Rac1 and Nectin3 are essential for PCP-directed axon guidance in the peripheral auditory system.

Our sense of hearing is critically dependent on the spiral ganglion neurons (SGNs) that connect the sound receptors in the organ of Corti (OC) to the cochlear nuclei of the hindbrain. Type I SGNs innervate inner hair cells (IHCs) to transmit sound signals, while type II SGNs (SGNIIs) innervate outer hair cells (OHCs) to detect moderate-to-intense sound. During development, SGNII afferents make a characteristic 90-degree turn toward the base of the cochlea and innervate multiple OHCs. It has been shown that the Planar Cell Polarity (PCP) pathway acts non-autonomously to mediate environmental cues in the cochlear epithelium for SGNII afferent turning towards the base. However, the underlying mechanisms are unknown. Here, we present evidence that PCP signaling regulates multiple downstream effectors to influence cell adhesion and the cytoskeleton in cochlear supporting cells (SCs), which serve as intermediate targets of SGNII afferents. We show that the core PCP gene Vangl2 regulates the localization of the small GTPase Rac1 and the cell adhesion molecule Nectin3 at SC-SC junctions through which SGNII afferents travel. Through in vivo genetic analysis, we also show that loss of Rac1 or Nectin3 partially phenocopied SGNII peripheral afferent turning defects in Vangl2 mutants, and that Rac1 plays a non-autonomous role in this process in part by regulating PCP protein localization at the SC-SC junctions. Additionally, epistasis analysis indicates that Nectin3 and Rac1 likely act in the same genetic pathway to control SGNII afferent turning. Together, these experiments identify Nectin3 and Rac1 as novel regulators of PCP-directed SGNII axon guidance in the cochlea. Significance statement: Planar Cell Polarity (PCP) signaling plays a non-autonomous role in the guidance of type II spiral ganglion neuron (SGNII) afferent projections that innervate cochlear hair cells. However, little is known about the underlying mechanisms. Here, we identify the small GTPase Rac1 and the cell adhesion molecule Nectin3 as two downstream effectors of PCP signaling in SGNII afferent guidance. We show that PCP signaling regulates Rac1 and Nectin3 localization in cochlear supporting cells that serve as intermediate targets for SGNII afferents and that Rac1 and Nectin3 likely act in the same genetic pathway to non-autonomously regulate SGNII afferent guidance. These findings significantly advance our understanding of auditory circuit assembly and shed light on PCP-directed axon guidance mechanisms.

The primitive streak and cellular principles of building an amniote body through gastrulation.

The primitive streak, a transient embryonic structure, marks bilateral symmetry in mammalian and avian embryos and helps confer anterior-posterior and dorsal-ventral spatial information to early differentiating cells during gastrulation. Its recapitulation in vitro may facilitate derivation of tissues and organs with in vivo­like complexity. Proper understanding of the primitive streak and what it entails in human development is key to achieving such research objectives. Here we provide an overview of the primitive streak and conclude that this structure is neither conserved nor necessary for gastrulation or early lineage diversification. We offer a model in which the primitive streak is viewed as part of a morphologically diverse yet molecularly conserved process of spatial coordinate acquisition. We predict that recapitulation of the primitive streak is dispensable for development in vitro.

Scribble mutation disrupts convergent extension and apical constriction during mammalian neural tube closure.

Morphogenesis of the vertebrate neural tube occurs by elongation and bending of the neural plate, tissue shape changes that are driven at the cellular level by polarized cell intercalation and cell shape changes, notably apical constriction and cell wedging. Coordinated cell intercalation, apical constriction, and wedging undoubtedly require complex underlying cytoskeletal dynamics and remodeling of adhesions. Mutations of the gene encoding Scribble result in neural tube defects in mice, however the cellular and molecular mechanisms by which Scrib regulates neural cell behavior remain unknown. Analysis of Scribble mutants revealed defects in neural tissue shape changes, and live cell imaging of mouse embryos showed that the Scrib mutation results in defects in polarized cell intercalation, particularly in rosette resolution, and failure of both cell apical constriction and cell wedging. Scrib mutant embryos displayed aberrant expression of the junctional proteins ZO-1, Par3, Par6, E- and N-cadherins, and the cytoskeletal proteins actin and myosin. These findings show that Scribble has a central role in organizing the molecular complexes regulating the morphomechanical neural cell behaviors underlying vertebrate neurulation, and they advance our understanding of the molecular mechanisms involved in mammalian neural tube closure.

