Defensive and offensive behaviours in a Kleefstra syndrome mouse model.

Kleefstra syndrome in humans is characterized by a general delay in development, intellectual disability and autistic features. The mouse model of this disease (Ehmt1±) expresses anxiety, autistic-like traits, and aberrant social interactions with non-cagemates. To investigate how Ehmt1± mice behave with unfamiliar conspecifics, we allowed adult, male animals to freely interact for 10 min in a neutral, novel environment within a host-visitor setting. In trials where the Ehmt1± mice were hosts, there were defensive and offensive behaviors. Our key finding was that Ehmt1± mice displayed defensive postures, attacking and biting; in contrast, wild-type (WT) interacting with other WT did not enact such behaviors. Further, if there was a fight between an Ehmt1± and a WT mouse, the Ehmt1± animal was the most aggressive and always initiated these behaviors.

Short exposure to glyphosate induces locomotor, craniofacial, and bone disorders in zebrafish (Danio rerio) embryos.

Glyphosate [N-(phosphonomethyl)glycine] is the active ingredient in widely used broad-spectrum herbicides. Even though the toxicity mechanism of this herbicide in vertebrates is poorly understood, evidence suggests that glyphosate is an endocrine disruptor capable of producing morphological anomalies as well as cardiotoxic and neurotoxic effects. We used the zebrafish model to assess the effects of early life glyphosate exposure on the development of cartilage and bone tissues and organismal responses. We found functional alterations, including a reduction in the cardiac rate, significant changes in the spontaneous tail movement pattern, and defects in craniofacial development. These effects were concomitant with alterations in the level of the estrogen receptor alpha osteopontin and bone sialoprotein. We also found that embryos exposed to glyphosate presented spine deformities as adults. These developmental alterations are likely induced by changes in protein levels related to bone and cartilage formation.

Canine DVL2 variant contributes to brachycephalic phenotype and caudal vertebral anomalies.

A frameshift deletion variant in the Wnt pathway gene dishevelled 2 (DVL2) is associated with a truncated, kinked tail ("screw tail") in English Bulldogs, French Bulldogs and Boston Terriers. These breeds are also characterized by distinctive morphological traits, including a wide head, flat face and short-limbed dwarfism, which are characteristic of Robinow syndrome in humans, caused by defects in genes such as DVL1 and DVL3. Based on these phenotypic and genetic similarities, it has previously been hypothesized that the canine DVL2 variant results in a syndromic phenotype called the Robinow-like syndrome. In our study, we investigated the distribution of the DVL2 variant in 1954 dogs from 15 breeds, identifying breeds with allele variation and enabling the dissection of the genotype-phenotype correlation for the first time. With CT examinations in American Staffordshire Terriers, we confirmed that the DVL2 allele is associated with caudal vertebral malformations and a brachycephalic phenotype. We also hypothesize that the variant may be linked to additional health conditions, including brachycephalic obstructive airway syndrome and congenital heart defects. Altogether, our study strengthens the role of DVL2 as one of the contributors to the "bulldog type" morphology and features on the spectrum of human Robinow syndrome.

De novo ZIC2 frameshift variant associated with frontonasal dysplasia in a Limousin calf.

BACKGROUND: Bovine frontonasal dysplasias like arhinencephaly, synophthalmia, cyclopia and anophthalmia are sporadic congenital facial malformations. In this study, computed tomography, necropsy, histopathological examinations and whole genome sequencing on an Illumina NextSeq500 were performed to characterize a stillborn Limousin calf with frontonasal dysplasia. In order to identify private genetic and structural variants, we screened whole genome sequencing data of the affected calf and unaffected relatives including parents, a maternal and paternal halfsibling. RESULTS: The stillborn calf exhibited severe craniofacial malformations. Nose and maxilla were absent, mandibles were upwardly curved and a median cleft palate was evident. Eyes, optic nerve and orbital cavities were not developed and the rudimentary orbita showed hypotelorism. A defect centrally in the front skull covered with a membrane extended into the intracranial cavity. Aprosencephaly affected telencephalic and diencephalic structures and cerebellum. In addition, a shortened tail was seen. Filtering whole genome sequencing data revealed a private frameshift variant within the candidate gene ZIC2 in the affected calf. This variant was heterozygous mutant in this case and homozygous wild type in parents, half-siblings and controls. CONCLUSIONS: We found a novel ZIC2 frameshift mutation in an aprosencephalic Limousin calf. The origin of this variant is most likely due to a de novo mutation in the germline of one parent or during very early embryonic development. To the authors' best knowledge, this is the first identified mutation in cattle associated with bovine frontonasal dysplasia.

