Martine Dunnwald

Van der Woude syndrome (VWS) is the most common syndromic form of orofacial clefts (OFCs). The heritability of VWS is largely (~70%) explained by variants in the IRF6 gene. Experimental studies to confirm genetic variant effects are essential but are time intensive and expensive. Advancing next-generation sequencing techniques has made it possible to identify genetic variation quicker than their causal effects in disease processes can be ascertained. Therefore, employing computational tools to reliably predict their causal effects is crucial for advancing translational research. We conducted Sanger sequencing of the IRF6 gene in 46 VWS cases across 34 families to identify likely causal missense variants. We then assessed structure-informed stability changes to the IRF6 protein when these variants were incorporated into the protein, using DDGun3D. We compared DDGun3D predictions to results from previous experimental studies. Among four variants previously shown to prevent functional rescue in zebrafish, only the variant IRF6(NM_006147.4):c.881T>C, shown to cause rupture of zebrafish embryo, exhibited a high free energy change (∆∆G_fold = 3.5 kcal/mol), indicating that substantial predicted thermodynamic destabilization does not capture all disruptive function of IRF6. Our study demonstrates the utility of a protein structure-based computational approach for functional variant interpretation, providing a complementary method for studying genetic variation. Integrating computational predictions with experimental data can enhance our understanding of genetic conditions. However, success depends on fully elucidating protein structures, which is an active area of research.

Orofacial clefts, including cleft palate (CP), are among the most common types of birth defects. CP results from a failure of palatal shelf fusion during development. Previous human studies have shown that mutations in the RhoA GTPase Activating Protein 29 (ARHGAP29) gene are linked to CP, yet the role and tissue-specific requirements for ARHGAP29 during palatogenesis remain unknown. Here, we use tissue-specific deletion of Arhgap29 in mice to provide the first direct evidence that ARHGAP29 is essential for palatal elevation and fusion. We demonstrate that ectodermal conditional loss of Arhgap29 induces a significant delay in palatogenesis at embryonic (E) day 14.5 and a significant, complete cleft of the secondary palate at E18.5 –which is not observed when Arhgap29 is lost later in development using K14-Cre and K6-Cre. Phenotypic analyses of palatal shelves at E14.5 reveal a disorganized and thicker epithelium. Loss of Arhgap29 increases palatal epithelial cell area and upregulates markers of contractility including alpha-smooth muscle actin and phospho-myosin regulatory light chain implicating cell morphology and contractility as potential drivers of CP.

The differentiation cascade that converts basal keratinocytes into suprabasal layers, including periderm, depends on the activity of transcription factors. Mutations in the genes encoding many of these transcription factors, including TP63, IRF6 and GRHL3 , disrupt periderm development. Such mutations can also interfere with embryonic fusion and septation events that depend on periderm development, including palatogenesis, digit separation and the formation of temporary epithelial fusions between digits, between eyelids, and between pinnae and the scalp. ZNF750 (Zfp750 in the mouse) is a transcription factor required for keratinocyte differentiation, but whether mutations in ZNF750 contribute risk for orofacial cleft, and the role of Zfp750 in periderm development, are unknown. To address these questions we sequenced ZNF750 in 5,659 individuals including 2,125 with nonsyndromic OFC. We identify 33 rare missense variants with frequencies less than 0.1% in gnomAD. Of these, about half are predicted to be damaging with in silico tools. Collectively, these missense variants are not overtransmitted from parents to children with OFCs. Two of the variants have lower activity than the reference variant in a zebrafish embryo-based assay but no phenotype in the corresponding murine model. However, in murine embryos homozygous for a frame-shift mutation in Zfp750 ( Zfp750 ^fs ) that we generated, palatal shelves are fused but intra-oral adhesions are present, a phenotype seen in murine mutants of several bonafide OFC genes. In addition, temporary epithelial fusions are absent in Zfp750 ^fs neonates. RNA sequencing of forelimbs from Zfp750 ^fs embryos reveals decreased expression of epidermal terminal differentiation genes, and both increased and decreased expression of distinct periderm genes. Immunofluorescence shows the consistent presence of periderm proteins within the oral adhesions in Zfp750 ^fs/fs embryos. Together these studies suggest that while mutations in ZNF750 are not a major contributor to OFC risk, Zfp750 does contribute to periderm-dependent morphogenic events.

In recent years, the importance of placental function for fetal neurodevelopment has become increasingly studied. This field, known as neuroplacentology, has greatly expanded possible etiologies of neurodevelopmental disorders by exploring the influence of placental function on brain development. It is also well‐established that brain development is influenced by craniofacial morphogenesis. However, there is less focus on the impact of the placenta on craniofacial development. Recent research suggests the functional influence of placental nutrients and hormones on craniofacial skeletal growth, such as prolactin, growth hormone, insulin‐like growth factor 1, vitamin D, sulfate, and calcium, impacting both craniofacial and brain development. Therefore, interactions between the placenta and both fetal neurodevelopment and craniofacial development likely influence the growth and morphology of the head as a whole. This review discusses the role of placental hormone production and nutrient delivery in the development of the fetal head—defined as craniofacial and brain tissue together—expanding on the more established focus on brain development to also include the skull (or cranium) and face.

