Dyrk1a is required for craniofacial development in Xenopus laevis.

Loss of function variations in the dual specificity tyrosine-phosphorylation-regulated kinase 1 A (DYRK1A) gene are associated with craniofacial malformations in humans. Here we characterized the effects of deficient DYRK1A in craniofacial development using a developmental model, Xenopus laevis. Dyrk1a mRNA and protein were expressed throughout the developing head and both were enriched in the branchial arches which contribute to the face and jaw. Consistently, reduced Dyrk1a function, using dyrk1a morpholinos and pharmacological inhibitors, resulted in orofacial malformations including hypotelorism, altered mouth shape, slanted eyes, and narrower face accompanied by smaller jaw cartilage and muscle. Inhibition of Dyrk1a function resulted in misexpression of key craniofacial regulators including transcription factors and members of the retinoic acid signaling pathway. Two such regulators, sox9 and pax3 are required for neural crest development and their decreased expression corresponds with smaller neural crest domains within the branchial arches. Finally, we determined that the smaller size of the faces, jaw elements and neural crest domains in embryos deficient in Dyrk1a could be explained by increased cell death and decreased proliferation. This study is the first to provide insight into why craniofacial birth defects might arise in humans with variants of DYRK1A.

Role of histone variants H2BC1 and H2AZ.2 in H2AK119ub nucleosome organization and Polycomb gene silencing.

Ubiquitination of histone H2A at lysine 119 residue (H2AK119ub) plays critical roles in a wide range of physiological processes, including Polycomb gene silencing 1,2 , replication 3-5 , DNA damage repair 6-10 , X inactivation 11,12 , and heterochromatin organization 13,14 . However, the underlying mechanism and structural basis of H2AK119ub remains largely elusive. In this study, we report that H2AK119ub nucleosomes have a unique composition, containing histone variants H2BC1 and H2AZ.2, and importantly, this composition is required for H2AK119ub and Polycomb gene silencing. Using the UAB domain of RSF1, we purified H2AK119ub nucleosomes to a sufficient amount and purity. Mass spectrometry analyses revealed that H2AK119ub nucleosomes contain the histone variants H2BC1 and H2AZ.2. A cryo-EM study resolved the structure of native H2AK119ub nucleosomes to a 2.6A resolution, confirming H2BC1 in one subgroup of H2AK119ub nucleosomes. Tandem GST-UAB pulldown, Flag-H2AZ.2, and HA-H2BC1 immunoprecipitation revealed that H2AK119ub nucleosomes could be separated into distinct subgroups, suggesting their composition heterogeneity and potential dynamic organization. Knockout or knockdown of H2BC1 or H2AZ.2 reduced cellular H2AK119ub levels, establishing H2BC1 and H2AZ.2 as critical determinants of H2AK119ub. Furthermore, genomic binding profiles of H2BC1 and H2AZ.2 overlapped significantly with H2AK119ub binding, with the most significant overlapping in the gene body and intergenic regions. Finally, assays in developing embryos reveal an interaction of H2AZ.2, H2BC1, and RING1A in vivo . Thus, this study revealed, for the first time, that the H2AK119ub nucleosome has a unique composition, and this composition is required for H2AK119ub and Polycomb gene silencing.

Dyrk1a is required for craniofacial development in Xenopus laevis.

Loss of function mutations in the dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A) gene are associated with craniofacial malformations in humans. Here we characterized the effects of deficient DYRK1A in craniofacial development using a developmental model, Xenopus laevis . Dyrk1a mRNA and protein was expressed throughout the developing head and was enriched in the branchial arches which contribute to the face and jaw. Consistently, reduced Dyrk1a function, using dyrk1a morpholinos and pharmacological inhibitors, resulted in orofacial malformations including hypotelorism, altered mouth shape, slanted eyes, and narrower face accompanied by smaller jaw cartilage and muscle. Inhibition of Dyrk1a function resulted in misexpression of key craniofacial regulators including transcription factors and members of the retinoic acid signaling pathway. Two such regulators, sox9 and pax3 are required for neural crest development and their decreased expression corresponds with smaller neural crest domains within the branchial arches. Finally, we determined that the smaller size of the faces, jaw elements and neural crest domains in embryos deficient in Dyrk1a could be explained by increased cell death and decreased proliferation. This study is the first to provide insight into why craniofacial birth defects might arise in humans with DYRK1A mutations.

