Cranial Muscles Research Articles

Clinical Experience With Patients With Spasmodic Dysphonia and Primary Meige Syndrome

IntroductionMeige syndrome (MS) is part of the group of segmental cranial dystonias, which affect more than two cranial muscle groups. Specifically, blepharospasm is associated with another cranial dystonia (oromandibular, cervical or laryngeal). The aim of this paper was to report our experience in patients with spasmodic dysphonia (SD) associated with primary MS. Material and methodsA retrospective study involving 8 patients between May 2010 and June 2015. Variables recorded were: age, sex, associated dystonia, electromyographic pattern in laryngeal muscles and treatment given. Outcomes after treatment were assessed using GRBAS(i) scale and VHI-30 questionnaire, always provided by the same examiner. ResultsFifty-six patients with MS were treated in the Neurology Department. Eight patients of 56 were diagnosed with SD (prevalence of 14%). All of our patients had adductor SD. The median age was 71 years. All the patients were treated with intralaryngeal botulinum toxin under electromyographic control. Clinically relevant improvements were found after treatment on both the GRBAS(i) scale and the VHI-30 questionnaire. ConclusionIn the study of SD, we should always rule out an association with MS. From the point of view of otorhinolaryngology, the joint use of the GRBAS(i) scale and the VHI-30 questionnaire are useful, reliable and efficient methods for assessing progress and response to treatment. Laryngeal infiltration under electromyographic control with botulinum toxin is the therapeutic alternative that provides better results. The management of SD associated with MS does not differ from isolated SD.

Musculoskeletal development of the Central African caecilian Idiocranium russeli (Amphibia: Gymnophiona: Indotyphlidae) and its bearing on the re-evolution of larvae in caecilian amphibians

Few detailed accounts of the developmental morphology of caecilian amphibians exist and recent studies have highlighted problems concerning the homology of some skull elements. We investigated the embryonic and post-hatching development of the skeleton and musculature of Idiocranium russeli, a possibly miniaturized caecilian endemic to Cameroon. Idiocranium has been suggested to be direct developing; our results strongly support this hypothesis. The external morphology of different embryonic stages, the ossification sequence, and the configuration of the cranial muscles of I. russeli indicate heterochronic shifts of adult traits into embryonic development, as well as the loss or absence of various larval and metamorphic traits. For example, the tentacle, which plesiomorphically develops during metamorphosis, is already fully developed in late embryos. The maxilla and the palatine, which fuse to form the maxillopalatine (during metamorphosis in most biphasic species), fuse well before hatching. Muscles exclusive to the larva, such as the m. interhyoideus and m. hyomandibularis, are absent during development, whereas adult muscles including the m. genioglossus and m. cephalodorsosubpharyngeus, form during embryonic development. A larval ceratobranchial IV is present and fuses to the ceratobranchial III very early in ontogeny. In its near complete absence of larval traits during development, I. russeli resembles the Indian indotyphlid Gegeneophis ramaswamii; this similarity complicates a straightforward explanation for the re-evolution of free-living larvae in Seychelles indotyphlid caecilians.

A quantitative physical model of the TMS-induced discharge artifacts in EEG.

The combination of Transcranial Magnetic Stimulation (TMS) with Electroencephalography (EEG) exposes the brain’s global response to localized and abrupt stimulations. However, large electric artifacts are induced in the EEG by the TMS, obscuring crucial stages of the brain’s response. Artifact removal is commonly performed by data processing techniques. However, an experimentally verified physical model for the origin and structure of the TMS-induced discharge artifacts, by which these methods can be justified or evaluated, is still lacking. We re-examine the known contribution of the skin in creating the artifacts, and outline a detailed model for the relaxation of the charge accumulated at the electrode-gel-skin interface due to the TMS pulse. We then experimentally validate implications set forth by the model. We find that the artifacts decay like a power law in time rather than the commonly assumed exponential. In fact, the skin creates a power-law decay of order 1 at each electrode, which is turned into a power law of order 2 by the reference electrode. We suggest an artifact removal method based on the model which can be applied from times after the pulse as short as 2 milliseconds onwards to expose the full EEG from the brain. The method can separate the capacitive discharge artifacts from those resulting from cranial muscle activation, demonstrating that the capacitive effect dominates at short times. Overall, our insight into the physical process allows us to accurately access TMS-evoked EEG responses that directly follow the TMS pulse, possibly opening new opportunities in TMS-EEG research.

