Endothelin B Receptor Mutant Exhibits Craniofacial Dysmorphology Resembling Domestication Syndrome


 Background: ETB-/- mutation is a major cause of HSCR, a neurocristopathy known for its enteric nervous system failure. Other than regulating ENCC migration, ETB mediates ET-1 clearance. Consequently, ETB may indirectly affect ET-1/ETA signaling, which controls CNCC migration and craniofacial development. Interestingly, it was hypothesized that “domestication syndrome” arise from changes in neural crest determining genes, including ETA and ETB. While ETA-/- animals are known to suffer severe dysmorphology resembling CATCH22 syndrome, we hypothesize that sl/sl rat, an ETB-/- HSCR model animal, may exhibit subtle craniofacial changes through indirect control. These features may share resemblance to those of domestication syndrome. Methods: Ten rat pups with an average age of 88 hours were anaesthetized with 5% isoflurane and culled via exsanguination. Tail tips were removed for genotyping. Head tissue were stained in 1.5% iodine for two weeks prior to micro-CT scanning. In vivo micro-CT scanning of cranial specimen was performed followed by ex vivo micro-CT scanning of 2 samples for image quality control. 3D visualization and analyses were performed using open-source program, Drishti. Cephalometric measurements were made based on selected craniofacial landmarks. Comparisons were made between sl/sl rats and the control group, which consisted of wild-type and heterozygotes. Results: Subtle reductions in facial measurements were seen in sl/sl rats when compared with the control group, ranging from 1.4% to 15%. These changes were observed in cranial, maxillary and mandibular parameters: total skull length, nasal length, nasal width, nasal cavity width, interorbital width, interlens distance, inner and outer canthal distance, maximal skull height, cranial length, intracranial length and width, interorbital width, and interzygomatic width. Consistently, craniofacial ratio indices showed sl/sl rat has a flatter cranium (skull height/skull length: 0.393 vs 0.413) and a shorter but broader nose (nasal-width/nasal-length: 0.794 vs 0.874). Additionally, subtle dystopia canthorum may be presented in sl/sl rat based on increased W index. While there was no discrepancy in dental number and morphology between the control and sl/sl groups, dimensional difference was detected. Conclusions: This study demonstrated subtle craniofacial changes are presented in ETB-/- HSCR model, supporting the idea that ETB regulates CNCC migration. The findings also implicate HSCR patient may have predisposing risks for conditions such as obstructive sleep apnea, cleft palate, or dental malocclusion. Lastly, these changes share resemblance with described domestication syndrome, supporting NCC-determining gene, ETB, may play a role in the formation of domestication.