Insights from the protein interaction Universe of the multifunctional "Goldilocks" kinase DYRK1A.

Human Dual specificity tyrosine (Y)-Regulated Kinase 1A (DYRK1A) is encoded by a dosage-dependent gene located in the Down syndrome critical region of human chromosome 21. The known substrates of DYRK1A include proteins involved in transcription, cell cycle control, DNA repair and other processes. However, the function and regulation of this kinase is not fully understood, and the current knowledge does not fully explain the dosage-dependent function of this kinase. Several recent proteomic studies identified DYRK1A interacting proteins in several human cell lines. Interestingly, several of known protein substrates of DYRK1A were undetectable in these studies, likely due to a transient nature of the kinase-substrate interaction. It is possible that the stronger-binding DYRK1A interacting proteins, many of which are poorly characterized, are involved in regulatory functions by recruiting DYRK1A to the specific subcellular compartments or distinct signaling pathways. Better understanding of these DYRK1A-interacting proteins could help to decode the cellular processes regulated by this important protein kinase during embryonic development and in the adult organism. Here, we review the current knowledge of the biochemical and functional characterization of the DYRK1A protein-protein interaction network and discuss its involvement in human disease.

Recognition of H2AK119ub plays an important role in RSF1-regulated early Xenopus development.

Polycomb group (PcG) proteins are key regulators of gene expression and developmental programs via covalent modification of histones, but the factors that interpret histone modification marks to regulate embryogenesis are less studied. We previously identified Remodeling and Spacing Factor 1 (RSF1) as a reader of histone H2A lysine 119 ubiquitination (H2AK119ub), the histone mark deposited by Polycomb Repressive Complex 1 (PRC1). In the current study, we used Xenopus laevis as a model to investigate how RSF1 affects early embryonic development and whether recognition of H2AK119ub is important for the function of RSF1. We showed that knockdown of Xenopus RSF1, rsf1, not only induced gastrulation defects as reported previously, but specific targeted knockdown in prospective neural precursors induced neural and neural crest defects, with reductions of marker genes. In addition, similar to knockdown of PRC1 components in Xenopus, the anterior-posterior neural patterning was affected in rsf1 knockdown embryos. Binding of H2AK119ub appeared to be crucial for rsf1 function, as a construct with deletion of the UAB domain, which is required for RSF1 to recognize the H2AK119ub nucleosomes, failed to rescue rsf1 morphant embryos and was less effective in interfering with early Xenopus development when ectopically expressed. Furthermore, ectopic deposition of H2AK119ub on the Smad2 target gene gsc using a ring1a-smad2 fusion protein led to ectopic recruitment of RSF1. The fusion protein was inefficient in inducing mesodermal markers in the animal region or a secondary axis when expressed in the ventral tissues. Taken together, our results reveal that rsf1 modulates similar developmental processes in early Xenopus embryos as components of PRC1 do, and that RSF1 acts at least partially through binding to the H2AK119ub mark via the UAB domain during development.

GBA Regulates EMT/MET and Chemoresistance in Squamous Cell Carcinoma Cells by Modulating the Cellular Glycosphingolipid Profile.

Glycosphingolipids (GSL) are plasma membrane components that influence molecular processes involved in cancer initiation, progression, and therapeutic responses. They also modulate receptor tyrosine kinases involved in EMT. Therefore, understanding the mechanisms that regulate GSLs in cancer has important therapeutic potential. One critical regulator of GSLs is the lysosomal glucosylceramidase ß1 (GBA) that catalyzes the last step in GSL degradation. We show that, in cancer, GBA copy number amplifications and increased expression are widespread. We show that depleting GBA in squamous cell carcinoma cell lines results in a mesenchymal-to-epithelial shift, decreased invasion and migration, increased chemotherapeutic sensitivity, and decreased activation of receptor tyrosine kinases that are involved in regulating EMT. Untargeted lipidomics shows that GBA depletion had significant effects on sphingolipids and GSLs, suggesting that increased GBA activity in cancer sustains EMT and chemoresistance by modulating receptor tyrosine kinase activity and signaling via effects on the cellular lipid profile.