Parallel evolution of regressive and constructive craniofacial traits across distinct populations of Astyanax mexicanus cavefish.

Life in complete darkness has driven the evolution of a suite of troglobitic features in the blind Mexican cavefish Astyanax mexicanus, such as eye and pigmentation loss. While regressive evolution is a hallmark of obligate cave-dwelling organisms, constructive (or augmented) traits commonly arise as well. The cavefish cranium has undergone extensive changes compared with closely-related surface fish. These alterations are rooted in both cranial bones and surrounding sensory tissues such as enhancements in the gustatory and lateral line systems. Cavefish also harbor numerous cranial bone asymmetries: fluctuating asymmetry of individual bones and directional asymmetry in a dorsal bend of the skull. This asymmetry is mirrored by the asymmetrical patterning of mechanosensory neuromasts. We explored the relationship between facial bones and neuromasts using in vivo fluorescent colabeling and microcomputed tomography. We found an increase in neuromast density within dermal bone boundaries across three distinct populations of cavefish compared to surface-dwelling fish. We also show that eye loss disrupts early neuromast patterning, which in turn impacts the development of dermal bones. While cavefish exhibit alterations in cranial bone and neuromast patterning, each population varied in the severity. This variation may reflect observed differences in behavior across populations. For instance, a bend in the dorsal region of the skull may expose neuromasts to water flow on the opposite side of the face, enhancing sensory input and spatial mapping in the dark.

A de novo variant in OTX2 in a lamb with otocephaly.

BACKGROUND: Otocephaly is a rare lethal malformation of the first branchial arch. While the knowledge on the causes of otocephaly in animals is limited, different syndromic forms in man are associated with variants of the PRRX1 and OTX2 genes. CASE PRESENTATION: A stillborn male lamb of the Istrian Pramenka sheep breed showed several congenital craniofacial anomalies including microstomia, agnathia, aglossia, and synotia. In addition, the lamb had a cleft palate, a small opening in the ventral neck region, a cystic oesophagus and two hepatic cysts. The brain was normally developed despite the deformed shape of the head. Taken together the findings led to a diagnosis of otocephaly. Whole-genome sequencing was performed from DNA of the affected lamb and both parents revealing a heterozygous single nucleotide variant in the OTX2 gene (Chr7: 71478714G > A). The variant was absent in both parents and therefore due to a de novo mutation event. It was a nonsense variant, XM_015097088.2:c.265C > T; which leads to an early premature stop codon and is predicted to truncate more than 70% of the OTX2 open reading frame (p.Arg89*). CONCLUSIONS: The genetic findings were consistent with the diagnosis of the otocephaly and provide strong evidence that the identified loss-of-function variant is pathogenic due to OTX2 haploinsufficiency. The benefits of trio-based whole-genome sequencing as an emerging tool in veterinary pathology to confirm diagnosis are highlighted.

Dose addition in chemical mixtures inducing craniofacial malformations in zebrafish (Danio rerio) embryos.

A challenge in cumulative risk assessment is to model hazard of mixtures. EFSA proposed to only combine chemicals linked to a defined endpoint, in so-called cumulative assessment groups, and use the dose-addition model as a default to predict combined effects. We investigated the effect of binary mixtures of compounds known to cause craniofacial malformations, by assessing the effect in the head skeleton (M-PQ angle) in 120hpf zebrafish embryos. We combined chemicals with similar mode of action (MOA), i.e. the triazoles cyproconazole, triadimefon and flusilazole; next, reference compounds cyproconazole or triadimefon were combined with dissimilar acting compounds, TCDD, thiram, VPA, prochloraz, fenpropimorph, PFOS, or endosulfan. These mixtures were designed as (near) equipotent combinations of the contributing compounds, in a range of cumulative concentrations. Dose-addition was assessed by evaluation of the overlap of responses of each of the 14 tested binary mixtures with those of the single compounds. All 10 test compounds induced an increase of the M-PQ angle, with varying potency and specificity. Mixture responses as predicted by dose-addition did not deviate from the observed responses, supporting dose-addition as a valid assumption for mixture risk assessment. Importantly, dose-addition was found irrespective of MOA of contributing chemicals.

External and computed tomography analysis of a strophocephalic lamb.