and paraxial mesoderm-derived arch core acts as a chemoattractant for the ET A -expressing ectomesenchymal cells to appropriate position prior to end-organ differentiation (12). Therefore, disruptions or hypostimulations in ET-1/ECE-1/ET A pathway leads to CNCC migration failure and severe craniofacial defects ultimately result in mechanical asphyxia post birth (12,14,15). Additionally, the ectomesenchymal cells originated from cardiac neural crest cells (CaNCC) migrate to pharyngeal arches 3, 4, and 6 to form the aortic pulmonary system; disruptions in the ET-1/ET A system can thus cause ventricular septal defects and aortic hypoplasia or interruptions (14).
Interestingly, the multi-genetic "domestication syndrome" also exhibits mild variant craniofacial dysmorphology seen in velocardiofacial syndrome and Waardenburg syndrome (WS); both of which arise from mutations in the endothelin system (16,17). Domestication syndrome was a concept rst introduced by Charles Darwin in 1868 to describe a range of behavioral, morphological and physiological traits difference between the domesticated animals and their wild forbearers (18). Since then, the genetics and implications of this have been the focus of extensive researches (19). The domestication syndrome refers to a combination of traits observed in domesticated animals including increased docility, tameness and prolonged juvenile behavior, depigmentation, ear and tail form, shorter nose, reduced tooth size, alterations in adrenocorticotropic hormone levels, altered concentrations of several neurotransmitters, and a reduction in total brain size and particular brain regions (19)(20)(21). Many of the similar characteristics are also observed in HSCR-associated syndromes, suggesting potential common etiology. Indeed, recent researches have suggested that features of domestication may arise from alterations in neural crest cells and neural crest determining genes, including RET, GDNF, SOX10, MITF, PAX3, ET-1, ET-3, ET A , and ET B genes, all of which play crucial roles in neural crest cell speci cation, migration, and post-migratory interaction (10,(22)(23)(24)(25)(26)(27)(28). Consistently, animals with these dysfunctional genes may display a range of abnormalities, ranging from mild domestication syndrome to debilitating or lethal neurocristopathy such as HSCR or Waardenburg syndromes (19,27).
Although we do not expect ET B -knockout mutants to exhibit gross dysmorphic features from the migration failure of cephalic neural crest cell (CNCC) as seen in ET-1/ET A mutants, we ponder the potential impact of elevated ET-1 on the development of HSCR subjects. To study the potential changes associated with HSCR, we adopted spotting-lethal (sl/sl) rat as our study model. sl/sl rat is an autosomal recessive ET B −/− mutant with high adherence to Mendelian inheritance.
Pathogenically, sl/sl rat carries a naturally-occurring 301 base-pair deletions in ET B gene resulting ET-3/ET B signaling dysfunction (9). Phenotypically, sl/sl rat exhibits features resembling HSCR and WS-IV patients including: histologically-con rmed aganglionic bowel, pigmentation defects, and hearing de cits (9,29). Based on the colony data of 475 rats, we observed high genetic penetrance of intestinal aganglionosis in up to 95% of homozygous sl/sl rats. Additionally, Gariepy et al (2000) showed sl/sl rats having approximately six-times higher ET-1 level than that of wild-type rats due to impaired clearance from the absence of functional ET B (30). This was also supported by the consistent nding that ET B inhibition by BQ-788 caused signi cant elevation of ET-1 levels (31). Consequently, we suspect potential developmental changes in craniofacial features to occur in sl/sl rats due to elevated ET-1 levels and subsequent changes in ET A -mediated signaling.
Given sl/sl rat is a single-gene HSCR model with high genetic penetrance, changes observed in mutants can provide further clari cations to the ET B functions and potential impairments associated with HSCR.
In addition to skin depigmentation, we ponder sl/sl rats may display similar characteristics of domestications, including craniofacial changes.
This study aims to extrapolate the impact of ET B on the craniofacial morphology by quantitatively comparing the external morphology of sl/sl rat to that of control group. All measurements are made on three-dimensional (3-D) rendering of structurally preserved micro-CT data. We hypothesized that sl/sl rats may possess altered craniofacial morphology resembling domestication features, including smaller a shorter and atter face.

Compliance with Ethical practice
All animal tissues used in this study were handled with strict compliance to ACT Health Human Research Ethics Committee (ACTH-HREC) and Australian National University Animal Experimentation Ethics Committee (ANU-AEEC), project number A2011/67. Additionally, this report is reported in accordance with ARRIVE guidelines.

Tissue preparation and staining.
Ten rat pups with an average age of 88 hours were used this study. They were derived from the crossbreeding between the heterozygous carriers of the ET B mutation. Their coat pattern, age, gender, weight, and colonic appearance were recorded prior to culling. The culling process involved overanesthetizing the rats with 5% iso urane followed by thoracotomy and abdominal aortomy. Fivemillimeter tail tips of each rat were resected and stored for subsequent genotyping.
To facilitate successful micro-CT scanning with adequate tissue differentiation, diffusion staining with iodine solution was performed prior to scanning. A rhomboid-shaped craniotomy of ve-millimeter in diameter was created on parietal bones to facilitate tissue staining. The rat samples were rst washed in 10% PBS solution for 30 minutes followed by 4% formalin xation for 24 hours. Next, formalin was replaced by progressive ethanol (EtOH) series: 20%, 50%, 70% and 90% for 24 hours each to replace the formalin. Finally, the ethanol-xed rat bodies were stained in 1.5% iodine solution for a minimum of 7 days prior to micro-CT scanning (32).