Jak2 and Jaw Muscles Are Required for Buccopharyngeal Membrane Perforation during Mouth Development.

The mouth is a central feature of our face, without which we could not eat, breathe, or communicate. A critical and early event in mouth formation is the creation of a "hole" which connects the digestive system and the external environment. This hole, which has also been called the primary or embryonic mouth in vertebrates, is initially covered by a 1-2 cell layer thick structure called the buccopharyngeal membrane. When the buccopharyngeal membrane does not rupture, it impairs early mouth functions and may also lead to further craniofacial malformations. Using a chemical screen in an animal model (Xenopus laevis) and genetic data from humans, we determined that Janus kinase 2 (Jak2) has a role in buccopharyngeal membrane rupture. We have determined that decreased Jak2 function, using antisense morpholinos or a pharmacological antagonist, caused a persistent buccopharyngeal membrane as well as the loss of jaw muscles. Surprisingly, we observed that the jaw muscle compartments were connected to the oral epithelium that is continuous with the buccopharyngeal membrane. Severing such connections resulted in buccopharyngeal membrane buckling and persistence. We also noted puncta accumulation of F-actin, an indicator of tension, in the buccopharyngeal membrane during perforation. Taken together, the data has led us to a hypothesis that muscles are required to exert tension across the buccopharyngeal membrane, and such tension is necessary for its perforation.

Genome-wide analysis of copy-number variation in humans with cleft lip and/or cleft palate identifies COBLL1, RIC1, and ARHGEF38 as clefting genes.

Cleft lip with or without cleft palate (CL/P) is a common birth defect with a complex, heterogeneous etiology. It is well established that common and rare sequence variants contribute to the formation of CL/P, but the contribution of copy-number variants (CNVs) to cleft formation remains relatively understudied. To fill this knowledge gap, we conducted a large-scale comparative analysis of genome-wide CNV profiles of 869 individuals from the Philippines and 233 individuals of European ancestry with CL/P with three primary goals: first, to evaluate whether differences in CNV number, amount of genomic content, or amount of coding genomic content existed within clefting subtypes; second, to assess whether CNVs in our cohort overlapped with known Mendelian clefting loci; and third, to identify unestablished Mendelian clefting genes. Significant differences in CNVs across cleft types or in individuals with non-syndromic versus syndromic clefts were not observed; however, several CNVs in our cohort overlapped with known syndromic and non-syndromic Mendelian clefting loci. Moreover, employing a filtering strategy relying on population genetics data that rare variants are on the whole more deleterious than common variants, we identify several CNV-associated gene losses likely driving non-syndromic clefting phenotypes. By prioritizing genes deleted at a rare frequency across multiple individuals with clefts yet enriched in our cohort of individuals with clefts compared to control subjects, we identify COBLL1, RIC1, and ARHGEF38 as clefting genes. CRISPR-Cas9 mutagenesis of these genes in Xenopus laevis and Danio rerio yielded craniofacial dysmorphologies, including clefts analogous to those seen in human clefting disorders.

E-liquids and vanillin flavoring disrupts retinoic acid signaling and causes craniofacial defects in Xenopus embryos.

