Robert J. Lipinski

The 46th Annual David W. Smith (DWS) Workshop on Malformations and Morphogenesis returned to the University of Wisconsin–Madison, a site of singular historical resonance for dysmorphology. The program unfolded across six themes—orofacial morphogenesis, gastrulation and early embryogenesis, tooth morphogenesis/dentition, genitourinary malformations, chromatin remodeling, and cardiovascular disorders—anchored by keynotes and emphasis lectures from Drs. Paul Trainor, Cigall Kadoch, Ophir Klein, Neil Chi, Robert Lipinski, Margaret Adam, and the Founder's Talk by Dr. Marilyn Jones. This narrative report follows the chronological flow of the meeting, providing fuller detail for anchor lectures and synthetic integration of presentations.

The prenatal environment contributes to the risk for congenital conditions, including birth defects, developmental disorders, and diseases that manifest in later life. However, our current understanding of prenatal exposures and their impact on disease risk is extremely limited. DNA methylation (DNAm) is a promising biomarker of prenatal exposures because this epigenetic mechanism is developmentally active, environmentally responsive, and imparts chemically stable marks that can be quantified with increasing accuracy and precision. However, development and utilization of DNAm biomarkers are impeded by inadequate understanding of how environmentally responsive prenatal DNAm changes persist across progenitor cell populations and pre- and postnatal development. This review synthesizes current evidence on the impact of the prenatal environment on DNAm, including specific dietary and chemical influences, and persistence of these changes across life stages. We then evaluate the suitability of common surrogate tissues (blood, saliva, and extra-embryonic tissues) from a developmental cell lineage framework for their applicability in prenatal exposure research and outline key considerations in selecting surrogate tissues for epigenome-wide association studies. Finally, using orofacial cleft etiopathogenesis as a model, we illustrate the conceptual application of DNAm biomarkers and highlight the need for longitudinal studies and comparative analysis of target and surrogate tissues. By identifying key knowledge gaps and proposing actionable strategies to address them, this review is directed at advancing the use of DNAm biomarkers in resolving how prenatal exposures contribute to human disease.

Chemical co-exposures are important contributors to adverse biological responses yet remain poorly understood, especially in the context of prenatal development. Sonic Hedgehog (Shh) signaling is an essential developmental pathway that is sensitive to small-molecule disruption and directly linked to common and etiologically complex human birth defects. Numerous mechanistically diverse small-molecule Shh pathway antagonists have been identified, but their interactions in pathway disruption have received minimal attention. We established a tractable co-culture model in which autonomous SHH ligand production initiates this complex inter- and intracellular signal transduction cascade and culminates in activation of a GLI-responsive luminescent reporter. Compounds reported to target SHH ligand processing (RU-SKI 43, AY 9944, U18666A), SMO-mediated signal transduction (cyclopamine, vismodegib, piperonyl butoxide, cannabidiol), and GLI transcription factors (GANT 61, arsenic trioxide) reduced Shh pathway-driven reporter activity with AC50 values in the low micromolar range or below. We then evaluated chemical interactions among Shh pathway inhibitors using isobolographic analysis. Co-exposure assays revealed additive interactions from combined SMO and GLI inhibition, whereas disruption of SMO and cholesterol dynamics synergistically decreased Shh pathway activity. Unexpectedly, piperonyl butoxide synergized with other SMO inhibitors, and further characterization of piperonyl butoxide’s impacts on Shh signaling supported an additional mechanism of inhibition independent of SMO. In zebrafish embryos, combined exposure to piperonyl butoxide and cyclopamine also produced a synergistic increase in craniofacial dysmorphogenesis. These findings demonstrate the importance of tractable models that recapitulate complex signal transduction pathways to empirically test for additive and synergistic chemical interactions in risk assessment.

Background: Teratogens and other environmental factors influence human birth defect risk, but our understanding of how they reach the developing conceptus is surprisingly limited. The placenta is often invoked as a key mediator of teratogenicity by acting as a physical barrier that can block or regulate the transfer of harmful substances to the embryo or fetus. Methods: In this review, we compare the timing of teratogen susceptibility with the development of the placenta. Teratogenicity data from multiple published studies were plotted on a unified multi‐species developmental timeline to relate findings from animal models to human developmental timing. Results: The critical periods for most teratogen‐induced structural birth defects, including fetal alcohol syndrome‐related defects, neural tube defects, orofacial clefts, and limb malformations translate to the 3rd to 6th week of human embryonic development, while the human hemochorial placenta matures later, between 8 and 12 weeks of pregnancy. Conclusions: This developmental chronology challenges the seemingly pervasive notion that placental transfer capacity plays a major role in mediating teratogenicity and highlights the need to further investigate the barrier capacity of the structures that surround and protect the developing embryo (e.g., trophoblast, yolk sac) prior to formation of the definitive placenta, and when the embryo is most sensitive to teratogenic insult.

