Developmental regulation of cellular metabolism is required for intestinal elongation and rotation.

Malrotation of the intestine is a prevalent birth anomaly, the etiology of which remains poorly understood. Here, we show that late-stage exposure of Xenopus embryos to atrazine, a widely used herbicide that targets electron transport chain (ETC) reactions, elicits intestinal malrotation at high frequency. Interestingly, atrazine specifically inhibits the cellular morphogenetic events required for gut tube elongation, including cell rearrangement, differentiation and proliferation; insufficient gut lengthening consequently reorients the direction of intestine rotation. Transcriptome analyses of atrazine-exposed intestines reveal misexpression of genes associated with glycolysis and oxidative stress, and metabolomics shows that atrazine depletes key glycolytic and tricarboxylic acid cycle metabolites. Moreover, cellular bioenergetics assays indicate that atrazine blocks a crucial developmental transition from glycolytic ATP production toward oxidative phosphorylation. Atrazine-induced defects are phenocopied by rotenone, a known ETC Complex I inhibitor, accompanied by elevated reactive oxygen species, and rescued by antioxidant supplementation, suggesting that malrotation may be at least partly attributable to redox imbalance. These studies reveal roles for metabolism in gut morphogenesis and implicate defective gut tube elongation and/or metabolic perturbations in the etiology of intestinal malrotation.

Morphoelastic models discriminate between different mechanisms of left-right asymmetric stomach morphogenesis.

The mechanisms by which the vertebrate stomach undergoes its evolutionarily conserved leftward bending remain incompletely understood. Although the left and right sides of the organ are known to possess different gene expression patterns and undergo distinct morphogenetic events, the physical mechanisms by which these differences generate morphological asymmetry remain unclear. Here, we develop a continuum model of asymmetric stomach morphogenesis. Using a morphoelastic framework, we investigate the morphogenetic implications of a variety of hypothetical, tissue-level growth differences between the left and right sides of a simplified tubular organ. Simulations reveal that, of the various differential growth mechanisms tested, only one category is consistent with the leftward stomach curvature observed in wild-type embryos: equal left and right volumetric growth rates, coupled with transversely isotropic tissue thinning on the left side. Simulating this mechanism in a defined region of the model over a longer period of growth leads to mature stomach-like curvatures.

Rare variants in CAPN2 increase risk for isolated hypoplastic left heart syndrome.

Hypoplastic left heart syndrome (HLHS) is a severe congenital heart defect (CHD) characterized by hypoplasia of the left ventricle and aorta along with stenosis or atresia of the aortic and mitral valves. HLHS represents only ∼4%-8% of all CHDs but accounts for ∼25% of deaths. HLHS is an isolated defect (i.e., iHLHS) in 70% of families, the vast majority of which are simplex. Despite intense investigation, the genetic basis of iHLHS remains largely unknown. We performed exome sequencing on 331 families with iHLHS aggregated from four independent cohorts. A Mendelian-model-based analysis demonstrated that iHLHS was not due to single, large-effect alleles in genes previously reported to underlie iHLHS or CHD in >90% of families in this cohort. Gene-based association testing identified increased risk for iHLHS associated with variation in CAPN2 (p = 1.8 × 10-5), encoding a protein involved in functional adhesion. Functional validation studies in a vertebrate animal model (Xenopus laevis) confirmed CAPN2 is essential for cardiac ventricle morphogenesis and that in vivo loss of calpain function causes hypoplastic ventricle phenotypes and suggest that human CAPN2707C>T and CAPN21112C>T variants, each found in multiple individuals with iHLHS, are hypomorphic alleles. Collectively, our findings show that iHLHS is typically not a Mendelian condition, demonstrate that CAPN2 variants increase risk of iHLHS, and identify a novel pathway involved in HLHS pathogenesis.

Normal Table of Xenopus development: a new graphical resource.