Resting cranial and upper cervical muscle activity is increased in patients with migraine

ObjectiveTo compare comprehensive measures of scalp-recorded muscle activity in migraineurs and controls. MethodWe used whole-of-head high-density scalp electrical recordings, independent component analysis (ICA) and spectral slope of the derived components, to define muscle (electromyogram-containing) components. After projecting muscle components back to scalp, we quantified scalp spectral power in the frequency range, 52–98 Hz, reflecting muscle activation. We compared healthy subjects (n = 65) and migraineurs during a non-headache period (n = 26). We also examined effects due to migraine severity, gender, scalp-region and task (eyes-closed and eyes-open). We could not examine the effect of pre-ictal versus inter-ictal versus post-ictal as this information was not available in the pre-existing dataset. ResultsThere was more power due to muscle activity (mean ± SEM) in migraineurs than controls (respectively, −13.61 ± 0.44 dB versus −14.73 ± 0.24 dB, p = 0.028). Linear regression showed no relationship between headache frequency and muscle activity in any combination of region and task. There was more power during eyes-open than eyes-closed (respectively, −13.42 ± 0.34 dB versus −14.92 ± 0.34 dB, p = 0.002). ConclusionsThere is an increase in cranial and upper cervical muscle activity in non-ictal migraineurs versus controls. This raises questions of the role of muscle in migraine, and the possible differentiation of non-ictal phases. SignificanceThis provides preliminary evidence to date of possible cranial muscle involvement in migraine.

Temporal changes in macrophage phenotype after peripheral nerve injury

BackgroundMacrophages play a key role in peripheral nerve repair and demonstrate complex phenotypes that are highly dependent on microenvironmental cues.MethodsWe determined temporal changes in macrophage gene expression over time using RNA sequencing after fluorescence-activated cell sorting (FACS) macrophage populations from injured peripheral nerve. We identified key upstream regulators and dominant pathways using ingenuity pathway analysis and confirmed these changes with NanoString technology. We then investigate the effects of extreme polarizers of macrophage phenotype (IL4 and IFNγ) on nerve regeneration. We determined macrophage gene expression in vivo at the site of peripheral nerve injury with NanoString technology, and assessed recovery from sciatic nerve injury by cranial tibial muscle weights and retrograde labeling motor neurons in mice with deletion of IL4 or IFNγ receptors.ResultsWe demonstrate that IL4R and IFNγR deletions provide complementary responses to polarization, and alter expression of genes associated with angiogenesis and axonal extension, but do not influence recovery from peripheral nerve transection at 8 weeks after repair.ConclusionsOverall, this study provides a framework to evaluate the phenotype of macrophages over time, and provides a broader and more precise assessment of gene expression changes than has previously been commonly used. This data suggests ways in which polarization may be modulated to improve repair.

Anatomy and physiology of swallowing process

Food intake and swallowing are complicated and intriguing series of movements involving voluntary and involuntary activities of cranial and spinal nerves and muscles. They have two most important functions, that is, food passage from the oral cavity to stomach and airway protection. Tongue, buccinators, and hyoid bone and its muscular attachments are anatomic structures for swallowing of special interests. The swallowing process of liquid is commonly divided into oral preparatory, oral propulsive, pharyngeal, and esophageal stages according to the location of the bolus. The movement of the food in the oral cavity and to the oropharynx differs between eating solid food and drinking liquid.

Complex pattern of variation in neurocranial ontogeny revealed by CT-scanning.