Environmental teratogens such as smoking are known risk factors for developmental disorders such as cleft palate. While smoking rates have declined, a new type of smoking, called vaping is on the rise. Vaping is the use of e-cigarettes to vaporize and inhale an e-liquid containing nicotine and food-like flavors. There is the potential that, like smoking, vaping could also pose a danger to the developing human. Rather than waiting for epidemiological and mammalian studies, we have turned to an aquatic developmental model, Xenopus laevis, to more quickly assess whether e-liquids contain teratogens that could lead to craniofacial malformations. Xenopus, like zebrafish, has the benefit of being a well-established developmental model and has also been effective in predicting whether a chemical could be a teratogen. We have determined that embryonic exposure to dessert flavored e-liquids can cause craniofacial abnormalities, including an orofacial cleft in Xenopus. To better understand the underlying mechanisms contributing to these defects, transcriptomic analysis of the facial tissues of embryos exposed to a representative dessert flavored e-liquid vapor extract was performed. Analysis of differentially expressed genes in these embryos revealed several genes associated with retinoic acid metabolism or the signaling pathway. Consistently, retinoic acid receptor inhibition phenocopied the craniofacial defects as those embryos exposed to the vapor extract of the e-liquid. Such malformations also correlated with a group of common differentially expressed genes, two of which are associated with midface birth defects in humans. Further, e-liquid exposure sensitized embryos to forming craniofacial malformations when they already had depressed retinoic acid signaling. Moreover, 13-cis-retinoic acid treatment could significantly reduce the e-liquid induced malformation in the midface. Such results suggest the possibility of an interaction between retinoic acid signaling and e-liquid exposure. One of the most popular and concentrated flavoring chemicals in dessert flavored e-liquids is vanillin. Xenopus embryos exposed to this chemical closely resembled embryos exposed to dessert-like e-liquids and a retinoic acid receptor antagonist. In summary, we determined that e-liquid chemicals, in particular vanillin, can cause craniofacial defects potentially by dysregulating retinoic acid signaling. This work warrants the evaluation of vanillin and other such flavoring additives in e-liquids on mammalian development.

Using an aquatic model, Xenopus laevis, to uncover the role of chromodomain 1 in craniofacial disorders.

The chromodomain family member chromodomain 1 (CHD1) has been shown to have numerous critical molecular functions including transcriptional regulation, splicing, and DNA repair. Complete loss of function of this gene is not compatible with life. On the other hand, missense and copy number variants of CHD1 can result in intellectual disabilities and craniofacial malformations in human patients including cleft palate and Pilarowski-Bjornsson Syndrome. We have used the aquatic developmental model organism Xenopus laevis, to determine a specific role for Chd1 in such cranioafcial disorders. Protein and gene knockdown techniques in Xenopus, including antisense oligos and mosaic Crispr/Cas9-mediated mutagenesis, recapitulated the craniofacial defects observed in humans. Further analysis indicated that embryos deficient in Chd1 had defects in cranial neural crest development and jaw cartilage morphology. Additionally, flow cytometry and immunohistochemistry revealed that decreased Chd1 resulted in increased in apoptosis in the developing head. Together, these experiments demonstrate that Chd1 is critical for fundamental processes and cell survival in craniofacial development. We also presented evidence that Chd1 is regulated by retinoic acid signaling during craniofacial development. Expression levels of chd1 mRNA, specifically in the head, were increased by RAR agonist exposure and decreased upon antagonist treatment. Subphenotypic levels of an RAR antagonist and Chd1 morpholinos synergized to result in orofacial defects. Further, RAR DNA binding sequences (RAREs) were detected in chd1 regulatory regions by bioinformatic analysis. In summary, by combining human genetics and experiments in an aquatic model we now have a better understanding of the role of CHD1 in craniofacial disorders.

Desmoplakin is required for epidermal integrity and morphogenesis in the Xenopus laevis embryo.

Desmoplakin (Dsp) is a unique and critical desmosomal protein, that is integral to epidermal development. However, it is unclear whether this protein is required specifically for epidermal morphogenesis. Using morpholinos or Crispr/Cas9 mutagenesis we decreased the function of Dsp in frog embryos to better understand its role during epidermal development. Dsp morphant and mutant embryos had developmental defects such as epidermal fragility that mimicked what has been reported in mammals. Most importantly, we also uncovered a novel function for Dsp in the morphogenesis of the epidermis in X. laevis. In particular, Dsp is required during the process of radial intercalation where basally located cells move into the outer epidermal layer. Once inserted these newly intercalated cells expand their apical surface and then they differentiate into specific epidermal cell types. Decreased levels of Dsp resulted in the failure of the radially intercalating cells to expand their apical surface, thereby reducing the number of differentiated multiciliated and secretory cells. Such defects correlate with changes in E-cadherin levels and actin and microtubule localization which could explain the defects in apical expansion. A mutated form of Dsp that maintains cell-cell adhesion but eliminates the connections to the cytoskeleton results in the same epidermal morphogenesis defect. These results suggest a specific role for Dsp in the apical expansion of cells during radial intercalation. We have developed a novel system, in the frog, to demonstrate for the first time that desmosomes not only protect against mechanical stress but are also critical for epidermal morphogenesis.