CONTEXT: The Museum of Anatomy and Embryology Louis Deroubaix attached to the Laboratory of Anatomy, Biomecanics and Organogenesis, ULB, Brussels, possesses in its liquid collections a cephalic extremity of a lamb suffering from strophocephaly. The origins have not been determined. The trunk and the limbs are resected. MATERIAL AND METHODS: The piece has been studied and photographed. A volumic computed tomography acquisition has been performed with a Siemens Volume Zoom. For pedagogic and museological purposes, surface reconstructions and 3D printing have been obtained. RESULTS: An otocephaly is observed. Both ears are located in place of the oral cavity. The mandible is welded to the braincase. The eyeballs are close together (synophtalmia) which confirms the presence of a cyclotocephaly. They are surmounted by a rudimentary snout rather than a proboscis. The presence of this muzzle allows the anomaly to be classified as a strophocephaly, a malformation already described in sheeps. CT slices of the brain show a semi-lobar holoprosencephaly with incomplete division of the cerebral hemispheres and ventricules. DISCUSSION AND CONCLUSION: The CT examination allows the facial anomalies to be allocated to a holoprosencephaly. The singularity of this case, compared to the human cyclotocephalies, is the presence of a differentiated muzzle rather than a simple proboscis. The holoprosencephaly is uncomplete. Such anomalies have been associated with an entire absence of cerebral differentiation but with a complete absence of the muzzle. The tridimensional printing represents an interesting educational tool easily transportable in contrast to the original specimen.

Whole genome variant association across 100 dogs identifies a frame shift mutation in DISHEVELLED 2 which contributes to Robinow-like syndrome in Bulldogs and related screw tail dog breeds.

Domestic dog breeds exhibit remarkable morphological variations that result from centuries of artificial selection and breeding. Identifying the genetic changes that contribute to these variations could provide critical insights into the molecular basis of tissue and organismal morphogenesis. Bulldogs, French Bulldogs and Boston Terriers share many morphological and disease-predisposition traits, including brachycephalic skull morphology, widely set eyes and short stature. Unlike other brachycephalic dogs, these breeds also exhibit vertebral malformations that result in a truncated, kinked tail (screw tail). Whole genome sequencing of 100 dogs from 21 breeds identified 12.4 million bi-allelic variants that met inclusion criteria. Whole Genome Association of these variants with the breed defining phenotype of screw tail was performed using 10 cases and 84 controls and identified a frameshift mutation in the WNT pathway gene DISHEVELLED 2 (DVL2) (Chr5: 32195043_32195044del, p = 4.37 X 10-37) as the most strongly associated variant in the canine genome. This DVL2 variant was fixed in Bulldogs and French Bulldogs and had a high allele frequency (0.94) in Boston Terriers. The DVL2 variant segregated with thoracic and caudal vertebral column malformations in a recessive manner with incomplete and variable penetrance for thoracic vertebral malformations between different breeds. Importantly, analogous frameshift mutations in the human DVL1 and DVL3 genes cause Robinow syndrome, a congenital disorder characterized by similar craniofacial, limb and vertebral malformations. Analysis of the canine DVL2 variant protein showed that its ability to undergo WNT-induced phosphorylation is reduced, suggesting that altered WNT signaling may contribute to the Robinow-like syndrome in the screwtail breeds.

A de novo germline mutation of DLX3 in a Brown Swiss calf with tricho-dento-osseus-like syndrome.

OBJECTIVE: A novel congenital disorder affecting a calf was observed, and its phenotype and genetic mutation identified. ANIMAL: A six-month-old female Brown Swiss calf. METHODS: Diagnostic investigation and whole genome sequencing of a case parent trio was performed. RESULTS: The calf had a dull kinky coat with mild hypotrichosis, and teeth with brown staining and enamel defects. Histological examination of skin biopsies was compatible with a follicular dysplasia. Radiography and computed tomography revealed thickening of the skull bones and large pulp cavities with a marked thinning of enamel affecting all teeth. A de novo germline mutation affecting the distal-less homeobox gene (DLX3) was identified. The 10 bp frameshift mutation in exon 3 of the bovine DLX3 gene is predicted to replace the second C-terminal transactivation domain of the wild-type protein by a recoded peptide of 99 amino acids without any sequence similarity. CONCLUSION AND CLINICAL IMPORTANCE: A causative mutation for a sporadic phenotype resembling human tricho-dento-osseous syndrome was identified after detection of a de novo germline mutation in the DLX3 gene.

Cichlid fishes as a model to understand normal and clinical craniofacial variation.

