to foster phosphorylation of the Wnt effector ß-catenin earmarking it for Lys-48-linked polyubiquitylation and proteasomal degradation. APC is conjugated with Lys-63-linked ubiquitin chains when it is bound to Axin, but it is unclear whether this modification promotes the APC-Axin interaction or confers upon APC an alternative function in the destruction complex. Here we identify HectD1 as a candidate E3 ubiquitin ligase that modifies APC with Lys-63 polyubiquitin. Knockdown of HectD1 diminished APC ubiquitylation, disrupted the APC-Axin interaction, and augmented Wnt3a-induced ß-catenin stabilization and signaling. These results indicate that HectD1 promotes the APC-Axin interaction to negatively regulate Wnt signaling.

The iron exporter ferroportin 1 is essential for development of the mouse embryo, forebrain patterning and neural tube closure.

Neural tube defects (NTDs) are some of the most common birth defects observed in humans. The incidence of NTDs can be reduced by peri-conceptional folic acid supplementation alone and reduced even further by supplementation with folic acid plus a multivitamin. Here, we present evidence that iron maybe an important nutrient necessary for normal development of the neural tube. Following implantation of the mouse embryo, ferroportin 1 (Fpn1) is essential for the transport of iron from the mother to the fetus and is expressed in the visceral endoderm, yolk sac and placenta. The flatiron (ffe) mutant mouse line harbors a hypomorphic mutation in Fpn1 and we have created an allelic series of Fpn1 mutations that result in graded developmental defects. A null mutation in the Fpn1 gene is embryonic lethal before gastrulation, hypomorphic Fpn1(ffe/ffe) mutants exhibit NTDs consisting of exencephaly, spina bifida and forebrain truncations, while Fpn1(ffe/KI) mutants exhibit even more severe NTDs. We show that Fpn1 is not required in the embryo proper but rather in the extra-embryonic visceral endoderm. Our data indicate that loss of Fpn1 results in abnormal morphogenesis of the anterior visceral endoderm (AVE). Defects in the development of the forebrain in Fpn1 mutants are compounded by defects in multiple signaling centers required for maintenance of the forebrain, including the anterior definitive endoderm (ADE), anterior mesendoderm (AME) and anterior neural ridge (ANR). Finally, we demonstrate that this loss of forebrain maintenance is due in part to the iron deficiency that results from the absence of fully functional Fpn1.

Mouse as a model for multifactorial inheritance of neural tube defects.

Neural tube defects (NTDs) such as spina bifida and anencephaly are some of the most common structural birth defects found in humans. These defects occur due to failures of neurulation, a process where the flat neural plate rolls into a tube. In spite of their prevalence, the causes of NTDs are poorly understood. The multifactorial threshold model best describes the pattern of inheritance of NTDs where multiple undefined gene variants interact with environmental factors to cause an NTD. To date, mouse models have implicated a multitude of genes as required for neurulation, providing a mechanistic understanding of the cellular and molecular pathways that control neurulation. However, the majority of these mouse models exhibit NTDs with a Mendelian pattern of inheritance. Still, many examples of multifactorial inheritance have been demonstrated in mouse models of NTDs. These include null and hypomorphic alleles of neurulation genes that interact in a complex fashion with other genetic mutations or environmental factors to cause NTDs. These models have implicated several genes and pathways for testing as candidates for the genetic basis of NTDs in humans, resulting in identification of putative pathogenic mutations in some patients. Mouse models also provide an experimental paradigm to gain a mechanistic understanding of the environmental factors that influence NTD occurrence, such as folic acid and maternal diabetes, and have led to the discovery of additional preventative nutritional supplements such as inositol. This review provides examples of how multifactorial inheritance of NTDs can be modeled in the mouse.

Does the cranial mesenchyme contribute to neural fold elevation during neurulation?