Pulsed actomyosin contractions in morphogenesis.

Cell and tissue shape changes are the fundamental elements of morphogenesis that drive normal development of embryos into fully functional organisms. This requires a variety of cellular processes including establishment and maintenance of polarity, tissue growth and apoptosis, and cell differentiation, rearrangement, and migration. It is widely appreciated that the cytoskeletal networks play an important role in regulating many of these processes and, in particular, that pulsed actomyosin contractions are a core cellular mechanism driving cell shape changes and cell rearrangement. In this review, we discuss the role of pulsed actomyosin contractions during developmental morphogenesis, advances in our understanding of the mechanisms regulating actomyosin pulsing, and novel techniques to probe the role of pulsed actomyosin processes in in vivo model systems.

Convergent extension in the amphibian, Xenopus laevis.

This review is a comprehensive analysis of the cell biology and biomechanics of Convergent Extension in Xenopus.

Convergent extension in mammalian morphogenesis.

Convergent extension is a fundamental morphogenetic process that underlies not only the generation of the elongated vertebrate body plan from the initially radially symmetrical embryo, but also the specific shape changes characteristic of many individual tissues. These tissue shape changes are the result of specific cell behaviors, coordinated in time and space, and affected by the physical properties of the tissue. While mediolateral cell intercalation is the classic cellular mechanism for producing tissue convergence and extension, other cell behaviors can also provide similar tissue-scale distortions or can modulate the effects of mediolateral cell intercalation to sculpt a specific shape. Regulation of regional tissue morphogenesis through planar polarization of the variety of underlying cell behaviors is well-recognized, but as yet is not well understood at the molecular level. Here, we review recent advances in understanding the cellular basis for convergence and extension and its regulation.

Tgif1 and Tgif2 Repress Expression of the RabGAP Evi5l.

Mouse embryos conditionally lacking Tgif1 and Tgif2 have holoprosencephaly and defects in left-right asymmetry. To identify pathways affected by loss of Tgif function during embryogenesis, we performed transcriptome profiling on whole mouse embryos. Among the genes with altered expression in embryos lacking Tgifs were a number with links to cilium function. One of these, Evi5l, encodes a RabGAP that is known to block the formation of cilia when overexpressed. Evi5l expression is increased in Tgif1; Tgif2-null embryos and in double-null mouse embryo fibroblasts (MEFs). Knockdown of Tgifs in a human retinal pigment epithelial cell line also increased EVI5L expression. We show that TGIF1 binds to a conserved consensus TGIF site 5' of the human and mouse Evi5l genes and represses Evi5l expression. In primary MEFs lacking both Tgifs, the number of cells with primary cilia was significantly decreased, and we observed a reduction in the transcriptional response to Shh pathway activation. Reducing Evi5l expression in MEFs lacking Tgifs resulted in a partial restoration of cilium numbers and in the transcriptional response to activation of the Shh pathway. In summary, this work shows that Tgifs regulate ciliogenesis and suggests that Evi5l mediates at least part of this effect.

Genetic and Molecular Analyses indicate independent effects of TGIFs on Nodal and Gli3 in neural tube patterning.

Holoprosencephaly (HPE) is a prevalent craniofacial developmental disorder that has both genetic and environmental causes. The gene encoding TG-interacting factor 1 (TGIF1) is among those that are routinely screened in HPE patients. However, the mechanisms by which TGIF1 variants cause HPE are not fully understood. TGIF1 is a transcriptional repressor that limits the output of the Transforming Growth Factor ß (TGFß)/Nodal signaling pathway, and HPE in patients with TGIF1 variants has been suggested to be due to increased Nodal signaling. Mice lacking both Tgif1 and its paralog, Tgif2, have HPE, and embryos lacking Tgif function do not survive past mid-gestation. Here, we show that in the presence of a Nodal heterozygous mutation, proliferation defects are rescued and a proportion of embryos lacking all Tgif function survive to late gestation. However, these embryos have a classic HPE phenotype, suggesting that this is a Nodal-independent effect of Tgif loss of function. Further, we show that the Gli3 gene is a direct target for repression by Tgifs, independent of TGFß/Nodal signaling, consistent with Tgif mutations causing HPE via Nodal-independent effects on the Sonic Hedgehog (Shh) pathway. Based on this work, we propose a model for distinct functions of Tgifs in the Nodal and Shh/Gli3 pathways during forebrain development.