We have made great strides towards understanding the etiology of craniofacial disorders, especially for 'simple' Mendelian traits. However, the facial skeleton is a complex trait, and the full spectrum of genetic, developmental, and environmental factors that contribute to its final geometry remain unresolved. Forward genetic screens are constrained with respect to complex traits due to the types of genes and alleles commonly identified, developmental pleiotropy, and limited information about the impact of environmental interactions. Here, we discuss how studies in an evolutionary model - African cichlid fishes - can complement traditional approaches to understand the genetic and developmental origins of complex shape. Cichlids exhibit an unparalleled range of natural craniofacial morphologies that model normal human variation, and in certain instances mimic human facial dysmorphologies. Moreover, the evolutionary history and genomic architecture of cichlids make them an ideal system to identify the genetic basis of these phenotypes via quantitative trait loci (QTL) mapping and population genomics. Given the molecular conservation of developmental genes and pathways, insights from cichlids are applicable to human facial variation and disease. We review recent work in this system, which has identified lbh as a novel regulator of neural crest cell migration, determined the Wnt and Hedgehog pathways mediate species-specific bone morphologies, and examined how plastic responses to diet modulate adult facial shapes. These studies have not only revealed new roles for existing pathways in craniofacial development, but have identified new genes and mechanisms involved in shaping the craniofacial skeleton. In all, we suggest that combining work in traditional laboratory and evolutionary models offers significant potential to provide a more complete and comprehensive picture of the myriad factors that are involved in the development of complex traits.

Utilizing the chicken as an animal model for human craniofacial ciliopathies.

The chicken has been a particularly useful model for the study of craniofacial development and disease for over a century due to their relatively large size, accessibility, and amenability for classical bead implantation and transplant experiments. Several naturally occurring mutant lines with craniofacial anomalies also exist and have been heavily utilized by developmental biologist for several decades. Two of the most well known lines, talpid(2) (ta(2)) and talpid(3) (ta(3)), represent the first spontaneous mutants to have the causative genes identified. Despite having distinct genetic causes, both mutants have recently been identified as ciliopathic. Excitingly, both of these mutants have been classified as models for human craniofacial ciliopathies: Oral-facial-digital syndrome (ta(2)) and Joubert syndrome (ta(3)). Herein, we review and compare these two models of craniofacial disease and highlight what they have revealed about the molecular and cellular etiology of ciliopathies. Furthermore, we outline how applying classical avian experiments and new technological advances (transgenics and genome editing) with naturally occurring avian mutants can add a tremendous amount to what we currently know about craniofacial ciliopathies.

Brain, Craniofacial, and Dental Lesions of a Free-ranging Gray Wolf (Canis lupus) Implicated in a Human Attack in Minnesota, USA.

We describe significant brain, craniofacial, and dental lesions in a free-ranging wolf (Canis lupus) involved in a human attack. On postmortem examination, the wolf presented asymmetric atrophy and bone remodeling affecting the mandible, incisive, maxilla, lacrimal, palatine, frontal, and ethmoid bones. There was an asymmetrical skeletal malocclusion and dental abnormalities including rotated, malpositioned, partially erupted teeth, and an odontogenic cyst associated with an unerupted canine tooth. Brain changes were bilateral loss and atrophy of extensive cortex regions including olfactory bulb, peduncles, and tract, and the frontal lobe. We highlight the relevance of a thorough postmortem examination of wildlife to elucidate disease-based abnormal behavior as the reason for human-animal conflict.

Investigation of an outbreak of craniofacial deformity in yellow-eyed penguin (Megadyptes antipodes) chicks.

AIM: To investigate an outbreak of severe craniofacial deformity in yellow-eyed penguin (Megadyptes antipodes, hoiho) chicks at a single breeding site on the Otago Peninsula in the South Island of New Zealand. METHODS: Morbidity and mortality of yellow-eyed penguins breeding on the coastal regions of Otago was monitored from November 2008 to March 2009. Dead chicks and unhatched eggs were recovered and examined. Between October and December 2008 32 eggs were recorded at 17 nests in the Okia Reserve. Eleven chicks survived to about 90 days of age, of which eight were found to have moderate to severe craniofacial deformity. The six most severe chicks were subject to euthanasia and examined in detail at necropsy, and the remaining two affected chicks were released to the wild after a period of care in a rehabilitation centre. Post-mortem samples were analysed for inorganic and organic toxins. RESULTS: The six deformed chicks all had severe shortening of the mandible and maxilla by 20-50 mm. The rostral and caudal regions of the skull were approximately 40 and 80% of normal length, respectively. Other, more variable lesions included cross bill deformity, malformed bill keratin, microphthalmia with misshapen scleral ossicles and oral soft tissue excess thought to be secondary to bony malformations. During the same year, mild sporadic bill deformities were also reported in 10 unrelated chicks from >167 chicks at other breeding sites on the southern Otago coast. Concentrations of organic toxins and heavy metals in body tissues from affected chicks were apparently similar to those in unaffected chicks on other beaches. CONCLUSIONS: No cause of this outbreak of craniofacial deformity could be established although the high prevalence at a single site suggests that it was due to an unidentified local teratogen.

