Craniofacial developmental biology in the single-cell era.

The evolution of a unique craniofacial complex in vertebrates made possible new ways of breathing, eating, communicating and sensing the environment. The head and face develop through interactions of all three germ layers, the endoderm, ectoderm and mesoderm, as well as the so-called fourth germ layer, the cranial neural crest. Over a century of experimental embryology and genetics have revealed an incredible diversity of cell types derived from each germ layer, signaling pathways and genes that coordinate craniofacial development, and how changes to these underlie human disease and vertebrate evolution. Yet for many diseases and congenital anomalies, we have an incomplete picture of the causative genomic changes, in particular how alterations to the non-coding genome might affect craniofacial gene expression. Emerging genomics and single-cell technologies provide an opportunity to obtain a more holistic view of the genes and gene regulatory elements orchestrating craniofacial development across vertebrates. These single-cell studies generate novel hypotheses that can be experimentally validated in vivo. In this Review, we highlight recent advances in single-cell studies of diverse craniofacial structures, as well as potential pitfalls and the need for extensive in vivo validation. We discuss how these studies inform the developmental sources and regulation of head structures, bringing new insights into the etiology of structural birth anomalies that affect the vertebrate head.

Phyllotactic patterning of gerbera flower heads.

Phyllotaxis, the distribution of organs such as leaves and flowers on their support, is a key attribute of plant architecture. The geometric regularity of phyllotaxis has attracted multidisciplinary interest for centuries, resulting in an understanding of the patterns in the model plants Arabidopsis and tomato down to the molecular level. Nevertheless, the iconic example of phyllotaxis, the arrangement of individual florets into spirals in the heads of the daisy family of plants (Asteraceae), has not been fully explained. We integrate experimental data and computational models to explain phyllotaxis in Gerbera hybrida We show that phyllotactic patterning in gerbera is governed by changes in the size of the morphogenetically active zone coordinated with the growth of the head. The dynamics of these changes divides the patterning process into three phases: the development of an approximately circular pattern with a Fibonacci number of primordia near the head rim, its gradual transition to a zigzag pattern, and the development of a spiral pattern that fills the head on the template of this zigzag pattern. Fibonacci spiral numbers arise due to the intercalary insertion and lateral displacement of incipient primordia in the first phase. Our results demonstrate the essential role of the growth and active zone dynamics in the patterning of flower heads.

Analysis of lamprey meis genes reveals that conserved inputs from Hox, Meis and Pbx proteins control their expression in the hindbrain and neural tube.

Meis genes are known to play important roles in the hindbrain and neural crest cells of jawed vertebrates. To explore the roles of Meis genes in head development during evolution of vertebrates, we have identified four meis genes in the sea lamprey genome and characterized their patterns of expression and regulation, with a focus on the hindbrain and pharynx. Each of the lamprey meis genes displays temporally and spatially dynamic patterns of expression, some of which are coupled to rhombomeric domains in the developing hindbrain and select pharyngeal arches. Studies of Meis loci in mouse and zebrafish have identified enhancers that are bound by Hox and TALE (Meis and Pbx) proteins, implicating these factors in the direct regulation of Meis expression. We examined the lamprey meis loci and identified a series of cis-elements conserved between lamprey and jawed vertebrate meis genes. In transgenic reporter assays we demonstrated that these elements act as neural enhancers in lamprey embryos, directing reporter expression in appropriate domains when compared to expression of their associated endogenous meis gene. Sequence alignments reveal that these conserved elements are in similar relative positions of the meis loci and contain a series of consensus binding motifs for Hox and TALE proteins. This suggests that ancient Hox and TALE-responsive enhancers regulated expression of ancestral vertebrate meis genes in segmental domains in the hindbrain and have been retained in the meis loci during vertebrate evolution. The presence of conserved Meis, Pbx and Hox binding sites in these lamprey enhancers links Hox and TALE factors to regulation of lamprey meis genes in the developing hindbrain, indicating a deep ancestry for these regulatory interactions prior to the divergence of jawed and jawless vertebrates.

Dynamic mRNA and miRNA expression of the head during early development in bighead carp (Hypophthalmichthys nobilis).

