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Type 1 diabetes is a challenging chronic condition and can lead to diabetes-related distress and disengagement. Historically disadvantaged, racially and ethnically diverse young adults (YAs) with type 1 diabetes experience higher blood glucose levels and encounter more barriers to care than their White counterparts. Current research shows that telehealth may provide a route for improving psychosocial issues and diabetes care among YAs.

Early surgical intervention in infants with complex CHD results in significant disruptions to their respiratory, gastrointestinal, and nervous systems, which are all instrumental to the development of safe and efficient oral feeding skills. Standardised assessments or treatment protocols are not currently available for this unique population, requiring the clinician to rely on knowledge based on neonatal literature. Clinicians need to be skilled at evaluating and analysing these systems to develop an appropriate treatment plan to improve oral feeding skill and safety, while considering post-operative recovery in the infant with complex CHD. Supporting the family to re-establish their parental role during the hospitalisation and upon discharge is critical to reducing parental stress and oral feeding success.

Background: Type one diabetes (T1D) management is challenging for adolescents and young adults (AYAs) due to physiological changes, psychosocial challenges, and increasing independence, resulting in increased diabetes distress and hemoglobin A1c (HbA1c). Alternative care models that engage AYAs and improve diabetes-related health outcomes are needed. Methods: A 15-month study evaluated an adaptation of the Colorado Young Adults with T1D (CoYoT1) Care model. CoYoT1 Care includes person-centered care, virtual peer groups, and physician training delivered via telehealth. AYAs (aged 16-25 years) were partially randomized to CoYoT1 or standard care, delivered via telehealth or in-person. As the study was ending, the COVID-19 pandemic forced all AYAs to transition to primarily telehealth appointments. This secondary analysis compares changes in clinic attendance, T1D-related distress, HbA1c, and device use between those who attended more than 50% of diabetes clinic visits via telehealth and those who attended more sessions in-person throughout the course of the study. Results: Out of 68 AYA participants, individuals (n = 39, 57%) who attended most (>50%) study visits by telehealth completed more diabetes care visits (3.3 visits) than those (n = 29, 43%) who primarily attended visits in-person (2.5 visits; P = .007). AYAs who primarily attended visits via telehealth maintained stable physician-related distress, while those who attended more in-person visits reported increases in physician-related distress ( P = .04). Conclusions: Greater usage of telehealth improved AYA engagement with their care, resulting in increased clinic attendance and reduced physician-related diabetes distress. A person-centered care model delivered via telehealth effectively meets the needs of AYAs with T1D.

Background: Diabetes technologies, such as insulin pumps and continuous glucose monitors (CGM), have been associated with improved glycemic control and increased quality of life for young people with type 1 diabetes (T1D); however, few young people use these devices, especially those from minority ethnic groups. Current literature predominantly focuses on white patients with private insurance and does not report experiences of diverse pediatric patients with limited resources. Methods: To explore potential differences between Latinx and non-Latinx patients, English- and Spanish-speaking young people with T1D ( n = 173, ages 11-25 years) were surveyed to assess attitudes about and barriers to diabetes technologies using the Technology Use Attitudes and Barriers to Device Use questionnaires. Results: Both English- and Spanish-speaking participants who identified as Latinx were more likely to have public insurance ( P = .0001). English-speaking Latinx participants reported higher Hemoglobin A1c values ( P = .003), less CGM use ( P = .002), and more negative attitudes about technology (generally, P = .003; and diabetes-specific, P Conclusions: Latinx English-speaking participants had less positive attitudes toward general and diabetes technology than Latinx Spanish-speaking and non-Latinx English-speaking peers, and differences in CGM use were associated with socioeconomic status. Additional work is needed to design and deliver diabetes interventions that are of interest to and supportive of patients from diverse ethnic and language backgrounds.