The central nervous system is derived from the neural plate, which undergoes a series of complex morphogenetic events resulting in formation of the neural tube in a process known as neurulation. The cellular behaviors driving neurulation in the cranial region involve forces generated by the neural tissue itself as well as the surrounding epithelium and mesenchyme. Of interest, the cranial mesenchyme underlying the neural plate undergoes stereotypical rearrangements hypothesized to drive elevation of the neural folds. As the neural folds rise, the hyaluronate-rich extracellular matrix greatly expands resulting in increased space between individual cranial mesenchyme cells. Based on inhibitor studies, expansion of the extracellular matrix has been implicated in driving neural fold elevation; however, because the surrounding neural and epidermal ectoderm were also affected by inhibitor exposure, these studies are inconclusive. Similarly, treatment of neurulating embryos with teratogenic doses of retinoic acid results in altered organization of the cranial mesenchyme, but alterations in surrounding tissues are also observed. The strongest evidence for a critical role for the cranial mesenchyme in neural fold elevation comes from studies of genes expressed exclusively in the cranial mesenchyme that when mutated result in exencephaly associated with abnormal organization of the cranial mesenchyme. Twist is the best studied of these and is expressed in both the paraxial mesoderm and neural crest derived cranial mesenchyme. In this article, we review the evidence implicating the cranial mesenchyme in providing a driving force for neural fold elevation to evaluate whether there are sufficient data to support this hypothesis.

Parental obesity-induced changes in developmental programming.

Many studies support the link between parental obesity and the predisposition to develop adult-onset metabolic syndromes that include obesity, high blood pressure, dyslipidemia, insulin resistance, and diabetes in the offspring. As the prevalence of obesity increases in persons of childbearing age, so does metabolic syndrome in their descendants. Understanding how parental obesity alters metabolic programs in the progeny, predisposing them to adult-onset metabolic syndrome, is key to breaking this cycle. This review explores the basis for altered metabolism of offspring exposed to overnutrition by focusing on critical developmental processes influenced by parental obesity. We draw from human and animal model studies, highlighting the adaptations in metabolism that occur during normal pregnancy that become maladaptive with obesity. We describe essential phases of development impacted by parental obesity that contribute to long-term alterations in metabolism in the offspring. These encompass gamete formation, placentation, adipogenesis, pancreas development, and development of brain appetite control circuits. Parental obesity alters the developmental programming of these organs in part by inducing epigenetic changes with long-term consequences on metabolism. While exposure to parental obesity during any of these phases is sufficient to alter long-term metabolism, offspring often experience multiple exposures throughout their development. These insults accumulate to increase further the susceptibility of the offspring to the obesogenic environments of modern society.

The visceral yolk sac endoderm provides for absorption of nutrients to the embryo during neurulation.

Neural tube defects (NTDs) represent some of the most common congenital malformations in humans. The causes of NTDs are complex with both genetic and environmental contributing factors. Periconception nutrition is an important environmental factor influencing the penetrance of NTDs. NTDs arise from failure to close the neural tube completely during development, an event that occurs before establishment of the chorioallantoic placenta. During neurulation, nutrients are absorbed by histotrophic mechanisms and absorbed by endocytosis in the endoderm-derived cell layer of the visceral yolk sac (VYS). Here we review the histotrophic mechanisms by which nutrients are delivered to the human embryo during this critical time period. Because more detailed studies on the molecular mechanisms regulating uptake of nutrients have been performed using rodent models, most importantly mouse and rat models, we will also review nutrient uptake in these model organisms to set the stage for presentation of experimental data that have provided valuable information about how nutrients are delivered to the neurulating embryo.

Hsp90 and complex birth defects: A plausible mechanism for the interaction of genes and environment.

How genes and environment interact to cause birth defects is not well understood, but key to developing new strategies to modify risk. The threshold model has been proposed to represent this complex interaction. This model stipulates that while environmental exposure or genetic mutation alone may not result in a defect, factors in combination increase phenotypic variability resulting in more individuals crossing the disease threshold where birth defects manifest. Many environmental factors that contribute to birth defects induce widespread cellular stress and misfolding of proteins. Yet, the impact of the stress response on the threshold model is not typically considered in discephering the etiology of birth defects. This mini-review will explore a potential mechanism for gene-environment interactions co-opted from studies of evolution. This model stipulates that heat shock proteins that mediate the stress response induced by environmental factors can influence the number of individuals that cross disease thresholds resulting in increased incidence of birth defects. Studies in the field of evolutionary biology have demonstrated that heat shock proteins and Hsp90 in particular provide a link between environmental stress, genotype and phenotype. Hsp90 is a highly expressed molecular chaperone that assists a wide variety of protein clients with folding and conformational changes needed for proper function. Hsp90 also chaperones client proteins with potentially deleterious amino acid changes to suppress variation caused by genetic mutations. However, upon exposure to stress, Hsp90 abandons its normal physiological clients and is diverted to assist with the misfolded protein response. This can impact the activity of signaling pathways that involve Hsp90 clients as well as unmask suppressed protein variation, essentially creating complex traits in a single step. In this capacity Hsp90 acts as an evolutionary capacitor allowing stored variation to accumulate and then become expressed in times of stress. This mechanism provides a substrate which natural selection can act upon at the population level allowing survival of the species with selective pressure. However, at the level of the individual, this mechanism can result in simultaneous expression of deleterious variants as well as reduced activity of a variety of Hsp90 chaperoned pathways, potentially shifting phenotypic variability over the disease threshold resulting in birth defects.