Normal tables of development are essential for studies of embryogenesis, serving as an important resource for model organisms, including the frog Xenopus laevis. Xenopus has long been used to study developmental and cell biology, and is an increasingly important model for human birth defects and disease, genomics, proteomics and toxicology. Scientists utilize Nieuwkoop and Faber's classic 'Normal Table of Xenopus laevis (Daudin)' and accompanying illustrations to enable experimental reproducibility and reuse the illustrations in new publications and teaching. However, it is no longer possible to obtain permission for these copyrighted illustrations. We present 133 new, high-quality illustrations of X. laevis development from fertilization to metamorphosis, with additional views that were not available in the original collection. All the images are available on Xenbase, the Xenopus knowledgebase (http://www.xenbase.org/entry/zahn.do), for download and reuse under an attributable, non-commercial creative commons license. Additionally, we have compiled a 'Landmarks Table' of key morphological features and marker gene expression that can be used to distinguish stages quickly and reliably (https://www.xenbase.org/entry/landmarks-table.do). This new open-access resource will facilitate Xenopus research and teaching in the decades to come.

Single-minded 2 is required for left-right asymmetric stomach morphogenesis.

The morphogenesis of left-right (LR) asymmetry is a crucial phase of organogenesis. In the digestive tract, the development of anatomical asymmetry is first evident in the leftward curvature of the stomach. To elucidate the molecular events that shape this archetypal laterality, we performed transcriptome analyses of the left versus right sides of the developing stomach in frog embryos. Besides the known LR gene pitx2, the only gene found to be expressed asymmetrically throughout all stages of curvature was single-minded 2 (sim2), a Down Syndrome-related transcription factor and homolog of a Drosophila gene (sim) required for LR asymmetric looping of the fly gut. We demonstrate that sim2 functions downstream of LR patterning cues to regulate key cellular properties and behaviors in the left stomach epithelium that drive asymmetric curvature. Our results reveal unexpected convergent cooption of single-minded genes during the evolution of LR asymmetric morphogenesis, and have implications for dose-dependent roles of laterality factors in non-laterality-related birth defects.

The twists and turns of left-right asymmetric gut morphogenesis.

Many organs develop left-right asymmetric shapes and positions that are crucial for normal function. Indeed, anomalous laterality is associated with multiple severe birth defects. Although the events that initially orient the left-right body axis are beginning to be understood, the mechanisms that shape the asymmetries of individual organs remain less clear. Here, we summarize new evidence challenging century-old ideas about the development of stomach and intestine laterality. We compare classical and contemporary models of asymmetric gut morphogenesis and highlight key unanswered questions for future investigation.

Exome sequencing of family trios from the National Birth Defects Prevention Study: Tapping into a rich resource of genetic and environmental data.

BACKGROUND: The National Birth Defects Prevention Study (NBDPS) is a multisite, population-based, case-control study of genetic and nongenetic risk factors for major structural birth defects. Eligible women had a pregnancy affected by a birth defect or a liveborn child without a birth defect between 1997 and 2011. They were invited to complete a telephone interview to collect pregnancy exposure data and were mailed buccal cell collection kits to collect specimens from themselves, their child (if living), and their child's father. Over 23,000 families representing more than 30 major structural birth defects provided DNA specimens. METHODS: To evaluate their utility for exome sequencing (ES), specimens from 20 children with colonic atresia were studied. Evaluations were conducted on specimens collected using cytobrushes stored and transported in open versus closed packaging, on native genomic DNA (gDNA) versus whole genome amplified (WGA) products and on a library preparation protocol adapted to low amounts of DNA. RESULTS: The DNA extracted from brushes in open packaging yielded higher quality sequence data than DNA from brushes in closed packaging. Quality metrics of sequenced gDNA were consistently higher than metrics from corresponding WGA products and were consistently high when using a low input protocol. CONCLUSIONS: This proof-of-principle study established conditions under which ES can be applied to NBDPS specimens. Successful sequencing of exomes from well-characterized NBDPS families indicated that this unique collection can be used to investigate the roles of genetic variation and gene-environment interaction effects in birth defect etiologies, providing a valuable resource for birth defect researchers.

Vangl2 coordinates cell rearrangements during gut elongation.