In 2018, the Neurodevelopmental and Psychosocial Interventions Working Group of the Cardiac Neurodevelopmental Outcome Collaborative convened through support from an R13 grant from the National Heart, Lung, and Blood Institute to survey the state of neurodevelopmental and psychosocial intervention research in congenital heart disease and to propose a slate of critical questions and investigations required to improve outcomes for this growing population of survivors and their families. Prior research, although limited, suggests that individualized developmental care interventions delivered early in life are beneficial for improving a range of outcomes including feeding, motor and cognitive development, and physiological regulation. Interventions to address self-regulatory, cognitive, and social-emotional challenges have shown promise in other medical populations yet their applicability and effectiveness for use in individuals with congenital heart disease have not been examined. To move this field of research forward, we must strive to better understand the impact of neurodevelopmental and psychosocial intervention within the congenital heart disease population including adapting existing interventions for individuals with congenital heart disease. We must examine the ways in which dedicated cardiac neurodevelopmental follow-up programs bolster resilience and support children and families through the myriad transitions inherent to the experience of living with congenital heart disease. And we must ensure that interventions are person-/family-centered, inclusive of individuals from diverse cultural backgrounds as well as those with genetic/medical comorbidities, and proactive in their efforts to include individuals who are at highest risk but who may be traditionally less likely to participate in intervention trials.

Objective For youth with type 1 diabetes (T1D), the early adolescent period is associated with worsening diabetes management and high rates of negative psychosocial issues, including depressive symptoms and family conflict. Alternative clinical models may help improve both diabetes and psychosocial outcomes. Our study aims to investigate whether Team Clinic, a shared medical appointment model developed specifically for adolescents with T1D, will improve psychosocial outcomes for middle school-aged youth. Methods Youth with T1D, 11-13 years of age, and their caregivers, participated in a randomized controlled trial comparing Team Clinic to traditional clinic visits (control group). Diabetes characteristics were obtained at every visit. Participants and caregivers completed depression screening and family conflict questionnaires at baseline and end of study. Changes in mean scores on clinical and psychosocial outcomes from baseline to end of study were compared between groups using linear mixed-effects models. Results Eighty-six youth (51% female; 74% White; 10% Hispanic) completed at least one visit during the twelve-month study period. At the end of the study, control group participants reported increases in Emotional Problems compared to Team Clinic participants, including higher levels of Negative Mood/Physical Symptoms (p=0.02). Team Clinic participants reported reduced family conflict surrounding diabetes at study end, compared to control group participants (p=0.03). Caregivers did not report change in depressive symptoms or family conflict during the study. Hemoglobin A1C levels did not change over time in either group. Conclusions Participation in Team Clinic was associated with improved psychosocial outcomes in middle school-aged participants with T1D. This article is protected by copyright. All rights reserved.

Background: Managing T1D as a YA is challenging due to changing physiology, psychosocial demands, and increasing independence. Fewer than one-third of YA with T1D meet the ADA guidelines of quarterly diabetes visits and only one in seven attain the recommended hemoglobin A1c level of Methods: The CoYoT1 Care model was adapted for diverse, predominantly publicly insured, YA with T1D at a large urban hospital. A 15-month 4-arm randomized controlled trial collected data on 68 YA, ages 16-25. Participants received either CoYoT1 Care or Standard Care via in-person or Telehealth (TH) , prior to COVID-19; all in-person visits transitioned to TH in March 2020. CoYoT1 Care consists of patient-centered provider visits with bimonthly virtual T1D peer group sessions. Results: YA who attended >50% of study visits by TH attended more visits during the study period (3.4) than those who primarily attended in-person (2.6, p Conclusions: TH was successfully adapted for diverse YA with T1D, resulting in increased attendance and stable diabetes distress compared to in-person care. Participants overwhelmingly indicated that the benefits associated with TH, such as saving time and money traveling and parking, outweighed any technical challenges. Larger studies with longer term follow-up, not occurring during a global pandemic, are needed to refine TH’s role in patient-centered care of YA with T1D. Disclosure J.Raymond: None. J.L.Fogel: None. M.W.Reid: None. E.Salcedo-rodriguez: None. D.Fox: None. E.Pyatak: Research Support; Abbott Diabetes. Funding Donaghue Foundation