The neurocranium of hominid species has been largely studied with reference to the midsagittal plane, with variations being attributed to brain evolution. By contrast, there is limited information on variation in non-midsagittal regions, which are the points of insertion of muscles and bony structures related to mastication. This work aims to analyze ontogenetic changes and sexual dimorphism (SD) in midsagittal and non-midsagittal neurocranial structures from a contemporary human sample comprising 138 computed tomography (CT) cranial images of individuals ranging from infants to adults. Morphology of the vault and the base was assessed by registering landmarks and semilandmarks, which were analyzed by geometric morphometrics, and the endocranial volume (EV). The results of regressions and Kruskal-Wallis test indicate that the major size and shape changes in both midsagittal and non-midsagittal regions occur during infancy and juvenility; shape changes are also associated with an increase in EV. The size of the midsagittal vault, the shape of the non-midsagittal vault and the size of the base show an extension of ontogenetic trajectories. Sexes show similar changes in shape but different changes in size. We conclude that brain growth appears to be an important factor influencing the morphology of the neurocranium, at least during infancy and childhood. Subsequent changes may be attributed to osteogenic activity and the differential growth of the brain lobes. Masticatory-related bony structures and muscles may not be strong enough factors to induce independent modifications in non-midsagittal structures. The small influence of the cranial muscles would explain why the human neurocranium is a quite integrated structure.

Experiencia clínica en pacientes con síndrome de Meige primario y disfonía espasmódica

IntroductionMeige syndrome (MS) is part of the group of segmental cranial dystonias, which affect more than two cranial muscle groups. Specifically, blepharospasm is associated with another cranial dystonia (oromandibular, cervical or laryngeal). The aim of this paper was to report our experience in patients with spasmodic dysphonia (SD) associated with primary MS. Material and methodsA retrospective study involving 8 patients between May 2010 and June 2015. Variables recorded were: age, sex, associated dystonia, electromyographic pattern in laryngeal muscles and treatment given. Outcomes after treatment were assessed using GRBAS(i) scale and VHI-30 questionnaire, always provided by the same examiner. ResultsFifty-six patients with MS were treated in the Neurology Department. Eight patients of 56 were diagnosed with SD (prevalence of 14%). All of our patients had adductor SD. The median age was 71years. All the patients were treated with intralaryngeal botulinum toxin under electromyographic control. Clinically relevant improvements were found after treatment on both the GRBAS(i) scale and the VHI-30 questionnaire. ConclusionIn the study of SD, we should always rule out an association with MS. From the point of view of otorhinolaryngology, the joint use of the GRBAS(i) scale and the VHI-30 questionnaire are useful, reliable and efficient methods for assessing progress and response to treatment. Laryngeal infiltration under electromyographic control with botulinum toxin is the therapeutic alternative that provides better results. The management of SD associated with MS does not differ from isolated SD.

Editors' note: Teaching Video NeuroImages: Olivary enlargement and pharyngeal nystagmus

In “Teaching Video NeuroImages: Olivary enlargement and pharyngeal nystagmus,” authors Finlay and Yacovino described a 77-year-old woman with a pontine cavernoma and rhythmic contractions of the soft palate and upper larynx on videofluoroscopy. The accompanying MRI showed right inferior olivary nucleus hypertrophy. Dr. Bhattacharjee comments that the simultaneous palate–pharyngeal tremor may be due to their common innervation by the vagus nerve. He also asks about the interval between the cavernoma expansion, the radiologic signal changes, and the patient's symptoms. Authors Finlay and Yacovino clarify that the olivary hypertrophy was present on the patient's initial MRI and that the palatal tremor was diagnosed 36 months after that study. They also note that synchronic contraction additionally was seen in the bilateral eyelids in their patient, which does not support Dr. Bhattacharjee's theory of common vagus nerve innervation. Dr. Mendez-Guerrero suggests that the term pharyngeal nystagmus, coined a century ago, may be misleading as a central nystagmus now infers a vestibular nuclei or vestibular pathway impairment, which is not present in this disorder. He proposes using the term pharyngeal/laryngeal myorhythmia, denoting a slow (2–3 Hz) rhythmic movement disorder of the cranial musculature. The authors expand on the terminology mentioned by Dr. Mendez-Guerrero and on their use of Spencer's original nomenclature. In “Teaching Video NeuroImages: Olivary enlargement and pharyngeal nystagmus,” authors Finlay and Yacovino described a 77-year-old woman with a pontine cavernoma and rhythmic contractions of the soft palate and upper larynx on videofluoroscopy. The accompanying MRI showed right inferior olivary nucleus hypertrophy.