BACKGROUND: Head of fish species, an exquisitely complex anatomical system, is important not only for studying fish evolution and development, but also for economic values. Currently, although some studies have been made on fish growth and body shapes, very limited information is available on the molecular mechanism of head development. RESULTS: In this study, RNA sequencing (RNA-Seq) and small RNA sequencing (sRNA-Seq) technologies were used to conduct integrated analysis for the head of bighead carp at different development stages, including 1, 3, 5, 15 and 30 Dph (days post hatch). By RNA-Seq data, 26 pathways related to growth and bone formation were identified as the main physiological processes during early development. Coupling this to sRNA-Seq data, we picked out six key pathways that may be responsible for head development, namely ECM receptor interaction, TNF signaling pathway, osteoclast differentiation, PI3K-Akt signaling pathway, Neuroactive ligand-receptor interaction and Jak-STAT signaling pathway. Totally, 114 important candidate genes from the six pathways were obtained. Then we found the top 20 key genes according to the degree value by cytohubba, which regulated cell growth, skeletal formation and blood homeostasis, such as pik3ca, pik3r1, egfr, vegfa, igf1 and itga2b. Finally, we also acquired 19 key miRNAs playing multiple roles in the perfection of various tissues in the head (such as brain, eye and mouth) and mineralization of head bone system, such as let-7e, miR-142a-5p, miR-144-3p, miR-23a-3p and miR-223. CONCLUSIONS: Results of this study will be informative for genetic mechanisms of head development and also provide potential candidate targets for the interaction regulation during early growth in bighead carp.

Dynamics of Wnt activity on the acquisition of ectoderm potency in epiblast stem cells.

During embryogenesis, the stringent regulation of Wnt activity is crucial for the morphogenesis of the head and brain. The loss of function of the Wnt inhibitor Dkk1 results in elevated Wnt activity, loss of ectoderm lineage attributes from the anterior epiblast, and the posteriorisation of anterior germ layer tissue towards the mesendoderm. The modulation of Wnt signalling may therefore be crucial for the allocation of epiblast cells to ectoderm progenitors during gastrulation. To test this hypothesis, we examined the lineage characteristics of epiblast stem cells (EpiSCs) that were derived and maintained under different signalling conditions. We showed that suppression of Wnt activity enhanced the ectoderm propensity of the EpiSCs. Neuroectoderm differentiation of these EpiSCs was further empowered by the robust re-activation of Wnt activity. Therefore, during gastrulation, the tuning of the signalling activities that mediate mesendoderm differentiation is instrumental for the acquisition of ectoderm potency in the epiblast.

Bighead is a Wnt antagonist secreted by the Xenopus Spemann organizer that promotes Lrp6 endocytosis.

The Xenopus laevis embryo has been subjected to almost saturating screens for molecules specifically expressed in dorsal Spemann organizer tissue. In this study, we performed high-throughput RNA sequencing of ectodermal explants, called animal caps, which normally give rise to epidermis. We analyzed dissociated animal cap cells that, through sustained activation of MAPK, differentiate into neural tissue. We also microinjected mRNAs for Cerberus, Chordin, FGF8, BMP4, Wnt8, and Xnr2, which induce neural or other germ layer differentiations. The searchable database provided here represents a valuable resource for the early vertebrate cell differentiation. These analyses resulted in the identification of a gene present in frog and fish, which we call Bighead. Surprisingly, at gastrula, it was expressed in the Spemann organizer and endoderm, rather than in ectoderm as we expected. Despite the plethora of genes already mined from Spemann organizer tissue, Bighead encodes a secreted protein that proved to be a potent inhibitor of Wnt signaling in a number of embryological and cultured cell signaling assays. Overexpression of Bighead resulted in large head structures very similar to those of the well-known Wnt antagonists Dkk1 and Frzb-1. Knockdown of Bighead with specific antisense morpholinos resulted in embryos with reduced head structures, due to increased Wnt signaling. Bighead protein bound specifically to the Wnt coreceptor lipoprotein receptor-related protein 6 (Lrp6), leading to its removal from the cell surface. Bighead joins two other Wnt antagonists, Dkk1 and Angptl4, which function as Lrp6 endocytosis regulators. These results suggest that endocytosis plays a crucial role in Wnt signaling.

Coupling the roles of Hox genes to regulatory networks patterning cranial neural crest.