Background: VPG have been shown to improve psychosocial well-being in AYA with T1D, but it is unknown what aspects of VPG are most or least valued. Methods: CoYoT1 to California is a 15 month randomized controlled trial for patients ages 16-25 with T1D. AYA received Usual Care (n=28) or CoYoT1 Care (n=40) , which consisted of patient-centered provider visits and bimonthly VPG led by a YA with T1D. VPG were AYA-driven discussions focused on topics pertinent to AYA with T1D, emphasizing problem-solving and emotional support. At study end, VPG participants responded to a survey about their preferences for intervention features. Results: CoYoT1 Care patients were 40% female, 53% Latinx, and 72% publicly insured; and they attended 1.9 VPG each on average. AYA who attended at least one VPG participated in 4.1 VPG on average. The average session had 4.5 AYA present; each topic was covered by 9.6 AYA on average. Most survey respondents (75%) reported VPG were extremely or very valuable in supporting their T1D care. Seeing peers use diabetes technology and being supported by same-aged peers with T1D increased VPG value the most. Larger group size and not feeling comfortable sharing decreased value the most. Conclusion: Peer interactions may support unmet needs of AYA with T1D from diverse backgrounds. Further work will help optimize the design of VPG based on patient preferences. Disclosure D.I.Bisno: None. E.Pyatak: Research Support; Abbott Diabetes. M.W.Reid: None. D.Fox: None. J.L.Fogel: None. E.Salcedo-rodriguez: None. J.J.Flores garcia: None. A.Torres sanchez: None. J.Raymond: None. Funding The Donaghue Foundation

Management of type 1 diabetes mellitus for pediatric and young adult patients is well suited for telehealth. Diabetes management requires frequent communication with health care providers as well as the interpretation of many types of data that can be measured in the home and shared virtually to the provider by the patient. Telehealth technologies allow for a safe alternative and/or addition to in-person care for youth with diabetes. Telehealth increases access to health care, saves time and money, and results in improvements in rates of appointment adherence, patient satisfaction, and quality of life.

INTRODUCTION: Glycemic control is challenging for adolescents with type 1 diabetes (T1D). Team Clinic, a shared medical appointment model, has improved psychosocial outcomes in middle school patients with T1D. We aimed to evaluate the costs of delivering Team Clinic. METHOD: Participants were randomized into Team Clinic (n = 44) or usual care (n = 42) groups. RESULTS: We found no significant difference in 6-month total costs per subject ($3,204 [intervention] vs. $3,476 [control]. No significant differences were found in health care use, test strip use, or continuous glucose monitoring and/or pump. The intervention had more clinic visits (2.41 vs. 1.52 times) and a longer length of visit (2.34 vs. 0.74 hr, but no difference in provider time per patient per visit (median, 0.67 vs. 0.68 hr). DISCUSSION: The Team Clinic care model may help young adolescents with T1D improve psychosocial outcomes and increase completion of clinical visits without increasing costs.

Micronutrients are dietary components important for health and physiological function, and inadequate intake of these nutrients can contribute to poor health outcomes. The risk of inadequate micronutrient intake has been shown to be greater among low-income Hispanics and postpartum and lactating women. Therefore, we aimed to determine the risk of nutrient inadequacies based on preliminary evidence among postpartum, Hispanic women. Risk of micronutrient inadequacy for Hispanic women (29–45 years of age) from the Southern California Mother’s Milk Study (n = 188) was assessed using 24 h dietary recalls at 1 and 6 months postpartum and the estimated average requirement (EAR) fixed cut-point approach. Women were considered at risk of inadequate intake for a nutrient if more than 50% of women were consuming below the EAR. The Chronic Disease Risk Reduction (CDRR) value was also used to assess sodium intake. These women were at risk of inadequate intake for folate and vitamins A, D, and E, with 87.0%, 93.4%, 43.8%, and 95% of women consuming less than the EAR for these nutrients, respectively. Lastly, 71.7% of women consumed excess sodium. Results from this preliminary analysis indicate that Hispanic women are at risk of inadequate intake of important micronutrients for maternal and child health.