Micro-CT Scanning
We have chosen micro-CT scanning for its high image-resolution power to secure accurate and detailed anatomical information. The image scanning was performed using a commercially available in vivo micro-CT scanner (Caliper Quantum FX) followed by a validation scan with a custom-built ex vivo micro-CT system at the Department of Applied Mathematics of Australian National University (ANU). Due to its limited accesses, the latter was only used for image quality control. The terminology of "in vivo" and "ex vivo" described here are not to be confused with their standard de nitions in biomedical science but rather as descriptions of micro-CT system setups (29). The in vivo micro-CT system consists of a stationary sample positioned in between a rotational system of X-ray source and detector with the aim to reduce radiation exposure while acquiring satisfactory images. On the contrary, the ex vivo micro-CT system positions a rotating sample in between an adjustable X-ray source and a detector for maximal magni cation and image data with the high signal-to-noise ratios.
Genotyping was performed with following protocols. Cells of the isolated ve-millimeter rat-tail segments were lysed using Proteinase K in lysis buffer, which consisted of 100mM Tris pH 8, 5mM EDTA, 0.2% SDS, 200mM NaCl in distilled water. DNAs were extracted via standard protocols: vortex heating, supernatant separation via centrifuging, and washing and drying DNA pellet with 70% EtOH. The extracted DNA was quanti ed with spectrophotometry.
Next, PCR was completed using "Master-Mix" reagents composed of 10*PCR buffer, Qiagen-contained

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A selection of cranial, maxillary, mandibular, and dental structures were chosen for analysis. The anatomical landmarks in this study were made based on anthropometric and cephalometric references in previous studies (35)(36)(37)(38). These descriptions were outlined in Fig. 1 and Table 1. Based on these craniofacial landmarks, direct dimensional measurements, as outlined in Fig. 2 and Table 2, were completed using Drishti (33). Table 1: Description of cephalometric landmarks selected for dimensional analysis.
*: refers to labelling shown in Figure 1; (): same landmarks visualized in different planes.  Distance between the occipital point (4) and nasofrontal point (2) following the curvature of the skull.

External Nasal Width
Distance between the two lateral nasal points (5) 5 Inner intercanthal distance Distance between the inner canthal points (6) 6 External interorbital width Distance between the two orbital points (8) 7 Outer intercanthal distance Distance between the outer canthal points (7) Sagittal Cranial Cephalometric Measurements Distance between occipital point 2 (11) and posterior nasal spine point (10) 11

Nasal cavity Length
Distance between the posterior nasal spine (10) and the intranasal point (1) 12

Nasal cavity Width
Distance between the two internal nasal points (12) 13 Interlens distance Distance between the two external lens points (13) 14

Intracranial width
Distance between the two temporal points (14) 15 Orbit length Distance between the superior orbital point (15) and the inferior orbital points (17) 16 Orbit Width Distance between the occipital point (8) and the lateral occipital point (16).

Orbit Circumference
Distance measured between the superior, lateral, inferior and medial orbit points, following the border of the orbit. The angle at the gnathion point (30) bounded by the Gonion point (29) and the inferior incisive alveolar point (31) *: refers to numbers listed in Figure 2.
(): bracketed numbers in the measurement description refer to anatomical landmarks described in Table 1 and Figure 1.
Cephalometric points were identi ed in several cross-sectional planes. Surface measurements were obtained from the external morphology of the rat head. Maximal skull height and incisor measurements were taken in the midsagittal plane while mandibular measurements were taken in the parasagittal plane passing through the buccal mucosa. Axial and maxillary measurements were obtained in the para-axial planes where the points of maximal lens protrusion and the midpoint of the philtrum were found, respectively. Based on these direct measurements, a selection of ratio indices was calculated to assess the proportional changes in the craniofacial features. These indices include W index, nasal length/skull length ratio, nasal width/nasal length ratio, intracranial width/intracranial length ratio, skull height/skull length ratio, maxillary width/maxillary length ratio, mandibular length/skull length ratio, mandibular corpus-height/mandibular corpus-length ratio, and mandibular ramus/mandibular length ratio.
ET B mutation has been known to associate with WS-II and WS-IVa (39). A key clinical characteristic of WS-I is the presence of dystopia canthorum (16). To assess the presence of this phenotypic feature, the W formula, as de ned for human WS, was applied (Eq. 1) to model animal measurements. As the eyelids of the neonatal rats were still fused, lens distance was used as a substitute for papillary distance.