BACKGROUND: The embryonic gut tube undergoes extensive lengthening to generate the surface area required for nutrient absorption across the digestive epithelium. In Xenopus, narrowing and elongation of the tube is driven by radial rearrangements of its core of endoderm cells, a process that concomitantly opens the gut lumen and facilitates epithelial morphogenesis. How endoderm rearrangements are properly oriented and coordinated to achieve this complex morphogenetic outcome is unknown. RESULTS: We find that, prior to gut elongation, the core Wnt/PCP component Vangl2 becomes enriched at both the anterior and apical aspects of individual endoderm cells. In Vangl2-depleted guts, the cells remain unpolarized, down-regulate cell-cell adhesion proteins, and, consequently, fail to rearrange, leading to a short gut with an occluded lumen and undifferentiated epithelium. In contrast, endoderm cells with ectopic Vangl2 protein acquire abnormal polarity and adhesive contacts. As a result, endoderm cells also fail to rearrange properly and undergo ectopic differentiation, resulting in guts with multiple torturous lumens, irregular epithelial architecture, and variable intestinal topologies. CONCLUSIONS: Asymmetrical enrichment of Vangl2 in individual gut endoderm cells orients polarity and adhesion during radial rearrangements, coordinating digestive epithelial morphogenesis and lumen formation with gut tube elongation.

The left-right asymmetry of liver lobation is generated by Pitx2c-mediated asymmetries in the hepatic diverticulum.

Internal organs exhibit left-right asymmetric sizes, shapes and anatomical positions, but how these different lateralities develop is poorly understood. Here we use the experimentally tractable Xenopus model to uncover the morphogenetic events that drive the left-right asymmetrical lobation of the liver. On the right side of the early hepatic diverticulum, endoderm cells become columnar and apically constricted, forming an expanded epithelial surface and, ultimately, an enlarged right liver lobe. In contrast, the cells on the left side become rounder, and rearrange into a compact, stratified architecture that produces a smaller left lobe. Side-specific gain- and loss-of-function studies reveal that asymmetric expression of the left-right determinant Pitx2c elicits distinct epithelial morphogenesis events in the left side of the diverticulum. Surprisingly, the cellular events induced by Pitx2c during liver development are opposite those induced in other digestive organs, suggesting divergent cellular mechanisms underlie the formation of different lateralities.

Acetylcholinesterase plays a non-neuronal, non-esterase role in organogenesis.

Acetylcholinesterase (AChE) is crucial for degrading acetylcholine at cholinergic synapses. In vitro studies suggest that, in addition to its role in nervous system signaling, AChE can also modulate non-neuronal cell properties, although it remains controversial whether AChE functions in this capacity in vivo Here, we show that AChE plays an essential non-classical role in vertebrate gut morphogenesis. Exposure of Xenopus embryos to AChE-inhibiting chemicals results in severe defects in intestinal development. Tissue-targeted loss-of-function assays (via microinjection of antisense morpholino or CRISPR-Cas9) confirm that AChE is specifically required in the gut endoderm tissue, a non-neuronal cell population, where it mediates adhesion to fibronectin and regulates cell rearrangement events that drive gut lengthening and digestive epithelial morphogenesis. Notably, the classical esterase activity of AChE is dispensable for this activity. As AChE is deeply conserved, widely expressed outside of the nervous system, and the target of many environmental chemicals, these results have wide-reaching implications for development and toxicology.

Stomach curvature is generated by left-right asymmetric gut morphogenesis.

Left-right (LR) asymmetry is a fundamental feature of internal anatomy, yet the emergence of morphological asymmetry remains one of the least understood phases of organogenesis. Asymmetric rotation of the intestine is directed by forces outside the gut, but the morphogenetic events that generate anatomical asymmetry in other regions of the digestive tract remain unknown. Here, we show in mouse and Xenopus that the mechanisms that drive the curvature of the stomach are intrinsic to the gut tube itself. The left wall of the primitive stomach expands more than the right wall, as the left epithelium becomes more polarized and undergoes radial rearrangement. These asymmetries exist across several species, and are dependent on LR patterning genes, including Foxj1, Nodal and Pitx2 Our findings have implications for how LR patterning manifests distinct types of morphological asymmetries in different contexts.