Persistent Feeding and Swallowing Deficits in a Mouse Model of 22q11.2 Deletion Syndrome.

Disrupted development of oropharyngeal structures as well as cranial nerve and brainstem circuits may lead to feeding and swallowing difficulties in children with 22q11. 2 deletion syndrome (22q11DS). We previously demonstrated aspiration-based dysphagia during early postnatal life in the LgDel mouse model of 22q11DS along with disrupted oropharyngeal morphogenesis and divergent differentiation and function of cranial motor and sensory nerves. We now ask whether feeding and swallowing deficits persist in adult LgDel mice using methods analogous to those used in human patients to evaluate feeding and swallowing dysfunction. Compared to wild-type mice, videofluoroscopic swallow study revealed that LgDel mice have altered feeding and swallowing behaviors, including slower lick rates, longer inter-lick intervals, and longer pharyngeal transit times with liquid consistency. Transoral endoscopic assessment identified minor structural anomalies of the palate and larynx in one-third of the LgDel mice examined. Video surveillance of feeding-related behaviors showed that LgDel mice eat and drink more frequently. Furthermore, LgDel animals engage in another oromotor behavior, grooming, more frequently, implying that divergent craniofacial and cranial nerve structure and function result in altered oromotor coordination. Finally, LgDel mice have significantly increased lung inflammation, a potential sign of aspiration-based dysphagia, consistent with results from our previous studies of early postnatal animals showing aspiration-related lung inflammation. Thus, oromotor dysfunction, feeding, and swallowing difficulties and their consequences persist in the LgDel 22q11DS mouse model. Apparently, postnatal growth and/or neural plasticity does not fully resolve deficits due to anomalous hindbrain, craniofacial, and cranial nerve development that prefigure perinatal dysphagia in 22q11DS. This new recognition of persistent challenges with feeding and swallowing may provide opportunities for improved therapeutic intervention for adolescents and adults with 22q11DS, as well as others with a history of perinatal feeding and swallowing disorders.

Variations in maternal vitamin A intake modifies phenotypes in a mouse model of 22q11.2 deletion syndrome.

BACKGROUND: Vitamin A regulates patterning of the pharyngeal arches, cranial nerves, and hindbrain that are essential for feeding and swallowing. In the LgDel mouse model of 22q11.2 deletion syndrome (22q11DS), morphogenesis of multiple structures involved in feeding and swallowing are dysmorphic. We asked whether changes in maternal dietary Vitamin A intake can modify cranial nerve, hindbrain and pharyngeal arch artery development in the embryo as well as lung pathology that can be a sign of aspiration dysphagia in LgDel pups. METHODS: Three defined amounts of vitamin A (4, 10, and 16 IU/g) were provided in the maternal diet. Cranial nerve, hindbrain and pharyngeal arch artery development was evaluated in embryos and inflammation in the lungs of pups to determine the impact of altering maternal diet on these phenotypes. RESULTS: Reduced maternal vitamin A intake improved whereas increased intake exacerbated lung inflammation in LgDel pups. These changes were accompanied by increased incidence and/or severity of pharyngeal arch artery and cranial nerve V (CN V) abnormalities in LgDel embryos as well as altered expression of Cyp26b1 in the hindbrain. CONCLUSIONS: Our studies demonstrate that variations in maternal vitamin A intake can influence the incidence and severity of phenotypes in a mouse model 22q11.2 deletion syndrome.

Cell polarity pathways converge and extend to regulate neural tube closure.

Neural tube defects, such as spinabifida, craniorachischisis and anencephaly, are some of the most common birth defects in humans. Recent studies in mouse model systems suggest that craniorachischisis is associated with mutations in genes that regulate cell polarity. Using Xenopus as a model system, Wallingford and Harland have now shed light on the mechanism by which these pathways affect neural tube closure.