Patient Factors Associated with Access to Outpatient Pediatric General Surgical Care in a Rural State.

BACKGROUND: We sought to understand factors impacting timely access to outpatient pediatric general surgical care in a largely rural state. METHODS: We conducted a multi-site retrospective cohort study, evaluating patients <18 years referred for outpatient pediatric general surgical evaluation from 11/1/2017-7/31/2022. Outcomes included obtaining an appointment, completing an appointment, and undergoing an operation. Time to appointment and operation were calculated. Bivariate analysis and multivariable logistic regression were performed to evaluate for associations between patient factors and the primary outcomes, as well as delay to appointment. RESULTS: Of 5270 patients, mean age was 7.1 years (SD = 6) with 59% male. All patients obtained an appointment; 85% (n = 4498) completed an appointment within one year. Forty percent (n = 2092) underwent an operation. Mean times from referral to appointment and operation were 22.5 (SD = 33.4) and 81.5 days (SD = 137.5), respectively. Patients who identified as African American/Black (OR = 1.94, p < 0.001), had self-pay (OR = 6.33, p < 0.001), or lived >100 miles away (OR = 1.55, p < 0.001) were more likely to not complete appointments. Patients with high household income (OR = 0.70, p = 0.009) and private insurance (OR = 0.60, p < 0.001) were less likely to not complete appointments. Delay to appointment was associated with race (p = 0.020). Patients with private insurance (p < 0.001) and higher income (p = 0.020) were more likely to undergo operation. CONCLUSION: Fifteen percent of patients referred for outpatient pediatric general surgical evaluation did not complete an appointment within one year. Race, household resources, insurance, and travel distance were associated with completing appointments. Information about groups that have disparate access to care will inform interventions to improve this access. TYPE OF STUDY: Retrospective Cohort Study. LEVEL OF EVIDENCE: III.

Cellular transitions during cranial suture establishment in zebrafish.

Cranial sutures separate neighboring skull bones and are sites of bone growth. A key question is how osteogenic activity is controlled to promote bone growth while preventing aberrant bone fusions during skull expansion. Using single-cell transcriptomics, lineage tracing, and mutant analysis in zebrafish, we uncover key developmental transitions regulating bone formation at sutures during skull expansion. In particular, we identify a subpopulation of mesenchyme cells in the mid-suture region that upregulate a suite of genes including BMP antagonists (e.g. grem1a) and pro-angiogenic factors. Lineage tracing with grem1a:nlsEOS reveals that this mid-suture subpopulation is largely non-osteogenic. Moreover, combinatorial mutation of BMP antagonists enriched in this mid-suture subpopulation results in increased BMP signaling in the suture, misregulated bone formation, and abnormal suture morphology. These data reveal establishment of a non-osteogenic mesenchyme population in the mid-suture region that restricts bone formation through local BMP antagonism, thus ensuring proper suture morphology.

Adolescents' Perceptions of Screening, Brief Intervention, and Referral to Treatment Service at Pediatric Trauma Centers.

Objective: Screening, brief intervention, and referral to treatment (SBIRT) for adolescent alcohol and drug (AOD) use is recommended to occur with adolescents admitted to pediatric trauma centers. Most metrics on SBIRT service delivery only reference medical record documentation. In this analysis we examined changes in adolescents' perception of SBIRT services and concordance of adolescent-report and medical record data, among a sample of adolescents admitted before and after institutional SBIRT implementation. Methods: We implemented SBIRT for adolescent AOD use using the Science to Service Laboratory implementation strategy and enrolled adolescents at 9 pediatric trauma centers. The recommended clinical workflow was for nursing to screen, social work to provide adolescents screening positive with brief intervention and referral to their PCP for continued AOD discussions with those. Adolescents screening as high-risk also referred to specialty services. Adolescents were enrolled and contacted 30 days after discharge and asked about their perception of any SBIRT services received. Data were also extracted from enrolled patient's medical record. Results: There were 430 adolescents enrolled, with 424 that were matched to their EHR data and 329 completed the 30-day survey. In this sample, EHR documented screening increased from pre-implementation to post-implementation (16.3%-65.7%) and brief interventions increased (27.1%-40.7%). Adolescents self-reported higher rates of being asked about alcohol or drug use than in EHR data both pre- and post-implementation (80.7%-81%). Both EHR data and adolescent self-reported data demonstrated low referral back to PCP for continued AOD discussions. Conclusions: Implementation of SBIRT at pediatric trauma centers was not associated with change in adolescent perceptions of SBIRT, despite improved documentation of delivery of AOD screening and interventions. Adolescents perceived being asked about AOD use more often than was documented. Referral to PCP or specialty care for continued AOD discussion remains an area of needed attention. Trial registration: Clinicaltrials.gov NCT03297060.