Statistical Analysis
A total of ten rats were included in analysis and divided into a control and sl/sl group. The control group was composed of two wild-type (ET B +/+ ) and three heterozygous (ET B +/− ) rat pups and ve spotting lethal (ET B −/− ) rats. Data was assessed for normality and Student's t test was applied for comparison between the control and mutant groups. A result was considered statistically signi cant at p-value ≤ 0.05. Analysis was conducted using SPSS software.

Measures of statistical error
To ensure validity of the study, researchers undertaking measurements were blinded to the genotype of rats until nalization of data analysis. To ensure accuracy and reproducibility, all measurements were repeated by the same researcher two weeks after the initial analysis. The difference between the two measures was determined with random error calculation, Dahlberg's error (36,41). The Dahlberg statistic reported the average amount of disparity between the measurement sessions.

Results
The basic parameters of the rat pups were listed in Table 3. Control and sl/sl groups have respective average body-weights of 13.16 ± 0.81g and 11.40 ± 1.62g, p-value = 0.063, translating to a difference of 13%. There was a positive correlation between body-weight and the cephalometric measurements, with the control group generally having larger craniofacial measures than those of sl/sl rats, apart from average inferior incisor length, Tables 4 and 5. In addition to this positive correlation, sl/sl rat exhibited several morphological features different from the control group, as revealed by dimensional-ratio index, Table 6.

Cranial Measures
Fifteen craniofacial dimensions were taken in the surface, parasagittal, and axial planes to assess cranial structures, as reported in Table 4. The control rats have detectably larger, ranging from 1.5-14%, cranial dimensions than sl/sl rats in the following parameters: total skull length, nasal length, nasal width, nasal cavity width, interorbital width, interlens distance, inner and outer canthal distance, maximal skull height, cranial length, intracranial length and width, interorbital width, and interzygomatic width. While statistically signi cance was only found in the respective comparison of external nasal lengths and interlens distance between two groups, the trend of sl/sl having smaller features has been persistent across all ndings measured in various planes.

Orbital Measures
Seven measures were taken to assess the orbital size and positioning in the context of the face, Table 5. Orbital dimensions were taken from both eyes followed by averaging these ndings for comparison.
While the control rats exhibited larger orbits than the sl/sl rats in terms of length, width, and circumference, these differences were small, ranging from 0.03 to 3.12%. On the other hand, statistical signi cantly shorter interlens distance was noted in the sl/sl rats, 6.74% smaller with p-value = 0.015, suggesting medial displacement of the lens.

Maxillary measures
Six measures were made to assess changes in maxillary structure associated with sl/sl mutants. Maxillary dimensions of sl/sl rats group were 4.53-5.45% smaller than the control group, Table 5. This was re ected by the measurements of maxillary length, maxillary width and incisive foramen width, albeit statistical signi cance was not achieved. On the other hand, statistically signi cant size-reductions associated with sl/sl rat were seen in U6 intermolar width, U8 intermolar width, and molar plate length. These observations suggested sl/sl rats having a smaller mouth and a narrower mid-face in comparison to the control group.

Mandibular measures
To assess the lower facial changes associated with ET B mutation, ve mandibular measures were compared, including mandibular length, ramus height, corpus length, and corpus height, Table 5. While these measurements showed a general trend of subtle size-reduction in sl/sl rats, ranging from 1.69-2.91%, none of these measures reached statistical signi cance. Furthermore, comparison of mandibular plane angle showed little difference between the control and sl/sl groups. These ndings suggested ET B mutation may have little impact on mandibular development.