Purpose: The purpose of this study was to analyze the impact of virtual group appointments (VGA) on self-reported health-related outcomes and care activities for young adults (YA) with type 1 diabetes (T1D). Methods: Fifty-three YA (ages 18-25 years) with T1D participated in a randomized controlled trial (RCT) of the Colorado Young Adults with T1D (CoYoT1) Clinic intervention, encompassing telehealth (TH) with or without VGA. Both new patients ( n = 32) and those who participated in a pilot phase ( n = 26) were randomized to CoYoT1 Clinic (TH+VGA; n = 23) or TH-only ( n = 35) and followed for 1 year. YA completed the Diabetes Distress Scale (DDS), Diabetes Strengths and Resilience (D-STAR), Self-Efficacy in Diabetes (SED), Self-Management of Type 1 Diabetes in Adolescence (SMOD-A), Center for Epidemiologic Studies Depression (CES-D), and EuroQol (EQ-5D) scales at baseline and study end. Results: YA were 67% female, 84% white, 10% Latinx, and the mean age was 20.4 years old. At study end, participants in CoYoT1 Clinic reported significantly reduced diabetes distress compared to those in TH-only, who reported increased levels [Effect Size (ES) = −0.40, P = .02]. Specifically, CoYoT1 Clinic participants reported relative reductions in Physician (ES = −2.87, P = .02) and Regimen-related distress (ES = −0.35, P = .01). In addition, participants in CoYoT1 Clinic reported improved self-management of T1D-related problem solving (ES = 0.47, P = .051) and communication with care providers (ES = 0.39, P = .07). Conclusions: Virtual group attendance in CoYoT1 Clinic was associated with significant improvements in diabetes-related distress. Long-term exposure to VGA should be investigated in YA with T1D and other pediatric chronic conditions.

OBJECTIVES: Pediatric Non-Alcoholic Fatty Liver Disease is prevalent among Hispanics, which may partially result from a higher frequency of the C > G polymorphism in the patatin-like phospholipase 3 (PNPLA3) gene. Our prior work showed an interaction between the GG genotype and high dietary sugar, resulting in higher liver fat. Additionally, studies show that extreme sugar depletion improves liver fat in children with obesity. This trial aimed to test whether a clinical intervention for sugar reduction versus standard diet advice in Hispanic youth differentially impacts liver fat, liver fibrosis, body weight and composition, and whether effects differed by PNPLA3 genotype. METHODS: Hispanic youth with obesity (n = 105; 72% GG) were randomized to a healthy eating control group or a sugar reduction group (goal, 0.05). No treatment by PNPLA3 genotype interaction was observed for any endpoint (P > 0.05). While there was no significant change in fat mass in either study arm, secondary analyses revealed that participants who reduced fat mass, significantly reduced liver fat compared to those without fat mass reduction [−1.8% (−6.2, −3.4) vs. 0.3% (−1.0, 1.1), P

Congenital heart disease (CHD) is the most common birth defect for infants born in the United States, with approximately 36,000 affected infants born annually. While mortality rates for children with CHD have significantly declined, there is a growing population of individuals with CHD living into adulthood prompting the need to optimise long-term development and quality of life. For infants with CHD, pre- and post-surgery, there is an increased risk of developmental challenges and feeding difficulties. Feeding challenges carry profound implications for the quality of life for individuals with CHD and their families as they impact short- and long-term neurodevelopment related to growth and nutrition, sensory regulation, and social-emotional bonding with parents and other caregivers. Oral feeding challenges in children with CHD are often the result of medical complications, delayed transition to oral feeding, reduced stamina, oral feeding refusal, developmental delay, and consequences of the overwhelming intensive care unit (ICU) environment. This article aims to characterise the disruptions in feeding development for infants with CHD and describe neurodevelopmental factors that may contribute to short- and long-term oral feeding difficulties.