Reverse shock index multiplied by the motor component of the Glasgow Coma Scale predicts mortality and need for intervention in pediatric trauma patients.

BACKGROUND: Timely identification of high-risk pediatric trauma patients and appropriate resource mobilization may lead to improved outcomes. We hypothesized that reverse shock index times the motor component of the Glasgow Coma Scale (GCS) (rSIM) would perform equivalently to reverse shock index times the total GCS (rSIG) in the prediction of mortality and the need for intervention following pediatric trauma. METHODS: The 2017-2020 National Trauma Data Bank data sets were used. We included all patients 16 years or younger who had a documented prehospital and trauma bay systolic blood pressure, heart rate, and total GCS. We excluded all patients who arrived at the trauma center without vital signs and interfacility transport patients. Receiver operating characteristic curves were used to model the performance of each metric as a classifier with respect to our primary and secondary outcomes, and the area under the receiver operating characteristic curve (AUROC) was used for comparison. Our primary outcome was mortality before hospital discharge. Secondary outcomes included blood product administration or hemorrhage control intervention (surgery or angiography) <4 hours following hospital arrival and intensive care unit admission. RESULTS: After application of exclusion criteria, 77,996 patients were included in our analysis. Reverse shock index times GCS-motor and rSIG performed equivalently as predictors of mortality in the 1- to 2- ( p = 0.05) and 3- to 5-year-old categories ( p = 0.28), but rSIM was statistically outperformed by rSIG in the 6- to 12- (AUROC, 0.96 vs. 0.95; p = 0.04) and 13- to 16-year-old age categories (AUROC, 0.96 vs. 0.95; p < 0.01). Reverse shock index times GCS-motor and rSIG also performed similarly with respect to prediction of secondary outcomes. CONCLUSION: Reverse shock index times GCS-total and rSIM are both outstanding predictors of mortality following pediatric trauma. Statistically significant differences in favor of rSIG were noted in some age groups. Because of the simplicity of calculation, rSIM may be a useful tool for pediatric trauma triage. LEVEL OF EVIDENCE: Diagnostic Tests or Criteria; Level III.

Augmentation of BMP Signaling in Cranial Neural Crest Cells Leads to Premature Cranial Sutures Fusion through Endochondral Ossification in Mice.