Transcriptome analysis of Xenopus orofacial tissues deficient in retinoic acid receptor function.

BACKGROUND: Development of the face and mouth is orchestrated by a large number of transcription factors, signaling pathways and epigenetic regulators. While we know many of these regulators, our understanding of how they interact with each other and implement changes in gene expression during orofacial development is still in its infancy. Therefore, this study focuses on uncovering potential cooperation between transcriptional regulators and one important signaling pathway, retinoic acid, during development of the midface. RESULTS: Transcriptome analyses was performed on facial tissues deficient for retinoic acid receptor function at two time points in development; early (35 hpf) just after the neural crest migrates and facial tissues are specified and later (60 hpf) when the mouth has formed and facial structures begin to differentiate. Functional and network analyses revealed that retinoic acid signaling could cooperate with novel epigenetic factors and calcium-NFAT signaling during early orofacial development. At the later stage, retinoic acid may work with WNT and BMP and regulate homeobox containing transcription factors. Finally, there is an overlap in genes dysregulated in Xenopus embryos with median clefts with human genes associated with similar orofacial defects. CONCLUSIONS: This study uncovers novel signaling pathways required for orofacial development as well as pathways that could interact with retinoic acid signaling during the formation of the face. We show that frog faces are an important tool for studying orofacial development and birth defects.

E-cigarette aerosol exposure can cause craniofacial defects in Xenopus laevis embryos and mammalian neural crest cells.

Since electronic cigarette (ECIG) introduction to American markets in 2007, vaping has surged in popularity. Many, including women of reproductive age, also believe that ECIG use is safer than traditional tobacco cigarettes and is not hazardous when pregnant. However, there are few studies investigating the effects of ECIG exposure on the developing embryo and nothing is known about potential effects on craniofacial development. Therefore, we have tested the effects of several aerosolized e-cigarette liquids (e-cigAM) in an in vivo craniofacial model, Xenopus laevis, as well as a mammalian neural crest cell line. Results demonstrate that e-cigAM exposure during embryonic development induces a variety of defects, including median facial clefts and midface hypoplasia in two of e-cigAMs tested e-cigAMs. Detailed quantitative analyses of the facial morphology revealed that nicotine is not the main factor in inducing craniofacial defects, but can exacerbate the effects of the other e-liquid components. Additionally, while two different e-cigAMs can have very similar consequences on facial appearances, there are subtle differences that could be due to the differences in e-cigAM components. Further assessment of embryos exposed to these particular e-cigAMs revealed cranial cartilage and muscle defects and a reduction in the blood supply to the face. Finally, the expression of markers for vascular and cartilage differentiation was reduced in a mammalian neural crest cell line corroborating the in vivo effects. Our work is the first to show that ECIG use could pose a potential hazard to the developing embryo and cause craniofacial birth defects. This emphasizes the need for more testing and regulation of this new popular product.

Role of JNK during buccopharyngeal membrane perforation, the last step of embryonic mouth formation.

BACKGROUND: The buccopharyngeal membrane is a thin layer of cells covering the embryonic mouth. The perforation of this structure creates an opening connecting the external and the digestive tube which is essential for oral cavity formation. In humans, persistence of the buccopharyngeal membrane can lead to orofacial defects such as choanal atresia, oral synechiaes, and cleft palate. Little is known about the causes of a persistent buccopharyngeal membrane and, importantly, how this structure ruptures. RESULTS: We have determined, using antisense and pharmacological approaches, that Xenopus embryos deficient c-Jun N-terminal kinase (JNK) signaling have a persistent buccopharyngeal membrane. JNK deficient embryos have decreased cell division and increased cellular stress and apoptosis. However, altering these processes independently of JNK did not affect buccopharyngeal membrane perforation. JNK deficient embryos also have increased intercellular adhesion and defects in e-cadherin localization. Conversely, embryos with overactive JNK have epidermal fragility, increased E-cadherin internalization, and increased membrane localized clathrin. In the buccopharyngeal membrane, clathrin is colocalized with active JNK. Furthermore, inhibition of endocytosis results in a persistent buccopharyngeal membrane, mimicking the JNK deficient phenotype. CONCLUSIONS: The results of this study suggest that JNK has a role in the disassembly adherens junctions by means of endocytosis that is required during buccopharyngeal membrane perforation. Developmental Dynamics 246:100-115, 2017. © 2016 Wiley Periodicals, Inc.