Infant formula feeding, compared with human milk, has been associated with development of a distinct infant gut microbiome, but no previous study has examined effects of formula with added sugars. This work examined differences in gut microbiota among 91 Hispanic infants who consumed human milk [at breast (BB) vs. pumped in bottle (BP)] and 2 kinds of infant formula [(traditional lactose-based (TF) vs. lactose-reduced with added sugar (ASF)]. At 1 and 6 months, infant stool was collected to characterize gut microbiota. At 6 months, mothers completed 24-hour dietary recalls and questionnaires to determine infant consumption of human milk (BB vs. BP) or formula (TF vs. ASF). Linear regression models were used to determine associations of milk consumption type and microbial features at 6 months. Infants in the formula groups exhibited a significantly more ‘mature’ microbiome than infants in the human milk groups with the most pronounced differences observed between the ASF vs. BB groups. In the ASF group, we observed reduced log-normalized abundance of Bifidobacteriaceae (TF-BB Mean Difference = −0.71, ASF-BB Mean Difference = −1.10), and increased abundance of Lachnospiraceae (TF-BB Mean Difference = +0.89, ASF-BB Mean Difference = +1.20). We also observed a higher Community Phenotype Index of propionate, most likely produced by Lachnospiraceae, in the ASF group (TF-BB Mean Difference = +0.27, ASF-BB Mean Difference = +0.36). This study provides the first evidence that consumption of infant formula with added sugar may have a stronger association than birth delivery mode, infant caloric intake, and maternal BMI on the infant’s microbiome at 6 months of age.

Introduction: CTC is a 15-month intervention study evaluating a patient-centered, home telehealth care model for young adults (YA) ages 16-25 with T1D. The patient-centered CoYoT1 Care arm includes a shared decision-making tool completed by providers (n=3) and patients (n=47) prior to each visit. The present analysis examines how patients and providers collaborated to shape initial visit agendas. Methods: Six potential topics were each rated on a 1-5 scale, where higher values indicate greater importance. Patient and provider ratings within each topic were compared using paired t-tests. Next, ratings were compared between visits where a topic was discussed or not discussed using independent t-tests; patient and provider data were evaluated separately. Results: Blood sugar ranges and supply/medication issues were discussed most frequently. Providers assigned higher overall importance to emotional well-being than their patients (p=0.005), but this topic was only discussed when patients assigned higher importance (p=0.001). Also, despite higher provider ratings, blood sugar ranges were only discussed when patients assigned higher importance (p=0.003). Discussion: Consistent with the CoYoT1 Care model, patients and providers collaborated to shape visit agendas, slightly favoring the patient perspective. Topics discussed were those assigned higher importance by patients and providers. Disclosure J.J. Flores Garcia: None. E. Pyatak: None. M.W. Reid: None. J.L. Fogel: None. J. Diaz: None. J. Raymond: None.

Background Infants born with critical congenital heart disease (cCHD) who require surgical intervention in the newborn period are often hospitalized in a cardiac intensive care unit (CICU). Cardiac surgery and the CICU environment are traumatic to infants and their families. Infants are exposed to overwhelming stress, which can result in increased pain, physiologic instability, behavioral disorganization, disrupted attachment, and altered brain development. Individualized Family-centered Developmental Care (IFDC) is a model that can address the unique needs and developmental challenges of infants with cCHD. Purpose The purpose of this article is to (1) clearly describe the uniqueness of the infant with cCHD, including the medical, neurological, and parental challenges, and (2) propose methods to apply IFDC to support recovery of infants with cCHD in the CICU. Conclusions The experiences in the CICU shape the developing brain and alter recovery and healing, thus adversely impacting development. Individualized Family-centered Developmental Care is a promising model of care that nurses can integrate into the CICU to promote neuroprotection and development. Nurses can effectively integrate IFDC into the CICU by understanding the unique characteristics of infants with cCHD and applying IFDC interventions that include both maturity and recovery perspectives. Clinical implications The incorporation of IFDC interventions is essential for the infant with cCHD and should be a standard of care. Applying IFDC with a recovery perspective in all aspects of caregiving will provide opportunities for individualization of care and parent engagement, allowing infants in the CICU to recover from surgery while supporting both short- and long-term neurodevelopment.