Dental measures
Because dental development is dependent on CNCC migration, a process partially regulated by ET B signaling, dentitions of sl/sl rat were examined. As shown by Table 5, there was no discrepancy in terms of dentition number between the control and sl/sl groups. Additionally, we detected no difference in dental morphology between the two groups. On the other hand, the superior incisor length of sl/sl mutant was 10.83% smaller than the control rat while the opposite trend was true for inferior incisor length, 6.11% larger. While these ndings did not reach strict statistical signi cance, p-value < 0.05, they were consistent with the change patterns observed in the maxillary and mandibular measurements.

Comparison of Craniofacial indices
Ratio indices were calculated to determine the dimensional proportionality of individual craniofacial features.
Cranial dimensional ratios were rst compared, as shown by Table 6. While there was no difference in the intracranial-width/intracranial-length ratio between the control and sl/sl groups, the latter has smaller skull height/skull length ratio, indicating the presence of a atter head. Additionally, comparisons made on the indices of nasal length/skull length and nasal width/nasal length indices demonstrated a disproportionally shorter but wider nose in sl/sl rat. Furthermore, while the absolute maxillary and mandibular measurements were smaller in sl/sl group, its respective dimensional indices were similar to those of control group, Table 6, suggesting proportional changes.
Lastly, the W index was used to assess for the presence of dystopia canthorum, a major clinical sign of WS, occurring in up to 98% of WS-I. The W index of sl/sl rat was found to be greater than that of control group, 3.328 versus 3.245 with p-value = 0.017, Table 6.