Scientific associations exist to serve their members and advance their science. They also provide a platform for scientists to communicate their science and engage with the public, such as hosting free resources on their web pages and organizing outreach activities with local communities. Historically, scientific associations were often internally focused. Despite the common stereotype of exclusive clubs of gentleman naturalists, there are numerous examples since the 17th century of scientific associations encouraging public engagement. This became increasingly common, and throughout the last several decades, scientific associations like the American Association for Anatomy (AAA) have been working to make changes in how science, scientists, and the public work together to produce the best scientific outcomes. This viewpoint defines different levels of relationships between the scientist and the public and how they affect outcomes related to the public's trust in science and scientists. It then provides a historical perspective on how associations have contributed to the communication of science. Lastly, it discusses the role of associations in science communication and public engagement and whether it is important for associations and why. It concludes with examples of the strategic programs of AAA that demonstrate how scientific associations can support public engagement, resulting in benefits to the public, scientists, and the anatomical sciences.

Orofacial clefts, including cleft palate (CP), are among the most common types of birth defects. CP specifically, results from a failure of palatal shelf fusion during development. Previous studies have shown that mutations in RhoA GTPase Activating Protein 29 ( ARHGAP29) are linked to CP, yet the role and tissue-specific requirements for ARHGAP29 during palatogenesis remain unknown. Here, we use tissue-specific deletion of Arhgap29 in mice to provide the first direct evidence that ARHGAP29 is essential for proper palatal elevation and fusion. We demonstrate that ectodermal conditional loss of Arhgap29 induces a significant delay in the fusion of palatal shelves at embryonic (E) day 14.5 and an incomplete yet significantly penetrant cleft palate at E18.5 – neither of which are observed when Arhgap29 is lost later in development using K14-Cre. Phenotypic analyses of palatal shelves at E14.5 reveal a disorganized and thicker epithelium at the tip of the shelves. Loss of Arhgap29 increases palate epithelial cell area and upregulates alpha-smooth muscle actin and phospho-myosin regulatory light chain implicating cell morphology and contractility as drivers of CP. Summary statement: This study in mice is the first direct evidence that ARHGAP29 is essential for proper palatal elevation and fusion. Loss of Arhgap29 alters oral epithelial morphology and upregulates contractility proteins.

Background: RhoA GTPases play critical roles in actin cytoskeletal remodeling required for controlling a diverse range of cellular functions including cell proliferation, adhesion, migration and changes in cell shape, all required for cutaneous wound healing. RhoA cycles between an active GTP‐bound and an inactive GDP‐bound form, a process regulated by guanine nucleotide exchange factors (GEFs) and GTPase‐activating proteins (GAPs). ARHGAP29 is a GAP expressed in skin keratinocytes and is decreased in the absence of interferon regulator factor 6, a critical regulator of cell proliferation, migration, and wound healing. However, the role for ARHGAP29 in keratinocyte biology is unknown.Results: We generated ARHGAP29 knockdown keratinocyte cell lines and show they displayed increased filamentous actin, phospho‐myosin regulatory light chain, cell area and population doubling time. Furthermore, we found that ARHGAP29 knockdown keratinocytes displayed significant delays in scratch wound closure in both single and collective cell migration conditions; these delays were rescued by both adding back ARHGAP29 or adding a ROCK inhibitor to ARHGAP29 knockdown cells. In vivo, however, Arhgap29 heterozygotes or keratinocyte‐specific knockouts showed on‐time wound healing.Conclusions: These data demonstrate that ARHGAP29 is required for keratinocyte morphology, proliferation and migration in vitro but is dispensable during wound healing in vivo.

BACKGROUND RhoA GTPase plays critical roles in actin cytoskeletal remodeling required for controlling a diverse range of cellular functions including cell proliferation, cell adhesions, migration and changes in cell shape. RhoA cycles between an active GTP-bound and an inactive GDP-bound form, a process that is regulated by guanine nucleotide exchange factors (GEFs), and GTPase-activating proteins (GAPs). ARHGAP29 is a GAP expressed in keratinocytes of the skin and is decreased in the absence of Interferon Regulator Factor 6, a critical regulator of cell proliferation and migration. However, the role for ARHGAP29 in keratinocyte biology is unknown. RESULTS Novel ARHGAP29 knockdown keratinocyte cell lines were generated using both CRISPR/Cas9 and shRNA technologies. Knockdown cells exhibited significant reduction of ARHGAP29 protein (50-80%) and displayed increased filamentous actin (stress fibers), phospho-myosin light chain (contractility), cell area and population doubling time. Furthermore, we found that ARHGAP29 knockdown keratinocytes displayed significant delays in scratch wound closure in both single cell and collective cell migration conditions. Particularly, our results show a reduction in path lengths, speed, directionality and persistence in keratinocytes with reduced ARHGAP29. The delay in scratch closure was rescued by both adding back ARHGAP29 or adding a ROCK inhibitor to ARHGAP29 knockdown cells. CONCLUSIONS These data demonstrate that ARHGAP29 is required for keratinocyte morphology, proliferation and migration mediated through the RhoA pathway.

Morphogenesis requires a tight coordination between mechanical forces and biochemical signals to inform individual cellular behavior. For these developmental processes to happen correctly the organism requires precise spatial and temporal coordination of the adhesion, migration, growth, differentiation, and apoptosis of cells originating from the three key embryonic layers, namely the ectoderm, mesoderm, and endoderm. The cytoskeleton and its remodeling are essential to organize and amplify many of the signaling pathways required for proper morphogenesis. In particular, the interaction of the cell junctions with the cytoskeleton functions to amplify the behavior of individual cells into collective events that are critical for development. In this review we summarize the key morphogenic events that occur during the formation of the face and the palate, as well as the protein complexes required for cell-to-cell adhesions. We then integrate the current knowledge into a comprehensive review of how mutations in cell-to-cell adhesion genes lead to abnormal craniofacial development, with a particular focus on cleft lip with or without cleft palate.