Reader response: Teaching Video NeuroImages: Olivary enlargement and pharyngeal nystagmus.

We noted with interest the Teaching Video NeuroImage by Drs. Finlay and Yacovino.1 The authors examined the phenomenology of a rare slow movement disorder of pharynx and larynx due to a brainstem cavernoma using videofluoroscopy. The term pharyngeal nystagmus, beyond the initial description more than a century ago,2 may mislead readers. Central nystagmus implies a vestibular nuclei or vestibular pathway impairment3 that are not related to this movement disorder. Hence, pharyngeal nystagmus was known as palatine myoclonus/tremor, in which the dentate-rubro-olivary tract is commonly involved.4 This term is also problematic due to its phenomenologic differences with true myoclonus. Currently, this movement disorder is better considered part of the spectrum of myorhythmias: slow (2–3 Hz) rhythmic movement disorders of cranial musculature or limbs.5 We suggest that the term pharyngeal nystagmus be avoided and replaced by pharyngeal/laryngeal myorhythmia.

High-Yield Purification, Preservation, and Serial Transplantation of Human Satellite Cells.

SummaryInvestigation of human muscle regeneration requires robust methods to purify and transplant muscle stem and progenitor cells that collectively constitute the human satellite cell (HuSC) pool. Existing approaches have yet to make HuSCs widely accessible for researchers, and as a result human muscle stem cell research has advanced slowly. Here, we describe a robust and predictable HuSC purification process that is effective for each human skeletal muscle tested and the development of storage protocols and transplantation models in dystrophin-deficient and wild-type recipients. Enzymatic digestion, magnetic column depletion, and 6-marker flow-cytometric purification enable separation of 104 highly enriched HuSCs per gram of muscle. Cryostorage of HuSCs preserves viability, phenotype, and transplantation potential. Development of enhanced and species-specific transplantation protocols enabled serial HuSC xenotransplantation and recovery. These protocols and models provide an accessible system for basic and translational investigation and clinical development of HuSCs.

Tongue exercise and ageing effects on morphological and biochemical properties of the posterior digastric and temporalis muscles in a Fischer 344 Brown Norway rat model

ObjectiveThis study sought to examin effects of age and tongue exercise on the posterior digastric (opener) and the temporalis (closer). We hypothesized 1) age would result in differing morphological (cross sectional area) and biochemical (myosin heavy chain isoform) components of these muscles; 2) tongue exercise would result in coactivation of these muscles inducing a decrease in age-related differences between age groups. DesignYoung adult (9 months) and old (32 months) Fischer 344 Brown Norway rats were randomized into a tongue exercise or control group. Post-training, posterior digastric and temporalis muscles were harvested and analyzed using: 1) Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) to assess percent myosin heavy chain (MyHC) content; 2) Immunohistochemical staining to determine cross sectional area (CSA). ResultsA larger proportion of slowly contracting MyHC isoforms in the posterior digastric and temporalis muscles were found in old. No significant main effects for age or exercise in fiber size were found in posterior digastric muscle. An interaction between age and exercise for temporalis cross sectional area indicated the old exercise group had smaller average cross sectional area than all other groups. Conclusions findingssuggest that: 1) Increasing age induces biochemical changes in muscles of the jaw, specifically showing an increase the proportion of slower contracting MyHC isoforms; 2) Increasing age and tongue exercise induce a reduction in muscle fiber cross sectional area in the temporalis muscle only. However, continued study of these cranial muscle systems is warranted to better understand these changes that occur with age and exercise.