Craniosynostosis is a congenital anomaly characterized by the premature fusion of cranial sutures. Sutures are a critical connective tissue that regulates bone growth; their aberrant fusion results in abnormal shapes of the head and face. The molecular and cellular mechanisms have been investigated for a long time, but knowledge gaps remain between genetic mutations and mechanisms of pathogenesis for craniosynostosis. We previously demonstrated that the augmentation of bone morphogenetic protein (BMP) signaling through constitutively active BMP type 1A receptor (caBmpr1a) in neural crest cells (NCCs) caused the development of premature fusion of the anterior frontal suture, leading to craniosynostosis in mice. In this study, we demonstrated that ectopic cartilage forms in sutures prior to premature fusion in caBmpr1a mice. The ectopic cartilage is subsequently replaced by bone nodules leading to premature fusion with similar but unique fusion patterns between two neural crest-specific transgenic Cre mouse lines, P0-Cre and Wnt1-Cre mice, which coincides with patterns of premature fusion in each line. Histologic and molecular analyses suggest that endochondral ossification in the affected sutures. Both in vitro and in vivo observations suggest a greater chondrogenic capacity and reduced osteogenic capability of neural crest progenitor cells in mutant lines. These results suggest that the augmentation of BMP signaling alters the cell fate of cranial NCCs toward a chondrogenic lineage to prompt endochondral ossification to prematurely fuse cranial sutures. By comparing P0-Cre;caBmpr1a and Wnt1-Cre;caBmpr1a mice at the stage of neural crest formation, we found more cell death of cranial NCCs in P0-Cre;caBmpr1a than Wnt1-Cre;caBmpr1a mice at the developing facial primordia. These findings may provide a platform for understanding why mutations of broadly expressed genes result in the premature fusion of limited sutures. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Lower incidence of blunt cerebrovascular injury among young, properly restrained children: An ATOMAC multicenter study.

BACKGROUND: Motor vehicle collision (MVC) remains a leading cause of injury and death among children, but the proper use of child safety seats and restraints has lowered the risks associated with motor vehicle travel. Blunt cerebrovascular injury (BCVI) is rare but significant among children involved in MVC. This study reviewed the incidence of BCVI after MVC causing blunt injury to the head, face, or neck, comparing those that were properly restrained with those that were not. METHODS: A prospective, multi-institutional observational study of children younger than 15 years who sustained blunt trauma to the head, face, or neck (Abbreviated Injury Scale score >0) and presented at one of six level I pediatric trauma centers from 2017 to 2020 was conducted. Diagnosis of BCVI was made either by imaging or neurological symptoms at 2-week follow-up. Restraint status among those involved in MVC was compared for each age group. RESULTS: A total of 2,284 patients were enrolled at the 6 trauma centers. Of these, 521 (22.8%) were involved in an MVC. In this cohort, after excluding patients with missing data, 10 of 371 (2.7%) were diagnosed with a BCVI. For children younger than 12 years, none who were properly restrained suffered a BCVI (0 of 75 children), while 7 of 221 (3.2%) improperly restrained children suffered a BCVI. For children between 12 and 15 years of age, the incidence of BCVI was 2 of 36 (5.5%) for children in seat belts compared with 1 of 36 (2.8%) for unrestrained children. CONCLUSION: In this large multicenter prospectively screened pediatric cohort, the incidence of BCVI among properly restrained children under 12 years after MVC was infrequent, while the incidence was 3.2% among those without proper restraint. This effect was not seen among children older than 12 years. Restraint status in young children may be an important factor in BCVI screening. LEVEL OF EVIDENCE: Prognostic and Epidemiological; Level IV.

Diagnostic accuracy of screening tools for pediatric blunt cerebrovascular injury: An ATOMAC multicenter study.

BACKGROUND: Blunt cerebrovascular injury (BCVI) is rare but significant among children. There are three sets of BCVI screening criteria validated for adults (Denver, Memphis, and Eastern Association for the Surgery of Trauma criteria) and two that have been validated for use in pediatrics (Utah score and McGovern score), all of which were developed using retrospective, single-center data sets. The purpose of this study was to determine the diagnostic accuracy of each set of screening criteria in children using a prospective, multicenter pediatric data set. METHODS: A prospective, multi-institutional observational study of children younger than 15 years who sustained blunt trauma to the head, face, or neck and presented at one of six level I pediatric trauma centers from 2017 to 2020 was conducted. All patients were screened for BCVI using the Memphis criteria, but criteria for all five were collected for analysis. Patients underwent computed tomography angiography of the head or neck if the Memphis criteria were met at presentation or neurological abnormalities were detected at 2-week follow-up. RESULTS: A total of 2,284 patients at the 6 trauma centers met the inclusion criteria. After excluding cases with incomplete data, 1,461 cases had computed tomography angiography and/or 2-week clinical follow-up and were analyzed, including 24 cases (1.6%) with BCVI. Sensitivity, specificity, positive predictive value, and negative predictive value for each set of criteria were respectively 75.0, 87.5, 9.1, and 99.5 for Denver; 91.7, 71.1, 5.0, and 99.8 for Memphis; 79.2, 82.7, 7.1, and 99.6 for Eastern Association for the Surgery of Trauma; 45.8, 95.8, 15.5, and 99.1 for Utah; and 75.0, 89.5, 10.7, and 99.5 for McGovern. CONCLUSION: In this large multicenter pediatric cohort, the Memphis criteria demonstrated the highest sensitivity at 91.7% and would have missed the fewest BCVI, while the Utah score had the highest specificity at 95.8% but would have missed more than half of the injuries. Development of a tool, which narrows the Memphis criteria while maintaining its sensitivity, is needed for application in pediatric patients. LEVEL OF EVIDENCE: Diagnostic Test/Criteria; Level II.