Frogs as integrative models for understanding digestive organ development and evolution.

The digestive system comprises numerous cells, tissues and organs that are essential for the proper assimilation of nutrients and energy. Many aspects of digestive organ function are highly conserved among vertebrates, yet the final anatomical configuration of the gut varies widely between species, especially those with different diets. Improved understanding of the complex molecular and cellular events that orchestrate digestive organ development is pertinent to many areas of biology and medicine, including the regeneration or replacement of diseased organs, the etiology of digestive organ birth defects, and the evolution of specialized features of digestive anatomy. In this review, we highlight specific examples of how investigations using Xenopus laevis frog embryos have revealed insight into the molecular and cellular dynamics of digestive organ patterning and morphogenesis that would have been difficult to obtain in other animal models. Additionally, we discuss recent studies of gut development in non-model frog species with unique feeding strategies, such as Lepidobatrachus laevis and Eleutherodactylous coqui, which are beginning to provide glimpses of the evolutionary mechanisms that may generate morphological variation in the digestive tract. The unparalleled experimental versatility of frog embryos make them excellent, integrative models for studying digestive organ development across multiple disciplines.

Budgett's frog (Lepidobatrachus laevis): A new amphibian embryo for developmental biology.

The large size and rapid development of amphibian embryos has facilitated ground-breaking discoveries in developmental biology. Here, we describe the embryogenesis of the Budgett's frog (Lepidobatrachus laevis), an unusual species with eggs that are over twice the diameter of laboratory Xenopus, and embryos that can tolerate higher temperatures to develop into a tadpole four times more rapidly. In addition to detailing their early development, we demonstrate that, like Xenopus, these embryos are amenable to explant culture assays and can express exogenous transcripts in a tissue-specific manner. Moreover, the steep developmental trajectory and large scale of Lepidobatrachus make it exceptionally well-suited for morphogenesis research. For example, the developing organs of the Budgett's frog are massive compared to those of most model species, and are composed of larger individual cells, thereby affording increased subcellular resolution of early vertebrate organogenesis. Furthermore, we found that complete limb regeneration, which typically requires months to achieve in most vertebrate models, occurs in a matter of days in the Budgett's tadpole, which substantially accelerates the pace of experimentation. Thus, the unusual combination of the greater size and speed of the Budgett's frog model provides inimitable advantages for developmental studies-and a novel inroad to address the mechanisms of spatiotemporal scaling during evolution.

A single codon insertion in PICALM is associated with development of familial subvalvular aortic stenosis in Newfoundland dogs.

Familial subvalvular aortic stenosis (SAS) is one of the most common congenital heart defects in dogs and is an inherited defect of Newfoundlands, golden retrievers and human children. Although SAS is known to be inherited, specific genes involved in Newfoundlands with SAS have not been defined. We hypothesized that SAS in Newfoundlands is inherited in an autosomal dominant pattern and caused by a single genetic variant. We studied 93 prospectively recruited Newfoundland dogs, and 180 control dogs of 30 breeds. By providing cardiac screening evaluations for Newfoundlands we conducted a pedigree evaluation, genome-wide association study and RNA sequence analysis to identify a proposed pattern of inheritance and genetic loci associated with the development of SAS. We identified a three-nucleotide exonic insertion in phosphatidylinositol-binding clathrin assembly protein (PICALM) that is associated with the development of SAS in Newfoundlands. Pedigree evaluation best supported an autosomal dominant pattern of inheritance and provided evidence that equivocally affected individuals may pass on SAS in their progeny. Immunohistochemistry demonstrated the presence of PICALM in the canine myocardium and area of the subvalvular ridge. Additionally, small molecule inhibition of clathrin-mediated endocytosis resulted in developmental abnormalities within the outflow tract (OFT) of Xenopus laevis embryos. The ability to test for presence of this PICALM insertion may impact dog-breeding decisions and facilitate reduction of SAS disease prevalence in Newfoundland dogs. Understanding the role of PICALM in OFT development may aid in future molecular and genetic investigations into other congenital heart defects of various species.