EEG-like signals can be synthesized from surface representations of single motor units of facial muscles

Electrodes for recording electroencephalogram (EEG) are placed on or around cranial muscles; hence, their electrical activity may contaminate the EEG signal even at rest conditions. Due to its role in maintaining mandibular posture, tonic activity of temporalis muscle interferes with the EEG signal particularly at fronto-temporal locations at single motor unit (SMU) level. By obtaining surface representation of a motor unit, we can evaluate its interference in EEG and if we could sum surface representations of several tonically active motor units, we could estimate the overall myogenic contamination in EEG. Therefore, in this study, we followed re-composition (RC) approach and generated EEG-like artefact model using surface representations of single motor units (RC). Furthermore, we compared signal characteristics of RC signals with simultaneously recorded EEG signal at different locations in terms of power spectral density and coherence. First, we found that RC signal represented the power spectral distribution of an EMG signal. Second, RC signal reflected the discharge rate of a SMU giving the greatest surface representation amplitude and strongest interference appeared as distinguishable frequency peak on RC power spectra. Moreover, for strong interferences, RC also contaminated the EEG at F7 and other EEG electrodes. These findings are important to illustrate the susceptibility of EEG signal to myogenic artefacts even at rest and the research using EEG coherence comparisons should consider muscle activity while drawing conclusions about neuronal interactions and oscillations.

Why Muscles Look and Act the Way They Do: Uncovering the Nexus Among Form, Function, Behavior, and Evolution.

Why Muscles Look and Act the Way They Do: Uncovering the Nexus Among Form, Function, Behavior, and Evolution.

Behavioral Correlates of Cranial Muscle Functional Morphology.

This issue of the Anatomical Record is the first of a two-volume set that focuses on new investigations into behavioral correlates of muscle functional morphology. Much of the research on functional morphology and adaptation to specific functional niches focuses on the shapes of hard-tissues-bones and teeth. Investigations into soft-tissue anatomy tend to be predominantly descriptive with only brief allusion to ontogenetic or evolutionary origins of structures. When muscles are included in analyses of functional systems, their function tends to be oversimplified-usually considered a simple force vector connecting two osteological points, with the force treated as a constant derived from some simple calculation of muscle size. The goal of these special issues is to present a series of studies that take a more elaborate look at how muscles can be viewed from a functional perspective in studies searching for morphological correlates of behavior. This first volume focuses on the behavioral correlates of cranial muscles-starting with a paper about the mimetic musculature of primates and ending with a series of papers on the masticatory muscles of many lineages of vertebrates. The next issue of the Anatomical Record (March 2018) includes our papers on the behavioral correlates of postcranial muscles. Taken together, we hope you agree that this series presents valuable insights into these form/function relationships using both traditional approaches+ and cutting-edge techniques. Anat Rec, 301:197-201, 2018. © 2018 Wiley Periodicals, Inc.

Three-dimensional movements of the pectoral fin during yaw turns in the Pacific spiny dogfish, Squalus suckleyi.

ABSTRACTFish pectoral fins move in complex ways, acting as control surfaces to affect force balance during swimming and maneuvering. Though objectively less dynamic than their actinopterygian relatives, shark pectoral fins undergo complex conformational changes and movements during maneuvering. Asynchronous pectoral fin movement is documented during yaw turning in at least two shark species but the three-dimensional (3D) rotation of the fin about the body axes is unknown. We quantify the 3D actuation of the pectoral fin base relative to the body axes. We hypothesized that Pacific spiny dogfish rotate pectoral fins with three degrees of freedom relative to the body during volitional turning. The pectoral fin on the inside of the turn is consistently protracted, supinated and depressed. Additionally, turning angular velocity increased with increasing fin rotation. Estimated drag on the fin increased and the shark decelerated during turning. Based on these findings, we propose that Pacific spiny dogfish uses drag-based turning during volitional swimming. Post-mortem muscle stimulation revealed depression, protraction and supination of the pectoral fin through stimulation of the ventral and cranial pterygoideus muscles. These data confirm functional hypotheses about pectoral fin musculature and suggest that Pacific spiny dogfish actively rotate pectoral fins to facilitate drag-based turning.This article has an associated First Person interview with the first author of the paper.