Embryonic requirements for Tcf12 in the development of the mouse coronal suture.

A major feature of Saethre-Chotzen syndrome is coronal craniosynostosis, the fusion of the frontal and parietal bones at the coronal suture. It is caused by heterozygous loss-of-function mutations in either of the bHLH transcription factors TWIST1 and TCF12. Although compound heterozygous Tcf12; Twist1 mice display severe coronal synostosis, the individual role of Tcf12 had remained unexplored. Here, we show that Tcf12 controls several key processes in calvarial development, including the rate of frontal and parietal bone growth, and the boundary between sutural and osteogenic cells. Genetic analysis supports an embryonic requirement for Tcf12 in suture formation, as combined deletion of Tcf12 in embryonic neural crest and mesoderm, but not in postnatal suture mesenchyme, disrupts the coronal suture. We also detected asymmetric distribution of mesenchymal cells on opposing sides of the wild-type frontal and parietal bones, which prefigures later bone overlap at the sutures. In Tcf12 mutants, reduced asymmetry is associated with bones meeting end-on-end, possibly contributing to synostosis. Our results support embryonic requirements of Tcf12 in proper formation of the overlapping coronal suture.

The developing mouse coronal suture at single-cell resolution.

Sutures separate the flat bones of the skull and enable coordinated growth of the brain and overlying cranium. The coronal suture is most commonly fused in monogenic craniosynostosis, yet the unique aspects of its development remain incompletely understood. To uncover the cellular diversity within the murine embryonic coronal suture, we generated single-cell transcriptomes and performed extensive expression validation. We find distinct pre-osteoblast signatures between the bone fronts and periosteum, a ligament-like population above the suture that persists into adulthood, and a chondrogenic-like population in the dura mater underlying the suture. Lineage tracing reveals an embryonic Six2+ osteoprogenitor population that contributes to the postnatal suture mesenchyme, with these progenitors being preferentially affected in a Twist1+/-; Tcf12+/- mouse model of Saethre-Chotzen Syndrome. This single-cell atlas provides a resource for understanding the development of the coronal suture and the mechanisms for its loss in craniosynostosis.

Hypothermia as an Outcome Predictor Tool in Pediatric Trauma: A Propensity-Matched Analysis.