Background: Neurodevelopmental disorders (NDDs) are common, highly variable, and etiologically complex. Identifying environmental factors that adversely impact prenatal brain development is a direct path to NDD prevention. Small molecule disruption of the Sonic hedgehog (Shh) signaling pathway, a key regulator of craniofacial morphogenesis, can lead to overt face and forebrain malformations that produce profound neurological deficits. However, whether environmental disruption of Shh signaling can cause subtle neurodevelopmental outcomes in the absence of overt facial malformations was unknown.Methods: We developed a dietary model of Shh signaling inhibition using the specific Shh pathway antagonist vismodegib. C57BL/6J mice were fed control chow or chow containing 25, 75, or 225 ppm vismodegib from gestational day (GD)4 through GD12 to target Shh signaling during craniofacial morphogenesis. Impacts of Shh pathway disruption on face and forebrain development were examined in exposed embryos and fetuses, and behavioral characteristics were assessed in adult mice.Results: Exposure to chow containing 225 ppm vismodegib resulted in abnormal forebrain patterning at GD11, face and brain malformations at GD17, and early postnatal mortality, while lower treatment groups appeared phenotypically normal. Adult mice exposed to 25 and 75 ppm vismodegib outperformed control mice on repeated rotarod sessions, but treated mice did not significantly differ from control animals in open field exploration, marble burying, olfactory discrimination and detection, or fear conditioning assays.Conclusions: Under the examined conditions, prenatal Shh disruption did not produce robust neurobehavioral differences in the absence of craniofacial malformations.

Orofacial clefts of the lip and palate are widely recognized to result from complex gene–environment interactions, but inadequate understanding of environmental risk factors has stymied development of prevention strategies. We interrogated the role of DNA methylation, an environmentally malleable epigenetic mechanism, in orofacial development. Expression of the key DNA methyltransferase enzyme DNMT1 was detected throughout palate morphogenesis in the epithelium and underlying cranial neural crest cell (cNCC) mesenchyme, a highly proliferative multipotent stem cell population that forms orofacial connective tissue. Genetic and pharmacologic manipulations of DNMT activity were then applied to define the tissue- and timing-dependent requirement of DNA methylation in orofacial development. cNCC-specific Dnmt1 inactivation targeting initial palate outgrowth resulted in OFCs, while later targeting during palatal shelf elevation and elongation did not. Conditional Dnmt1 deletion reduced cNCC proliferation and subsequent differentiation trajectory, resulting in attenuated outgrowth of the palatal shelves and altered development of cNCC-derived skeletal elements. Finally, we found that the cellular mechanisms of cleft pathogenesis observed in vivo can be recapitulated by pharmacologically reducing DNA methylation in multipotent cNCCs cultured in vitro. These findings demonstrate that DNA methylation is a crucial epigenetic regulator of cNCC biology, define a critical period of development in which its disruption directly causes OFCs, and provide opportunities to identify environmental influences that contribute to OFC risk.

Background Frem1 has been linked to human face shape variation, dysmorphology, and malformation, but little is known about its regulation and biological role in facial development. Results During midfacial morphogenesis in mice, we observed Frem1 expression in the embryonic growth centers that form the median upper lip, nose, and palate. Expansive spatial gradients of Frem1 expression in the cranial neural crest cell (cNCC) mesenchyme of these tissues suggested transcriptional regulation by a secreted morphogen. Accordingly, Frem1 expression paralleled that of the conserved Sonic Hedgehog (Shh) target gene Gli1 in the cNCC mesenchyme. Suggesting direct transcriptional regulation by Shh signaling, we found that Frem1 expression is induced by SHH ligand stimulation or downstream pathway activation in cNCCs and observed GLI transcription factor binding at the Frem1 transcriptional start site during midfacial morphogenesis. Finally, we found that FREM1 is sufficient to induce cNCC proliferation in a concentration-dependent manner and that Shh pathway antagonism reduces Frem1 expression during pathogenesis of midfacial hypoplasia. Conclusions By demonstrating that the Shh signaling pathway regulates Frem1 expression in cNCCs, these findings provide novel insight into the mechanisms underlying variation in midfacial morphogenesis.