The neural crest is a transient population of cells that forms within the developing central nervous system and migrates away to generate a wide range of derivatives throughout the body during vertebrate embryogenesis. These cells are of evolutionary and clinical interest, constituting a key defining trait in the evolution of vertebrates and alterations in their development are implicated in a high proportion of birth defects and craniofacial abnormalities. In the hindbrain and the adjacent cranial neural crest cells (cNCCs), nested domains of Hox gene expression provide a combinatorial'Hox-code' for specifying regional properties in the developing head. Hox genes have been shown to play important roles at multiple stages in cNCC development, including specification, migration, and differentiation. However, relatively little is known about the underlying gene-regulatory mechanisms involved, both upstream and downstream of Hox genes. Furthermore, it is still an open question as to how the genes of the neural crest GRN are linked to Hox-dependent pathways. In this review, we describe Hox gene expression, function and regulation in cNCCs with a view to integrating these genes within the emerging gene regulatory network for cNCC development. We highlight early roles for Hox1 genes in cNCC specification, proposing that this may be achieved, in part, by regulation of the balance between pluripotency and differentiation in precursor cells within the neuro-epithelium. We then describe what is known about the regulation of Hox gene expression in cNCCs and discuss this from the perspective of early vertebrate evolution.

Craniofacial, orofacial and dental disorders: the role of the RAS/ERK pathway.

Deviations from the precisely coordinated programme of human head development can lead to craniofacial and orofacial malformations often including a variety of dental abnormalities too. Although the aetiology is still unknown in many cases, during the last decades different intracellular signalling pathways have been genetically linked to specific disorders. Among these pathways, the RAS/extracellular signal-regulated kinase (ERK) signalling cascade is the focus of this review since it encompasses a large group of genes that when mutated cause some of the most common and severe developmental anomalies in humans. We present the components of the RAS/ERK pathway implicated in craniofacial and orodental disorders through a series of human and animal studies. We attempt to unravel the specific molecular targets downstream of ERK that act on particular cell types and regulate key steps in the associated developmental processes. Finally we point to ambiguities in our current knowledge that need to be clarified before RAS/ERK-targeting therapeutic approaches can be implemented.

Mechanistic insights from the LHX1-driven molecular network in building the embryonic head.

Development of an embryo is driven by a series of molecular instructions that control the differentiation of tissue precursor cells and shape the tissues into major body parts. LIM homeobox 1 (LHX1) is a transcription factor that plays a major role in the development of the embryonic head of the mouse. Loss of LHX1 function disrupts the morphogenetic movement of head tissue precursors and impacts on the function of molecular factors in modulating the activity of the WNT signaling pathway. LHX1 acts with a transcription factor complex to regulate the transcription of target genes in multiple phases of development and in a range of embryonic tissues of the mouse and Xenopus. Determining the interacting factors and transcriptional targets of LHX1 will be key to unraveling the ensemble of factors involved in head development and building a head gene regulatory network.

Odd-Paired: The Drosophila Zic Gene.

Zinc finger in the cerebellum (Zic) proteins are a family of transcription factors with multiple roles during development, particularly in neural tissues. The founding member of the Zic family is the Drosophila odd-paired (opa) gene. The Opa protein has a DNA binding domain containing five Cys2His2-type zinc fingers and has been shown to act as a sequence-specific DNA binding protein. Opa has significant homology to mammalian Zic1, Zic2, and Zic3 within the zinc finger domain and in two other conserved regions outside that domain. opa was initially identified as a pair-rule gene, part of the hierarchy of genes that establish the segmental body plan of the early Drosophila embryo. However, its wide expression pattern during embryogenesis indicates it plays additional roles. Embryos deficient in opa die before hatching with aberrant segmentation but also with defects in larval midgut formation. Post-embryonically, opa plays important roles in adult head development and circadian rhythm. Based on extensive neural expression, opa is predicted to be involved in many aspects of neural development and behavior, like other proteins of the Zic family. Consensus DNA binding sites have been identified for Opa and have been shown to activate transcription in vivo. However, there is evidence Opa may serve as a transcriptional regulator in the absence of direct DNA binding, as has been seen for other Zic proteins.

LKB1 signaling in cephalic neural crest cells is essential for vertebrate head development.

Head development in vertebrates proceeds through a series of elaborate patterning mechanisms and cell-cell interactions involving cephalic neural crest cells (CNCC). These cells undergo extensive migration along stereotypical paths after their separation from the dorsal margins of the neural tube and they give rise to most of the craniofacial skeleton. Here, we report that the silencing of the LKB1 tumor suppressor affects the delamination of pre-migratory CNCC from the neural primordium as well as their polarization and survival, thus resulting in severe facial and brain defects. We further show that LKB1-mediated effects on the development of CNCC involve the sequential activation of the AMP-activated protein kinase (AMPK), the Rho-dependent kinase (ROCK) and the actin-based motor protein myosin II. Collectively, these results establish that the complex morphogenetic processes governing head formation critically depends on the activation of the LKB1 signaling network in CNCC.