Constraints on Mammalian forelimb development: insights from developmental disparity.

Tetrapod limb development has been studied extensively for decades, yet the strength and role of developmental constraints in this process remains unresolved. Mammals exhibit a particularly wide array of limb morphologies associated with various locomotion modes and behaviors, providing a useful system for identifying periods of developmental constraint and conserved developmental mechanisms or morphologies. In this study, landmark-based geometric morphometrics are used to investigate levels and patterns of morphological diversity (disparity) among the developing forelimbs of four mammals with diverse limb morphologies: mice, opossums, horses, and pigs. Results indicate that disparity among the forelimbs of these species slightly decreases or stays the same from the appearance of the limb ridge to the bud stage, and increases dramatically from the paddle through tissue regression stages. Heterochrony exhibited by the precocial opossum limb was not found to drive these patterns of morphological disparity, suggesting that the low disparity of the middle stages of limb development (e.g., paddle stage) is driven by processes operating within the limb and is likely not a result of embryo-wide constraint.

Developmental origins of a novel gut morphology in frogs.

Phenotypic variation is a prerequisite for evolution by natural selection, yet the processes that give rise to the novel morphologies upon which selection acts are poorly understood. We employed a chemical genetic screen to identify developmental changes capable of generating ecologically relevant morphological variation as observed among extant species. Specifically, we assayed for exogenously applied small molecules capable of transforming the ancestral larval foregut of the herbivorous Xenopus laevis to resemble the derived larval foregut of the carnivorous Lepidobatrachus laevis. Appropriately, the small molecules that demonstrate this capacity modulate conserved morphogenetic pathways involved in gut development, including downregulation of retinoic acid (RA) signaling. Identical manipulation of RA signaling in a species that is more closely related to Lepidobatrachus, Ceratophrys cranwelli, yielded even more similar transformations, corroborating the relevance of RA signaling variation in interspecific morphological change. Finally, we were able to recover the ancestral gut phenotype in Lepidobatrachus by performing a reverse chemical manipulation to upregulate RA signaling, providing strong evidence that modifications to this specific pathway promoted the emergence of a lineage-specific phenotypic novelty. Interestingly, our screen also revealed pathways that have not yet been implicated in early gut morphogenesis, such as thyroid hormone signaling. In general, the chemical genetic screen may be a valuable tool for identifying developmental mechanisms that underlie ecologically and evolutionarily relevant phenotypic variation.

Jun N-terminal kinase maintains tissue integrity during cell rearrangement in the gut.

Tissue elongation is a fundamental morphogenetic process that generates the proper anatomical topology of the body plan and vital organs. In many elongating embryonic structures, tissue lengthening is driven by Rho family GTPase-mediated cell rearrangement. During this dynamic process, the mechanisms that modulate intercellular adhesion to allow individual cells to change position without compromising structural integrity are not well understood. In vertebrates, Jun N-terminal kinase (JNK) is also required for tissue elongation, but the precise cellular role of JNK in this context has remained elusive. Here, we show that JNK activity is indispensable for the rearrangement of endoderm cells that underlies the elongation of the Xenopus gut tube. Whereas Rho kinase is necessary to induce cell intercalation and remodel adhesive contacts, we have found that JNK is required to maintain cell-cell adhesion and establish parallel microtubule arrays; without JNK activity, the reorganizing endoderm dissociates. Depleting polymerized microtubules phenocopies this effect of JNK inhibition on endoderm morphogenesis, consistent with a model in which JNK regulates microtubule architecture to preserve adhesive contacts between rearranging gut cells. Thus, in contrast to Rho kinase, which generates actomyosin-based tension and cell movement, JNK signaling is required to establish microtubule stability and maintain tissue cohesion; both factors are required to achieve proper cell rearrangement and gut extension. This model of gut elongation has implications not only for the etiology of digestive tract defects, but sheds new light on the means by which intra- and intercellular forces are balanced to promote topological change, while preserving structural integrity, in numerous morphogenetic contexts.