Non-alcoholic fatty liver disease impacts 15.2% of Hispanic adolescents and can progress to a build-up of scared tissue called liver fibrosis. If diagnosed early, liver fibrosis may be reversible, so it is necessary to understand risk factors. The aims of this study in 59 Hispanic adolescents with obesity were to: (1) identify potential biological predictors of liver fibrosis and dietary components that influence liver fibrosis, and (2) determine if the association between dietary components and liver fibrosis differs by PNPLA3 genotype, which is highly prevalent in Hispanic adolescents and associated with elevated liver fat. We examined liver fat and fibrosis, genotyped for PNPLA3 gene, and assessed diet via 24-h diet recalls. The prevalence of increased fibrosis was 20.9% greater in males, whereas participants with the GG genotype showed 23.7% greater prevalence. Arachidonic acid was associated with liver fibrosis after accounting for sex, genotype, and liver fat (β = 0.072, p = 0.033). Intakes of several dietary types of unsaturated fat have different associations with liver fibrosis by PNPLA3 genotype after accounting for sex, caloric intake, and liver fat. These included monounsaturated fat (βCC/CG = −0.0007, βGG = 0.03, p-value = 0.004), polyunsaturated fat (βCC/CG = −0.01, βGG = 0.02, p-value = 0.01), and omega-6 (βCC/CG = −0.0102, βGG = 0.028, p-value = 0.01). Results from this study suggest that reduction of arachidonic acid and polyunsaturated fatty acid intake might be important for the prevention of non-alcoholic fatty liver disease progression, especially among those with PNPLA3 risk alleles.

For decades, the mechanism of skeletal patterning along a proximal-distal axis has been an area of intense inquiry. Here, we examine the development of the ribs, simple structures that in most terrestrial vertebrates consist of two skeletal elements—a proximal bone and a distal cartilage portion. While the ribs have been shown to arise from the somites, little is known about how the two segments are specified. During our examination of genetically modified mice, we discovered a series of progressively worsening phenotypes that could not be easily explained. Here, we combine genetic analysis of rib development with agent-based simulations to conclude that proximal-distal patterning and outgrowth could occur based on simple rules. In our model, specification occurs during somite stages due to varying Hedgehog protein levels, while later expansion refines the pattern. This framework is broadly applicable for understanding the mechanisms of skeletal patterning along a proximal-distal axis., eLife digest During animal development, the ribs grow from the back of the embryo around towards the chest. In fish, these bones simply terminate. Yet in land animals, cartilage forms at the end of the rib where it connects to the breastbone, or sternum. This encloses the chest cavity. Fogel, Lakeland et al. have now asked how the progenitor cells that develop into the ribs form these two skeletal elements – the bone element and the cartilage element – in land animals. Their approach involved genetic analysis in mice and a simple computing model. It revealed that two elements could form if the progenitor cells decide which element they will belong to based on the concentration of the diffusible protein called Hedgehog. This protein controls many aspects of animal development, and higher concentrations seem to bias the cells in a developing rib toward belonging to the bone element. Fogel, Lakeland et al. propose that this decision is locked-in early, before the rib grows outward and becomes more refined. An analysis using this simple model reproduces all the basic observations seen in the experiments with mice. The model also explains how processes like cell division and cell death control the growth of developing skeletal elements. These modeling techniques can be applied to many fields within biology, including research into the causes of birth defects, the mechanisms of tissue repair, and the evolution of skeletal diversity. An advantage to this modeling technique is that it uses only the information in each cell’s local environment to make decisions.

As more individuals from diverse backgrounds are diagnosed with Type 1 Diabetes (T1D), the need to address resulting disparities in diabetes outcomes among these populations also escalates. Although young adulthood proves challenging for all patients with diabetes, young adults (YA) from racial/ethnic minorities and low socioeconomic backgrounds face even greater T1D management obstacles. The poorer outcomes in these populations drive an urgent need for alternative care models to improve YA's engagement in their T1D clinical care and address barriers to improved health outcomes. Previous telemedicine initiatives for T1D have yielded positive diabetes care results, especially in YA, offering one promising way to reach this high-risk population. To serve these patients better, an established and successful home telehealth group appointment model, "CoYoT1 Clinic" (Colorado Young Adults with T1D), was adapted to provide care to YA with T1D at a large urban children's hospital in Southern California. At this location, ~70% of patients have public/no insurance, and 85% are racial/ethnic minorities. In this paper, we report the process of adapting the CoYoT1 Clinic model and designing a randomized controlled trial (RCT) to evaluate its efficacy. The adapted model uses meticulous study-design methods that incorporate patient advisors, quantitative and qualitative data collection, collaboration with local stakeholders, intervention development, and patient randomization into a factorial design analyzing telemedicine versus in-person and patient-centered versus standard care. The new model addresses the needs of high-risk YA in Southern California, with the goal of increasing access to care, improving follow-up frequency, and strengthening patient and provider satisfaction. The study is registered with ClinicalTrials.gov (Clinical Trials Number: NCT03793673).