Beyond the functional matrix hypothesis: a network null model of human skull growth for the formation of bone articulations.

Craniofacial sutures and synchondroses form the boundaries among bones in the human skull, providing functional, developmental and evolutionary information. Bone articulations in the skull arise due to interactions between genetic regulatory mechanisms and epigenetic factors such as functional matrices (soft tissues and cranial cavities), which mediate bone growth. These matrices are largely acknowledged for their influence on shaping the bones of the skull; however, it is not fully understood to what extent functional matrices mediate the formation of bone articulations. Aiming to identify whether or not functional matrices are key developmental factors guiding the formation of bone articulations, we have built a network null model of the skull that simulates unconstrained bone growth. This null model predicts bone articulations that arise due to a process of bone growth that is uniform in rate, direction and timing. By comparing predicted articulations with the actual bone articulations of the human skull, we have identified which boundaries specifically need the presence of functional matrices for their formation. We show that functional matrices are necessary to connect facial bones, whereas an unconstrained bone growth is sufficient to connect non-facial bones. This finding challenges the role of the brain in the formation of boundaries between bones in the braincase without neglecting its effect on skull shape. Ultimately, our null model suggests where to look for modified developmental mechanisms promoting changes in bone growth patterns that could affect the development and evolution of the head skeleton.

rbm47, a novel RNA binding protein, regulates zebrafish head development.

BACKGROUND: Vertebrate trunk induction requires inhibition of bone morphogenetic protein (BMP) signaling, whereas vertebrate head induction requires concerted inhibition of both Wnt and BMP signaling. RNA binding proteins play diverse roles in embryonic development and their roles in vertebrate head development remain to be elucidated. RESULTS: We first characterized the human RBM47 as an RNA binding protein that specifically binds RNA but not single-stranded DNA. Next, we knocked down rbm47 gene function in zebrafish using morpholinos targeting the start codon and exon-1/intron-1 splice junction. Down-regulation of rbm47 resulted in headless and small head phenotypes, which can be rescued by a wnt8a blocking morpholino. To further reveal the mechanism of rbm47's role in head development, microarrays were performed to screen genes differentially expressed in normal and knockdown embryos. epcam and a2ml were identified as the most significantly up- and down-regulated genes, respectively. The microarrays also confirmed up-regulation of several genes involved in head development, including gsk3a, otx2, and chordin, which are important regulators of Wnt signaling. CONCLUSIONS: Altogether, our findings reveal that Rbm47 is a novel RNA-binding protein critical for head formation and embryonic patterning during zebrafish embryogenesis which may act through a Wnt8a signaling pathway.

Integrative mRNA and miRNA Expression Profiles from Developing Zebrafish Head Highlight Brain-Preference Genes and Regulatory Networks.

Zebrafish is an emerging animal model for studying molecular mechanism underlying neurodevelopmental disorder due to its advantage characters. miRNAs are small non-coding RNAs that play a key role in brain development. Understanding of dynamic transcriptional and post-transcriptional molecules and their regulation during the head development is important for the study of neurodevelopmental disorder. In this study, we performed the high-throughput sequencing of mRNAs and miRNAs in developing zebrafish head from pharyngula to early larval stages and carried out bioinformatic analysis including differential expression and functional enrichment as well as joint analysis of miRNAs and mRNAs, and also compared with other related public sequencing datasets to aid our interpretation. A large number of differential expression genes with a large fold change were detected during the head development. Further clustering and functional enrichment analyses indicated that genes in late stage were most related with synaptic signaling. Overlap test analysis showed a significant enrichment of brain-preference and synapse-associated gene set in the head transcriptome compared with the whole embryo transcriptome. We also constructed miRNA-mRNA network for those brain-preference genes and focused on those densely connected network components. CRISPR-Cas9-mediated snap25b mutants led to embryonic development defects and decreases locomotor activity. Altogether, the present study provides developmental profiles of head-enriched mRNAs and miRNAs at three critical windows for nervous system development, which may contribute to the study of neurodevelopmental disorder.