OBJECTIVE: Hypothermia is an independent risk factor for mortality in adult trauma patients. Two small studies have shown similar results in pediatric trauma patients. Temperature is not included in any pediatric trauma assessment scores. This study sought to compare mortality and various descriptive outcomes between pediatric hypothermic and normothermic trauma patients. METHODS: Data were obtained from the National Trauma Database from 2009 to 2012. Patients meeting inclusion criteria were stratified by presence of isolated head injury, head injury with multiple trauma, and absence of head injury. These groups were then subdivided into hypothermic (temperature ≤36°C) and normothermic groups. We used propensity score matching to 1:1 match hypothermic and normothermic patients. Mortality, neurosurgical interventions, endotracheal intubation, blood transfusion, length of stay, laparotomy, thoracotomy, conversion of cardiac rhythm, and time receiving mechanical ventilation were evaluated. RESULTS: Data from 3,011,482 patients were obtained. There were 414,562 patients who met the inclusion criteria. In all patients meeting inclusion criteria, hypothermia was a significant risk factor in all outcomes measured. Following stratification and 1:1 matching, in all groups, hypothermia was associated with increased mortality (P < 0.0001), increased rate of endotracheal intubation (P < 0.0002), increased need for blood transfusion (P < 0.0025), and conversion of cardiac rhythm (P < 0.0027). CONCLUSION: Hypothermia has been shown to be a significant prognostic indicator in the pediatric trauma patient with further potential application. Future studies are indicated to evaluate the incorporation of hypothermia into the Pediatric Trauma Score not only to help predict injury severity and mortality but also to improve appropriate and expeditious patient transfer to pediatric trauma centers and potentially facilitate earlier intervention.

Resolving homology in the face of shifting germ layer origins: Lessons from a major skull vault boundary.

The vertebrate skull varies widely in shape, accommodating diverse strategies of feeding and predation. The braincase is composed of several flat bones that meet at flexible joints called sutures. Nearly all vertebrates have a prominent 'coronal' suture that separates the front and back of the skull. This suture can develop entirely within mesoderm-derived tissue, neural crest-derived tissue, or at the boundary of the two. Recent paleontological findings and genetic insights in non-mammalian model organisms serve to revise fundamental knowledge on the development and evolution of this suture. Growing evidence supports a decoupling of the germ layer origins of the mesenchyme that forms the calvarial bones from inductive signaling that establishes discrete bone centers. Changes in these relationships facilitate skull evolution and may create susceptibility to disease. These concepts provide a general framework for approaching issues of homology in cases where germ layer origins have shifted during evolution.

Development and Validation of a Controlled Vocabulary: An OWL Representation of Organizational Structures of Trauma Centers and Trauma Systems.

In trauma care and trauma care research there exists an implementation gap regarding a consistent controlled vocabulary to describe organizational aspects of trauma centers and trauma systems. This paper describes the development and evaluation of a controlled vocabulary for trauma care organizations. We give a detailed description of the involvement of domain experts in the domain analysis workflow and the authoring of definitions and additional term descriptions. Finally, the paper details the evaluation methodology to assess the initial version of the controlled vocabulary. The results of the evaluation show that our development process yields terms most of which find approval from domain experts not involved in the development. In addition, our evaluation tools resulted in valuable domain expert input to optimize the controlled vocabulary.

Altered bone growth dynamics prefigure craniosynostosis in a zebrafish model of Saethre-Chotzen syndrome.

Cranial sutures separate the skull bones and house stem cells for bone growth and repair. In Saethre-Chotzen syndrome, mutations in TCF12 or TWIST1 ablate a specific suture, the coronal. This suture forms at a neural-crest/mesoderm interface in mammals and a mesoderm/mesoderm interface in zebrafish. Despite this difference, we show that combinatorial loss of TCF12 and TWIST1 homologs in zebrafish also results in specific loss of the coronal suture. Sequential bone staining reveals an initial, directional acceleration of bone production in the mutant skull, with subsequent localized stalling of bone growth prefiguring coronal suture loss. Mouse genetics further reveal requirements for Twist1 and Tcf12 in both the frontal and parietal bones for suture patency, and to maintain putative progenitors in the coronal region. These findings reveal conservation of coronal suture formation despite evolutionary shifts in embryonic origins, and suggest that the coronal suture might be especially susceptible to imbalances in progenitor maintenance and osteoblast differentiation.

Culturing and Manipulation of O9-1 Neural Crest Cells.