Discussion
HSCR has traditionally been regarded as a single-organ disease despite multi-genetic involvement. ET B mutation is one of the major causes for HSCR. While ET B is known to exert direct control on ENCC migration through ET-3/ET B signaling, we found dysfunctional ET B can also lead to subtle craniofacial changes with resemblance to those of "domestication syndrome." These similarities include depigmentation, a atter head, a shorter but broader nose, and smaller teeth (19). Our nding therefore supports the long-held hypothesis that genetic alterations in the development of NCCs contribute, at least partially, to the formation of domestication syndrome (19). The culprit, in this instance, is the ET B mutation.
In this study, we reported craniofacial changes found in sl/sl rat, an autosomal recessive ET B −/− HSCR model with high genetic penetrance. While there was no direct signaling linkage between ET B and the development of CNCC documented in current literature, we found sl/sl rats exhibit reduced cranial, nasal, maxillary, and mandibular dimensions. As shown by Tables 4 and 5, sl/sl rat exhibited a smaller midface, a narrowed and shortened molar bed, a shortened muzzle, and a attened cranium when comparing to those of the control group. Albeit small, ranging from 5-15%, these observed changes in conjunction with previously described depigmentation and reduced adrenal size (Lopez Dee, unpublished data) of sl/sl rat support the notion that ET B may act as a mediator for domestication syndrome.
Additionally, dental changes resembling domestication were observed in sl/sl rats. Indeed, the incisor size was different between the sl/sl and control rats; superior incisor length was ~ 10% smaller whereas inferior incisor length was ~ 6% larger in the sl/sl rats. Although this observation was not fully consistent with the domestication features of global dental-size reduction, this variance may be explained by the young age of studied rats, 3.5 days. As rat's dental eruption occurs at eight to ten days postnatally and maturates between forty to fty days post-birth (42,43), the full dental features of domestication may not be appreciated prior to the maturation age. Nevertheless, given the domestication feature is in uenced by a combination of genetic and behavioral factors, our nding on sl/sl rat supports the notion that ET B can be a predisposing gene for dental changes. On the other hand, the presence of normal dental anatomy and molar teeth number in each quadrant by both the control and sl/sl groups suggest ET B 's impact may be small. While individual molar dimensions were not measured, size-reductions in maxillary molar plate and mandible were seen in sl/sl rats, Table 5. This decreased molar bed may predispose matured sl/sl rat to have decreased molar size and increased risks of molar overcrowding, features consistent with domestication. Overall, our ndings support ET B likely plays a modulating role in craniofacial development.
Craniofacial morphogenesis is known to be dictated by ET-1/ET A signaling, and hypostimulation of which leads to premature arrest of CNCC migration. This severe craniofacial dysmorphology seen in CATCH 22 or velocardiofacial syndrome results in mechanical asphyxia and early death (12). Although the subtle changes identi ed in sl/sl rats were not as drastic, they nevertheless provided supporting evidence that HSCR children may suffer subtle craniofacial maldevelopment. These facial feature changes not only may affect patients aesthetically, such as cleft-palate (44), but more importantly increases the health risks. These risks include development of obstructive sleep apnea or chronic sinusitis due to a shorter but wider nose in addition to dysphagia from a smaller oral ori ce; all of which can impair children's growth.
Furthermore, our ndings are consistent with the notion that ET B mediates the endocytic degradation of ET-1 and thereby indirectly affecting ET-1/ET A signaling. As reported by Gariepy et al (2000), sl/sl rat has 6-folds higher ET-1 due to impaired clearance (30). This likely hyperstimulates ET A pathway, as demonstrated by the increased vascular tone in ET B -de cient mice (45). Consequently, modulation rather than premature arrest of CNCC migration were likely to have led to the subtle craniofacial changes.
Moreover, ET B mutation is known to associate with WS, particularly in the pathogenesis of WS-II and WS- On the other hand, other than the nasal dimensional indices showing distinctive shorter but wider nose in sl/sl rat, the dimensional ratio indices made on cranium, maxilla, and mandible showed little difference between sl/sl and control groups, Table 6. These proportional reductions in facial features associated with sl/sl rats suggests ET B mutation exerts global inhibition on cephalic growth.
It is worth noting that the control group has larger body-size than sl/sl rats at culling despite similar age group, suggesting malnutrition from dysfunctional enteric system may contributes to the craniofacial difference observed. While this positive body-weight and cranial size is consistent with report made by Miller and German (1999) (48), it fails to explain the presence of non-uniform dimensional-ratio indices, Table 6. This suggests an additional independent factor is likely to have contributed to the formation of atter cranium and shorter muzzle in sl/sl rats.
The ndings of this study, if translatable to human HSCR patients, have several clinical implications for patient management. A genome-wide association study has recently identi ed ET B gene as a major susceptibility locus for craniofacial microcephaly (49), a fact that is consistent with the craniofacial alterations observed in the sl/sl rat. In addition to those described in syndromic HSCR (DS, MWS, and Goldberg-Shprintzen syndrome), additional craniofacial anomalies have also been reported in nonsyndromic HSCR patients, including cleft palate, broad nasal root, microcephaly, and hypertelorism (6,(50)(51)(52)(53)(54)(55). Our dimensional nding supports these reports with quantitative measures. Of the craniofacial alterations identi ed in the sl/sl rat, shorter but broader nose, smaller mid-face, and decreased length of the molar bed suggested HSCR patients may be predispose to obstructive sleep apnea and dental malocclusion (56); both of which instigate with adverse health outcomes and require intervention. Additionally, we may nd HSCR patients with higher risks of clinical midfacial hypoplasia based on animal ndings.
We acknowledge the power of this study can bene t from a larger sample size. Nevertheless, the presence of multiple craniofacial alterations in sl/sl rats cannot be overlooked. Additionally, while potential dose-dependent response of ET B was not assessed, this set-up was compliant with the autosomal recessive mendelian traits of sl/sl rat. Furthermore, the tissue-preparation and imaging protocols used in this study may be applicable to other model studies. Future research should aim to include a larger sample-size and corresponding morphological study on clinical HSCR patients.

Conclusion
Overall, this study demonstrated subtle craniofacial changes are presented in HSCR model and provided some support for the hypothesis of neural crest gene involvement in domestication. ET B may be one of these genes. It also contributed to the growing body of literature suggesting manifestations of HSCR are not limited to the enteric nervous system and may extend to include craniofacial malformations that increase risks for obstructive sleep apnea, cleft palate, or dental malocclusion. Con rmatory clinical studies are therefore warranted.

Declarations
Ethical approval and consent to participate All animals This research project was approved by both Australian Capital Territory Health Human Research Ethics Committee (ACTH-HREC) and Australian National University Animal Experimentation Ethics Committee (ANU-AEEC), project number A2011/67.

Consent to publish
Not applicable.
Availability of data and materials