Single-cell sequencing suggests a conserved function of Hedgehog-signalling in spider eye development.

BACKGROUND: Spiders evolved different types of eyes, a pair of primary eyes that are usually forward pointing, and three pairs of secondary eyes that are typically situated more posterior and lateral on the spider's head. The best understanding of arthropod eye development comes from the vinegar fly Drosophila melanogaster, the main arthropod model organism, that also evolved different types of eyes, the larval eyes and the ocelli and compound eyes of the imago. The gene regulatory networks that underlie eye development in this species are well investigated revealing a conserved core network, but also show several differences between the different types of eyes. Recent candidate gene approaches identified a number of conserved genes in arthropod eye development, but also revealed crucial differences including the apparent lack of some key factors in some groups of arthropods, including spiders. RESULTS: Here, we re-analysed our published scRNA sequencing data and found potential key regulators of spider eye development that were previously overlooked. Unlike earlier research on this topic, our new data suggest that Hedgehog (Hh)-signalling is involved in eye development in the spider Parasteatoda tepidariorum. By investigating embryonic gene expression in representatives of all main groups of spiders, we demonstrate that this involvement is conserved in spiders. Additionally, we identified genes that are expressed in the developing eyes of spiders, but that have not been studied in this context before. CONCLUSION: Our data show that single-cell sequencing represents a powerful method to gain deeper insight into gene regulatory networks that underlie the development of lineage-specific organs such as the derived set of eyes in spiders. Overall, we gained deeper insight into spider eye development, as well as the evolution of arthropod visual system formation.

Cell-specific occupancy dynamics between the pioneer-like factor Opa/ZIC and Ocelliless/OTX regulate early head development in embryos.

During development, embryonic patterning systems direct a set of initially uncommitted pluripotent cells to differentiate into a variety of cell types and tissues. A core network of transcription factors, such as Zelda/POU5F1, Odd-paired (Opa)/ZIC3 and Ocelliless (Oc)/OTX2, are conserved across animals. While Opa is essential for a second wave of zygotic activation after Zelda, it is unclear whether Opa drives head cell specification, in the Drosophila embryo. Our hypothesis is that Opa and Oc are interacting with distinct cis-regulatory regions for shaping cell fates in the embryonic head. Super-resolution microscopy and meta-analysis of single-cell RNAseq datasets show that opa's and oc's overlapping expression domains are dynamic in the head region, with both factors being simultaneously transcribed at the blastula stage. Additionally, analysis of single-embryo RNAseq data reveals a subgroup of Opa-bound genes to be Opa-independent in the cellularized embryo. Interrogation of these genes against Oc ChIPseq combined with in situ data, suggests that Opa is competing with Oc for the regulation of a subgroup of genes later in gastrulation. Specifically, we find that Oc binds to late, head-specific enhancers independently and activates them in a head-specific wave of zygotic transcription, suggesting distinct roles for Oc in the blastula and gastrula stages.

17q12 deletion syndrome mouse model shows defects in craniofacial, brain and kidney development, and glucose homeostasis.

17q12 deletion (17q12Del) syndrome is a copy number variant (CNV) disorder associated with neurodevelopmental disorders and renal cysts and diabetes syndrome (RCAD). Using CRISPR/Cas9 genome editing, we generated a mouse model of 17q12Del syndrome on both inbred (C57BL/6N) and outbred (CD-1) genetic backgrounds. On C57BL/6N, the 17q12Del mice had severe head development defects, potentially mediated by haploinsufficiency of Lhx1, a gene within the interval that controls head development. Phenotypes included brain malformations, particularly disruption of the telencephalon and craniofacial defects. On the CD-1 background, the 17q12Del mice survived to adulthood and showed milder craniofacial and brain abnormalities. We report postnatal brain defects using automated magnetic resonance imaging-based morphometry. In addition, we demonstrate renal and blood glucose abnormalities relevant to RCAD. On both genetic backgrounds, we found sex-specific presentations, with male 17q12Del mice exhibiting higher penetrance and more severe phenotypes. Results from these experiments pinpoint specific developmental defects and pathways that guide clinical studies and a mechanistic understanding of the human 17q12Del syndrome. This mouse mutant represents the first and only experimental model to date for the 17q12 CNV disorder. This article has an associated First Person interview with the first author of the paper.

Loss of Foxd4 Impacts Neurulation and Cranial Neural Crest Specification During Early Head Development.