Neural crest cells (NCCs) are migrating multipotent stem cells that can differentiate into different cell types and give rise to multiple tissues and organs. The O9-1 cell line is derived from the endogenous mouse embryonic NCCs and maintains its multipotency. However, under specific culture conditions, O9-1 cells can differentiate into different cell types and be utilized in a wide range of research applications. Recently, with the combination of mouse studies and O9-1 cell studies, we have shown that the Hippo signaling pathway effectors Yap and Taz play important roles in neural crest-derived craniofacial development. Although the culturing process for O9-1 cells is more complicated than that used for other cell lines, the O9-1 cell line is a powerful model for investigating NCCs in vitro. Here, we present a protocol for culturing the O9-1 cell line to maintain its stemness, as well as protocols for differentiating O9-1 cells into different cell types, such as smooth muscle cells and osteoblasts. In addition, protocols are described for performing gene loss-of-function studies in O9-1 cells by using CRISPR-Cas9 deletion and small interfering RNA-mediated knockdown.

Msx2 Supports Epidermal Competency during Wound-Induced Hair Follicle Neogenesis.

Cutaneous wounds in adult mammals typically heal by scarring. However, large full-thickness wounds undergo wound-induced hair follicle neogenesis (WIHN), a form of regeneration. Here, we show that WIHN requires transient expression of epidermal Msx2 in two phases: the wound margin early and the wound center late. Msx2 expression is present in the migrating epithelium during early wound healing and then presents in the epithelium and mesenchyme later in the wound center. WIHN is abrogated in germline and epithelial-specific Msx2 mutant mice. Unlike the full-length Msx2 promoter, a minimal Msx2 promoter fails activation in the wound center, suggesting complex regulation of Msx2 expression. The Msx2 promoter binding sites include Tcf/Lef, Jun/Creb, Pax3, and three SMAD sites. However, basal epithelial-induced BMP suppression by noggin overexpression did not affect WIHN. We propose that Msx2 signaling is required for the epidermis to acquire spatiotemporal competence during WIHN. Topologically, hair regeneration dominates in the wound center, coinciding with late Msx2 expression. Together, these results suggest that intrinsic Msx2 expression supports epithelial competency during hair follicle neogenesis. This work provides insight into endogenous mechanisms modulating competency of adult epidermal progenitors for mammalian ectodermal appendage neogenesis, and offers the target Msx2 for future regeneration-promoting therapies.

Impact of a statewide trauma system on the triage, transfer, and inpatient mortality of injured patients.

BACKGROUND: In 2009, Arkansas implemented a statewide trauma system to address the high rates of mortality and morbidity due to trauma. The principal objective of the Arkansas Trauma System is to transport patients to the appropriate facility based on the injuries of the patients. This study evaluated four metrics that were crucial to system health. These measures included: treatment location, scene triage, admission to nondesignated facilities, and inpatient mortality. Furthermore, the authors sought to quantify how the system is selective toward the severely injured regarding triage and treatment location. The authors hypothesized that system implementation should increase the proportion of patients, particularly the severely injured, treated at Level I/II facilities. The system should increase the proportion of patients, especially the severely injured, admitted to Level I/II facilities directly from the scene. The system should result in fewer patients admitted to nondesignated facilities. Lastly, system implementation should result in fewer inpatient deaths. METHODS: A pre-post study design was used for this evaluation. Data from the Arkansas Hospital Discharge data set (2007 through 2012) identified patients who were admitted as a result of their injuries. The ICD-MAP software was used to categorize those with and without severe injuries based on an Injury Severity Score of 16 or greater or head Abbreviated Injury Scale score of 3 or greater. RESULTS: The results indicate that while there was an overall increase in odds of patients being admitted to Level I/II facilities, those with severe injuries were associated with an even greater odds of admission to Level I/II facilities (p < 0.0001). System implementation was also associated with more severely injured patients admitted to Level I/II facilities from the scene. There were also fewer patients admitted to nondesignated hospitals after system implementation (p < 0.0001). System implementation was associated with fewer inpatient deaths (p = 0.02). CONCLUSION: Two years after implementation, the trauma system showed significant progress. The measures evaluated in this study are believed to support the effectiveness of the trauma system. LEVEL OF EVIDENCE: Therapeutic study, level IV.