Disgenesia troncoencefálica: más allá del síndrome de Moebius

Brainstem dysgenesis designates all those patients with congenital dysfunction of cranial nerves and muscle tone due to prenatal lesions or malformations of the brainstem. This generic term has the advantage over the eponyms Moebius 'expanded' or 'unrestricted', Robin, Cogan or Carey-Fineman-Ziter syndromes in that it has a less restrictive view and provides a frame work that enables a systematic approach to diagnosis and research of most developmental disorders involving the brainstem. The review of the literature and our experience shows that infants with a predominant rombencephalic involvement are due to brainstem prenatal disruptive vascular accidents, while cases with midbrain and cerebellar involvement and widespread malformative syndromes have most likely an underlying genetic cause. Due to phenotypic heterogeneity associated with brainstem dysgenesis, it is crucial to evaluate each case individually and to establish a specific therapeutic plan. Intervention programs should start soon after diagnosis and directed to improve functions needed for daily life activities. Even though the prognosis of patients with brainstem dysgenesis due to prenatal destructive lesions depends on the magnitude of the vascular territory involved, in most patients with brainstem dysgenesis, the prognosis is better than the initial clinical manifestations would indicate.

Cephalic muscle development in the Australian lungfish, Neoceratodus forsteri.

Lungfishes are the extant sister group of tetrapods. As such, they are important for the study of evolutionary processes involved in the water to land transition of vertebrates. The evolution of a true neck, that is, the complete separation of the pectoral girdle from the cranium, is one of the most intriguing morphological transitions known among vertebrates. Other salient changes involve new adaptations for terrestrial feeding, which involves both the cranium and its associated musculature. Historically, the cranium has been extensively investigated, but the development of the cranial muscles much less so. Here, we present a detailed study of cephalic muscle development in the Australian lungfish, Neoceratodus forsteri, which is considered to be the sister taxon to all other extant lungfishes. Neoceratodus shows several developmental patterns previously described in other taxa; the tendency of muscles to develop from anterior to posterior, from their region of origin toward insertion, and from lateral to ventral/medial (outside-in), at least in the branchial arches. The m.protractor pectoralis appears to develop as an extension of the most posterior m.levatores arcuum branchialium, supporting the hypothesis that the m.cucullaris and its derivatives (protractor pectoralis, levatores arcuum branchialium) are branchial muscles. We present a new hypothesis regarding the homology of the ventral branchial arch muscles (subarcualis recti and obliqui, transversi ventrales) in lungfishes and amphibians. Moreover, the morphology and development of the cephalic muscles confirms that extant lungfishes are neotenic and have been strongly influenced via paedomorphosis during their evolutionary history.

A new exceptionally preserved specimen of Dracaenosaurus (Squamata, Lacertidae) from the Oligocene of France as revealed by micro-computed tomography

ABSTRACTThe best-preserved material of Dracaenosaurus croizeti, an almost complete and previously unpublished skull with a few associated postcranial bones (stylopodium, zeugopodium, and cervical vertebra), is described. The material comes from the locality of Cournon, a late Oligocene site in south-central France. Micro-computed tomography applied to this specimen revealed previously unknown internal osteological characters. Among lacertids, this taxon represents a notable phenomenon: it is an extreme durophagous specialist. Many of the newly observed cranial character states reflect the lifestyle of this lizard, because animals with a hard-shelled diet display a specialized cranial morphology associated with more massive cranial muscles. One unique character for Lacertidae is observed: the parietal-supraoccipital contact is formed by a ventrally deep parietal crest that fits into a bifurcate ascending process of the supraoccipital. In fact, such a connection represents the opposite to the connection in modern members of Lacertidae. Phylogenetic analysis recovered Dracaenosaurus inside Gallotiinae, a clade that would consist of the mainly Oligocene genera Pseudeumeces and Dracaenosaurus, the Miocene genus Janosikia, and the extant Psammodromus and Gallotia. Our study supports previous phylogenetic results and provides an example of the achievement of large size in mainland members of the stem of Gallotia, previously exemplified by Janosikia and Pseudeumeces. The extreme amblyodonty of Dracaenosaurus also confirms the view that herbivory in Gallotia is derived and may be the result of insularity.SUPPLEMENTAL DATA—Supplemental materials are available for this article for free at www.tandfonline.com/UJVPCitation for this article: Čerňanský, A., A. Bolet, J. Müller, J.-C. Rage, M. Augé, and A. Herrel. 2017. A new exceptionally preserved specimen of Dracaenosaurus (Squamata, Lacertidae) from the Oligocene of France as revealed by micro-computed tomography. Journal of Vertebrate Paleontology. DOI: 10.1080/02724634.2017.1384738.

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.