The specification of anterior head tissue in the late gastrulation mouse embryo relies on signaling cues from the visceral endoderm and anterior mesendoderm (AME). Genetic loss-of-function studies have pinpointed a critical requirement of LIM homeobox 1 (LHX1) transcription factor in these tissues for the formation of the embryonic head. Transcriptome analysis of embryos with gain-of-function LHX1 activity identified the forkhead box gene, Foxd4, as one downstream target of LHX1 in late-gastrulation E7.75 embryos. Our analysis of single-cell RNA-seq data show Foxd4 is co-expressed with Lhx1 and Foxa2 in the anterior midline tissue of E7.75 mouse embryos, and in the anterior neuroectoderm (ANE) at E8.25 alongside head organizer genes Otx2 and Hesx1. To study the role of Foxd4 during early development we used CRISPR-Cas9 gene editing in mouse embryonic stem cells (mESCs) to generate bi-allelic frameshift mutations in the coding sequence of Foxd4. In an in vitro model of the anterior neural tissues derived from Foxd4-loss of function (LOF) mESCs and extraembryonic endoderm cells, expression of head organizer genes as well as Zic1 and Zic2 was reduced, pointing to a need for FOXD4 in regulating early neuroectoderm development. Mid-gestation mouse chimeras harbouring Foxd4-LOF mESCs displayed craniofacial malformations and neural tube closure defects. Furthermore, our in vitro data showed a loss of FOXD4 impacts the expression of cranial neural crest markers Twist1 and Sox9. Our findings have demonstrated that FOXD4 is essential in the AME and later in the ANE for rostral neural tube closure and neural crest specification during head development.

Development of cranial muscles in the actinopterygian fish Senegal bichir, Polypterus senegalus Cuvier, 1829.

Polypterus senegalus Cuvier, 1829 is one of the most basal living actinopterygian fish and a member of the Actinopterygii. We analyzed the spatial and temporal pattern of cranial muscle development of P. senegalus using whole-mount immunostaining and serial sectioning. We described the detailed structure of the external gill muscles which divided into dorsal and ventral parts after yolk exhaustion. The pattern of the division is similar to that of urodeles. We suggest that, the external gill muscles of P. senegalus are involved in spreading and folding of the external gill stem and the branches. The fibers of the external gill muscles appear postero-lateral to the auditory capsule. In addition, the facial nerve passes through the external gills. Therefore, the external gill muscles are probably derived from the m. constrictor hyoideus dorsalis. In contrast to previous studies, we described the mm. interhyoideus and hyohyoideus fibers as independent components in the yolk-sac larvae. The m. hyohyoideus fibers appear lateral to the edge of the ventral portion of the external gill muscles, which are probably derived from the m. constrictor hyoideus dorsalis. These findings suggest that the m. hyohyoidues is derived from the m. constrictor hyoideus dorsalis in P. senegalus. In other actinopterygians, the m. hyohyoideus is derived from the m. constrictor hyoideus ventralis; therefore, the homology of the m. hyohyoidues of P. senegalus and other actinopterygians remains unclear. J. Morphol. 278:450-463, 2017. © 2017 Wiley Periodicals, Inc.

Tongue Abnormalities Are Associated to a Maternal Folic Acid Deficient Diet in Mice.

It is widely accepted that maternal folic acid (FA) deficiency during pregnancy is a risk factor for abnormal development. The tongue, with multiple genes working together in a coordinated cascade in time and place, has emerged as a target organ for testing the effect of FA during development. A FA-deficient (FAD) diet was administered to eight-week-old C57/BL/6J mouse females for 2-16 weeks. Pregnant dams were sacrificed at gestational day 17 (E17). The tongues and heads of 15 control and 210 experimental fetuses were studied. In the tongues, the maximum width, base width, height and area were compared with width, height and area of the head. All measurements decreased from 10% to 38% with increasing number of weeks on maternal FAD diet. Decreased head and tongue areas showed a harmonic reduction (Spearman nonparametric correlation, Rho = 0.802) with respect to weeks on a maternal FAD diet. Tongue congenital abnormalities showed a 10.9% prevalence, divided in aglossia (3.3%) and microglossia (7.6%), always accompanied by agnathia (5.6%) or micrognathia (5.2%). This is the first time that tongue alterations have been related experimentally to maternal FAD diet in mice. We propose that the tongue should be included in the list of FA-sensitive birth defect organs due to its relevance in several key food and nutrition processes.