During neural tube closure, specialized regions called hinge points (HPs) display dynamic and polarized cell behaviors necessary for converting the neural plate into a neural tube. The molecular bases of such cell behaviors (e.g. apical constriction, basal nuclear migration) are poorly understood. We have identified a two-dimensional canonical BMP activity gradient in the chick neural plate that results in low and temporally pulsed BMP activity at the ventral midline/median hinge point (MHP). Using in vivo manipulations, high-resolution imaging and biochemical analyses, we show that BMP attenuation is necessary and sufficient for MHP formation. Conversely, BMP overexpression abolishes MHP formation and prevents neural tube closure. We provide evidence that BMP modulation directs neural tube closure via the regulation of apicobasal polarity. First, BMP blockade produces partially polarized neural cells, which retain contact with the apical and basal surfaces but where basolateral proteins (LGL) become apically localized and apical junctional proteins (PAR3, ZO1) become targeted to endosomes. Second, direct LGL misexpression induces ectopic HPs identical to those produced by noggin or dominant-negative BMPR1A. Third, BMP-dependent biochemical interactions occur between the PAR3-PAR6-aPKC polarity complex and phosphorylated SMAD5 at apical junctions. Finally, partially polarized cells normally occur at the MHP, their frequencies inversely correlated with the BMP activity gradient in the neural plate. We propose that spatiotemporal modulation of the two-dimensional BMP gradient transiently alters cell polarity in targeted neuronal cells. This ensures that the neural plate is flexible enough to be focally bent and shaped into a neural tube, while retaining overall epithelial integrity.

The clinical need for methods to repair and regenerate large cartilage and bone lesions persists. One way to make new headway is to study skeletal regeneration when it occurs naturally. Cartilage repair is typically slow and incomplete. However, an exception to this observation can be found in the costal cartilages, where complete repair has been reported in humans but the cellular and molecular mechanisms have not yet been characterized. In this study, we establish a novel animal model for cartilage repair using the mouse rib costal cartilage. We then use this model to test the hypothesis that the perichondrium, the dense connective tissue that surrounds the cartilage, is a tissue essential for repair. Our results show that full replacement of the resected cartilage occurs quickly (within 1 to 2 months) and properly differentiates but that repair occurs only in the presence of the perichondrium. We then provide evidence that the rib perichondrium contains a special niche that houses chondrogenic progenitors that possess qualities particularly suited for mediating repair. Label-retaining cells can be found within the perichondrium that can give rise to new chondrocytes. Furthermore, the perichondrium proliferates and thickens during the healing period and when ectopically placed can generate new cartilage. In conclusion, we have successfully established a model for hyaline cartilage repair in the mouse rib, which should be useful for gaining a more detailed understanding of cartilage regeneration and ultimately for developing methods to improve cartilage and bone repair in other parts of the skeleton.