Skeletal dysplasia with craniofacial deformity and disproportionate dwarfism in hair sheep of northeastern Brazil.

This paper reports a newly described form of skeletal dysplasia affecting Brazilian hair sheep of the Cabugi breed. This breed is characterized by having a short head and in some cases the animals are smaller and more compact than sheep of similar breeds. Lambs born with craniofacial abnormalities and dwarfism that die at 2-6 months of age are frequent in this breed. In a flock of 68 ewes and three rams of the Cabugi breed, 134 lambs were born over a 4-year period. Of these, 14 (10.4%) had marked cranial abnormalities and dwarfism and died or were humanely destroyed, 43 (32%) had a normal face and 77 (57.5%) had the short face characteristic of the breed. Dwarf lambs were much smaller than normal, with short legs, a domed head with retruded muzzle and protruded mandible, sternal deformities and exophthalmic eyes situated more laterally in the face than normal. Microscopical examination of long bones of the limbs, bones of the base of the skull and vertebrae showed no lesions. Bones from four affected lambs and one control lamb were macerated for morphometric examination. Although the length of the spinal cord was similar, there was disproportionate shortening of the appendicular bones, particularly the distal segments. Thus the disease was defined as a skeletal dysplasia characterized by craniofacial deformity and disproportionate dwarfism. It is suggested that the disease is inherited as an incomplete dominant trait. The shortened face, which is a feature of the Cabugi breed, may represent the heterozygous state and the more severe, often lethal, dwarfism may occur in homozygotes.

The gold standard of dental care: the juvenile horse.

Postpartum evaluation of the foal's head and mouth are performed to detect craniofacial malformations and other congenital defects. Detailed oral examination and diagnostic imaging can provide diagnostic and prognostic information about congenital abnormalities of the mouth or skull. Important abnormalities of foals include wry nose, cleft palate, overbite (parrot mouth), and underbite (monkey mouth, sow mouth). Tumors and cysts can be detected in young horses. In juvenile horses, primary dental care procedures include oral examination, management of sharp enamel points, management of deciduous teeth, and management of wolf teeth. Facial or jaw swellings are also important considerations.

Ovine craniofacial malformation: a morphometrical study.

Craniofacial malformation in 64 sheep was phenotypically described as mandibular distoclusion. Digital radiographs were examined in order to determine the degree of morphological changes in certain bones of the skull. Therefore, laterolateral standardised digital radiographs were used to determine anatomic reference points. Subsequently, five reference lines were defined and 16 linear and seven angular measurements were determined to describe malformations in the bones of the skull. Statistical analysis revealed a significant shortening of the rostral part of the corpus mandibulae and of the ramus mandibulae. However, the molar part of the mandible remained unchanged. These morphological changes caused premolar and molar malocclusion. No further craniofacial abnormalities, such as an elongation of the maxilla or of the incisive bone, were identified. In conclusion, the phenotypically observed mandibular distoclusion is caused by a shortening of specific parts of the mandible. This form of ovine craniofacial malformation is therefore best described as brachygnathia inferior.

Aprosencephaly with otocephaly in a lamb (Ovis aries).

Aprosencephaly is a rare condition in veterinary and human medicine characterized by the complete absence of telencephalon and diencephalon. Some cases are accompanied by a facial dysmorphism designated as otocephaly. A stillborn lamb had splanchnocranial anomalies that were classified by computed tomography, magnetic resonance imaging, and pathologic examination as aprosencephaly and otocephaly. The brain included parts of the cerebellum and brainstem but no telencephalon, diencephalon, or mesencephalon. The cerebellum had a structurally normal cortex with expression of neuronal nuclear antigen in the inner and doublecortin in the outer granular cell layers, as well as an irregularly situated nucleus dentatus. Aprosencephaly with otocephaly has been described in mice with heterozygous mutations in the Otx2 gene; however, no causative polymorphisms were detected in the Otx2 gene region of this lamb.