Injured Children Receive Twice the Radiation Dose at Nonpediatric Trauma Centers Compared With Pediatric Trauma Centers.

BACKGROUND: Use of cranial CT scans in children has been increasing, in part due to increased awareness of sports-related concussions. CT is the largest contributor to medical radiation exposure, a risk factor for cancer. Long-term cancer risks of CT scans can be two to three times higher for children than for adults because children are more radiosensitive and have a longer lifetime in which to accumulate exposure from multiple scans. STUDY AIM: To compare the radiation exposure injured children receive when imaged at nonpediatric hospitals (NPHs) versus pediatric hospitals. METHODS: Injured children younger than 18 years who received a CT scan at a referring hospital during calendar years (CYs) 2010 and 2013 were included. Patient-level factors included demographics, mode of transportation, and Injury Severity Score, and hospital-level factors included region of state, radiology services, and hospital type and size. Our primary outcome of interest was the effective radiation dose. RESULTS: Four hundred eighty-seven children were transferred to the pediatric trauma center during CYs 2010 and 2013, with a median age of 7.2 years (interquartile range 5-13). The median effective radiation dose received at NPHs was twice that received at the pediatric trauma center (3.8 versus 1.6 mSv, P < .001). Results were confirmed in independent and paired analyses, after controlling for mode of transportation, emergency department disposition, level of injury severity, and at the NPH trauma center level, hospital type, size, region, and radiology services location. CONCLUSION: NPHs have the potential to substantially reduce the medical radiation received by injured children. Pediatric CT protocols should be considered.

Requirement for Jagged1-Notch2 signaling in patterning the bones of the mouse and human middle ear.

Whereas Jagged1-Notch2 signaling is known to pattern the sensorineural components of the inner ear, its role in middle ear development has been less clear. We previously reported a role for Jagged-Notch signaling in shaping skeletal elements derived from the first two pharyngeal arches of zebrafish. Here we show a conserved requirement for Jagged1-Notch2 signaling in patterning the stapes and incus middle ear bones derived from the equivalent pharyngeal arches of mammals. Mice lacking Jagged1 or Notch2 in neural crest-derived cells (NCCs) of the pharyngeal arches display a malformed stapes. Heterozygous Jagged1 knockout mice, a model for Alagille Syndrome (AGS), also display stapes and incus defects. We find that Jagged1-Notch2 signaling functions early to pattern the stapes cartilage template, with stapes malformations correlating with hearing loss across all frequencies. We observe similar stapes defects and hearing loss in one patient with heterozygous JAGGED1 loss, and a diversity of conductive and sensorineural hearing loss in nearly half of AGS patients, many of which carry JAGGED1 mutations. Our findings reveal deep conservation of Jagged1-Notch2 signaling in patterning the pharyngeal arches from fish to mouse to man, despite the very different functions of their skeletal derivatives in jaw support and sound transduction.

Negative regulation of endothelin signaling by SIX1 is required for proper maxillary development.

Jaw morphogenesis is a complex event mediated by inductive signals that establish and maintain the distinct developmental domains required for formation of hinged jaws, the defining feature of gnathostomes. The mandibular portion of pharyngeal arch 1 is patterned dorsally by Jagged-Notch signaling and ventrally by endothelin receptor A (EDNRA) signaling. Loss of EDNRA signaling disrupts normal ventral gene expression, the result of which is homeotic transformation of the mandible into a maxilla-like structure. However, loss of Jagged-Notch signaling does not result in significant changes in maxillary development. Here we show in mouse that the transcription factor SIX1 regulates dorsal arch development not only by inducing dorsal Jag1 expression but also by inhibiting endothelin 1 (Edn1) expression in the pharyngeal endoderm of the dorsal arch, thus preventing dorsal EDNRA signaling. In the absence of SIX1, but not JAG1, aberrant EDNRA signaling in the dorsal domain results in partial duplication of the mandible. Together, our results illustrate that SIX1 is the central mediator of dorsal mandibular arch identity, thus ensuring separation of bone development between the upper and lower jaws.