Programmed cell death (PCD) occurs in adults to maintain normal tissue homeostasis and during embryological development to shape tissues and organs(1,2,6,7). During development, toxic chemicals or genetic alterations can cause an increase in PCD or change PCD patterns resulting in developmental abnormalities and birth defects(3-5). To understand the etiology of these defects, the study of embryos can be complemented with in vitro assays that use differentiating embryonic stem (ES) cells. Apoptosis is a well-studied form of PCD that involves both intrinsic and extrinsic signaling to activate the caspase enzyme cascade. Characteristic cell changes include membrane blebbing, nuclear shrinking, and DNA fragmentation. Other forms of PCD do not involve caspase activation and may be the end-result of prolonged autophagy. Regardless of the PCD pathway, dying cells need to be removed. In adults, the immune cells perform this function, while in embryos, where the immune system has not yet developed, removal occurs by an alternative mechanism. This mechanism involves neighboring cells (called "non-professional phagocytes") taking on a phagocytic role-they recognize the 'eat me' signal on the surface of the dying cell and engulf it(8-10). After engulfment, the debris is brought to the lysosome for degradation. Thus regardless of PCD mechanism, an increase in lysosomal activity can be correlated with increased cell death. To study PCD, a simple assay to visualize lysosomes in thick tissues and multilayer differentiating cultures can be useful. LysoTracker dye is a highly soluble small molecule that is retained in acidic subcellular compartments such as the lysosome(11-13). The dye is taken up by diffusion and through the circulation. Since penetration is not a hindrance, visualization of PCD in thick tissues and multi-layer cultures is possible(12,13). In contrast, TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) analysis(14), is limited to small samples, histological sections, and monolayer cultures because the procedure requires the entry/permeability of a terminal transferase. In contrast to Aniline blue, which diffuses and is dissolved by solvents, LysoTracker Red DND-99 is fixable, bright, and stable. Staining can be visualized with standard fluorescent or confocal microscopy in whole-mount or section using aqueous or solvent-based mounting media(12,13). Here we describe protocols using this dye to look at PCD in normal and sonic hedgehog null mouse embryos. In addition, we demonstrate analysis of PCD in differentiating ES cell cultures and present a simple quantification method. In summary, LysoTracker staining can be a great complement to other methods of detecting PCD.

BACKGROUND A critical event in neural tube closure is the formation of median hinge points (MHPs) and dorsolateral hinge points (DLHPs). Together, they buckle the ventral midline and elevate and juxtapose the neural folds for proper neural tube closure. Dynamic cell behaviors occur at hinge points (HPs), but their molecular regulation is largely unexplored. Bone morphogenetic proteins (BMPs) have been implicated in a variety of neural tube closure defects, although the underlying mechanisms are poorly understood. METHODS In this study, we used in vivo electroporations, high-resolution microscopy, and biochemical analyses to explore the role of BMP signaling in chick midbrain neural tube closure. RESULTS We identified a cell-cycle–dependent BMP gradient in the midbrain neural plate, which results in low-level BMP activity at the MHP. We show that although BMP signaling does not have a role in midbrain cell-fate specification, its attenuation is necessary and sufficient for MHP formation and midbrain closure. BMP blockade induces MHP formation by regulating apical constriction and basal nuclear migration. Furthermore, BMP signaling is critically important for maintaining epithelial organization by biochemically interacting with apicobasal polarity proteins (e.g., PAR3). As a result, prolonged BMP blockade disrupts apical junctions, desegregating the apical (PAR3+, ZO1+) and basolateral (LGL+) compartments. Direct apical LGL-GFP misexpression in turn is sufficient to induce ectopic HPs. CONCLUSIONS BMPs have a critical role in maintaining epithelial organization, a role that is conserved across species and tissue types. Its cell-cycle–dependent modulation in the neural plate dynamically regulates apicobasal polarity and helps to bend, shape, and close the neural tube. Birth Defects Research (Part A) 2012. © 2012 Wiley Periodicals, Inc.

Although Hedgehog (HH) signaling plays a critical role in patterning the ventral midbrain, its role in early midbrain specification is not known. We examined the midbrains of sonic hedgehog (Shh) and smoothened (Smo) mutant mice where HH signaling is respectively attenuated and eliminated. We show that some ventral (Evx1+) cell fates are specified in the Shh−/− mouse in a Ptc1- and Gli1-independent manner. HH-independent ventral midbrain induction was further confirmed by the presence of a Pax7-negative ventral midbrain territory in both Shh−/− and Smo−/− mice at and before embryonic day (E) 8.5. Midbrain signaling centers are severely disrupted in the Shh−/− mutant. Interestingly, dorsal markers are up-regulated (Wnt1, Gdf7, Pax7), down-regulated (Lfng), or otherwise altered (Zic1) in the Shh−/− midbrain. Together with the increased cell death seen specifically in Shh−/− dorsal midbrains (E8.5–E9), our results suggest specific regulation of dorsal patterning by SHH, rather than a simple deregulation due to its absence. Developmental Dynamics 237:1359-1372, 2008. © 2008 Wiley-Liss, Inc.