A photoactivatable small-molecule inhibitor for light-controlled spatiotemporal regulation of Rho kinase in live embryos.

To uncover the molecular mechanisms of embryonic development, the ideal loss-of-function strategy would be capable of targeting specific regions of the living embryo with both temporal and spatial precision. To this end, we have developed a novel pharmacological agent that can be light activated to achieve spatiotemporally limited inhibition of Rho kinase activity in vivo. A new photolabile caging group, 6-nitropiperonyloxymethyl (NPOM), was installed on a small-molecule inhibitor of Rho kinase, Rockout, to generate a 'caged Rockout' derivative. Complementary biochemical, cellular, molecular and morphogenetic assays in both mammalian cell culture and Xenopus laevis embryos validate that the inhibitory activity of the caged compound is dependent on exposure to light. Conveniently, this unique reagent retains many of the practical advantages of conventional small-molecule inhibitors, including delivery by simple diffusion in the growth medium and concentration-dependent tuneability, but can be locally activated by decaging with standard instrumentation. Application of this novel tool to the spatially heterogeneous problem of embryonic left-right asymmetry revealed a differential requirement for Rho signaling on the left and right sides of the primitive gut tube, yielding new insight into the molecular mechanisms that generate asymmetric organ morphology. As many aromatic/heterocyclic small-molecule inhibitors are amenable to installation of this caging group, our results indicate that photocaging pharmacological inhibitors might be a generalizable technique for engendering convenient loss-of-function reagents with great potential for wide application in developmental biology.

Heterotaxin: a TGF-ß signaling inhibitor identified in a multi-phenotype profiling screen in Xenopus embryos.

Disruptions of anatomical left-right asymmetry result in life-threatening heterotaxic birth defects in vital organs. We performed a small molecule screen for left-right asymmetry phenotypes in Xenopus embryos and discovered a pyridine analog, heterotaxin, which disrupts both cardiovascular and digestive organ laterality and inhibits TGF-ß-dependent left-right asymmetric gene expression. Heterotaxin analogs also perturb vascular development, melanogenesis, cell migration, and adhesion, and indirectly inhibit the phosphorylation of an intracellular mediator of TGF-ß signaling. This combined phenotypic profile identifies these compounds as a class of TGF-ß signaling inhibitors. Notably, heterotaxin analogs also possess highly desirable antitumor properties, inhibiting epithelial-mesenchymal transition, angiogenesis, and tumor cell proliferation in mammalian systems. Our results suggest that assessing multiple organ, tissue, cellular, and molecular parameters in a whole organism context is a valuable strategy for identifying the mechanism of action of bioactive compounds.

Photocaged morpholino oligomers for the light-regulation of gene function in zebrafish and Xenopus embryos.

Morpholino oligonucleotides, or morpholinos, have emerged as powerful antisense reagents for evaluating gene function in both in vitro and in vivo contexts. However, the constitutive activity of these reagents limits their utility for applications that require spatiotemporal control, such as tissue-specific gene disruptions in embryos. Here we report a novel and efficient synthetic route for incorporating photocaged monomeric building blocks directly into morpholino oligomers and demonstrate the utility of these caged morpholinos in the light-activated control of gene function in both cell culture and living embryos. We demonstrate that a caged morpholino that targets enhanced green fluorescent protein (EGFP) disrupts EGFP production only after exposure to UV light in both transfected cells and living zebrafish (Danio rerio) and Xenopus frog embryos. Finally, we show that a caged morpholino targeting chordin, a zebrafish gene that yields a distinct phenotype when functionally disrupted by conventional morpholinos, elicits a chordin phenotype in a UV-dependent manner. Our results suggest that photocaged morpholinos are readily synthesized and highly efficacious tools for light-activated spatiotemporal control of gene expression in multiple contexts.