Using frogs faces to dissect the mechanisms underlying human orofacial defects.

In this review I discuss how Xenopus laevis is an effective model to dissect the mechanisms underlying orofacial defects. This species has been particularly useful in studying the understudied structures of the developing face including the embryonic mouth and primary palate. The embryonic mouth is the first opening between the foregut and the environment and is critical for adult mouth development. The final step in embryonic mouth formation is the perforation of a thin layer of tissue covering the digestive tube called the buccopharyngeal membrane. When this tissue does not perforate in humans it can pose serious health risks for the fetus and child. The primary palate forms just dorsal to the embryonic mouth and in non-amniotes it functions as the roof of the adult mouth. Defects in the primary palate result in a median oral cleft that appears similar across the vertebrates. In humans, these median clefts are often severe and surgically difficult to repair. Xenopus has several qualities that make it advantageous for craniofacial research. The free living embryo has an easily accessible face and we have also developed several new tools to analyze the development of the region. Further, Xenopus is readily amenable to chemical screens allowing us to uncover novel gene-environment interactions during orofacial development, as well as to define underlying mechanisms governing such interactions. In conclusion, we are utilizing Xenopus in new and innovative ways to contribute to craniofacial research.

The role of folate metabolism in orofacial development and clefting.

Folate deficiency has been associated with numerous diseases and birth defects including orofacial defects. However, whether folate has a role in the face during early orofacial development has been unclear. The present study reveals that pharmacological and antisense oligonucleotide mediated inhibition of DHFR, an integral enzyme in the folate pathway, results in specific changes in the size and shape of the midface and embryonic mouth. Such defects are accompanied by a severe reduction in the muscle and cartilage jaw elements without significant change in neural crest pattern or global levels of methylation. We propose that the orofacial defects associated with DHFR deficient function are the result of decreased cell proliferation and increased cell death via DNA damage. In particular, localized apoptosis may also be depleting the cells of the face that express crucial genes for the differentiation of the jaw structures. Folate supplementation is widely known to reduce human risk for orofacial clefts. In the present study, we show that activating folate metabolism can reduce median oral clefts in the primary palate by increasing cell survival. Moreover, we demonstrate that a minor decrease in DHFR function exacerbates median facial clefts caused by RAR inhibition. This work suggests that folate deficiencies could be a major contributing factor to multifactorial orofacial defects.

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.

Quantification of orofacial phenotypes in Xenopus.

Xenopus has become an important tool for dissecting the mechanisms governing craniofacial development and defects. A method to quantify orofacial development will allow for more rigorous analysis of orofacial phenotypes upon abrogation with substances that can genetically or molecularly manipulate gene expression or protein function. Using two dimensional images of the embryonic heads, traditional size dimensions-such as orofacial width, height and area- are measured. In addition, a roundness measure of the embryonic mouth opening is used to describe the shape of the mouth. Geometric morphometrics of these two dimensional images is also performed to provide a more sophisticated view of changes in the shape of the orofacial region. Landmarks are assigned to specific points in the orofacial region and coordinates are created. A principle component analysis is used to reduce landmark coordinates to principle components that then discriminate the treatment groups. These results are displayed as a scatter plot in which individuals with similar orofacial shapes cluster together. It is also useful to perform a discriminant function analysis, which statistically compares the positions of the landmarks between two treatment groups. This analysis is displayed on a transformation grid where changes in landmark position are viewed as vectors. A grid is superimposed on these vectors so that a warping pattern is displayed to show where significant landmark positions have changed. Shape changes in the discriminant function analysis are based on a statistical measure, and therefore can be evaluated by a p-value. This analysis is simple and accessible, requiring only a stereoscope and freeware software, and thus will be a valuable research and teaching resource.