Protein tyrosine kinase 7 is essential for tubular morphogenesis of the Wolffian duct.

The Wolffian duct, the proximal end of the mesonephric duct, undergoes non-branching morphogenesis to achieve an optimal length and size for sperm maturation. It is important to examine the mechanisms by which the developing mouse Wolffian duct elongates and coils for without proper morphogenesis, male infertility will result. Here we show that highly proliferative epithelial cells divide in a random orientation relative to the elongation axis in the developing Wolffian duct. Convergent extension (CE)-like of cell rearrangements is required for elongating the duct while maintaining a relatively unchanged duct diameter. The Wolffian duct epithelium is planar polarized, which is characterized by oriented cell elongation, oriented cell rearrangements, and polarized activity of regulatory light chain of myosin II. Conditional deletion of protein tyrosine kinase 7 (PTK7), a regulator of planar cell polarity (PCP), from mesoderm results in loss of the PCP characteristics in the Wolffian duct epithelium. Although loss of Ptk7 does not alter cell proliferation or division orientation, it affects CE and leads to the duct with significantly shortened length, increased diameter, and reduced coiling, which eventually results in loss of sperm motility, a key component of sperm maturation. In vitro experiments utilizing inhibitors of myosin II results in reduced elongation and coiling, similar to the phenotype of Ptk7 knockout. This data suggest that PTK7 signaling through myosin II regulates PCP, which in turn ensures CE-like of cell rearrangements to drive elongation and coiling of the Wolffian duct. Therefore, PTK7 is essential for Wolffian duct morphogenesis and male fertility.

Tissue morphodynamics shaping the early mouse embryo.

Generation of the elongated vertebrate body plan from the initially radially symmetrical embryo requires comprehensive changes to tissue form. These shape changes are generated by specific underlying cell behaviors, coordinated in time and space. Major principles and also specifics are emerging, from studies in many model systems, of the cell and physical biology of how region-specific cell behaviors produce regional tissue morphogenesis, and how these, in turn, are integrated at the level of the embryo. New technical approaches have made it possible more recently, to examine the morphogenesis of the mouse embryo in depth, and to elucidate the underlying cellular mechanisms. This review focuses on recent advances in understanding the cellular basis for the early fundamental events that establish the basic form of the embryo.

Distinct apical and basolateral mechanisms drive planar cell polarity-dependent convergent extension of the mouse neural plate.

The mechanisms of tissue convergence and extension (CE) driving axial elongation in mammalian embryos, and in particular, the cellular behaviors underlying CE in the epithelial neural tissue, have not been identified. Here we show that mouse neural cells undergo mediolaterally biased cell intercalation and exhibit both apical boundary rearrangement and polarized basolateral protrusive activity. Planar polarization and coordination of these two cell behaviors are essential for neural CE, as shown by failure of mediolateral intercalation in embryos mutant for two proteins associated with planar cell polarity signaling: Vangl2 and Ptk7. Embryos with mutations in Ptk7 fail to polarize cell behaviors within the plane of the tissue, whereas Vangl2 mutant embryos maintain tissue polarity and basal protrusive activity but are deficient in apical neighbor exchange. Neuroepithelial cells in both mutants fail to apically constrict, leading to craniorachischisis. These results reveal a cooperative mechanism for cell rearrangement during epithelial morphogenesis.

Financial and employment impacts of serious injury: a qualitative study.

OBJECTIVES: To explore the financial and employment impacts following serious injury. DESIGN: Semi-structured telephone administered qualitative interviews with purposive sampling and thematic qualitative analysis. PARTICIPANTS: 118 patients (18-81 years) registered by the Victorian State Trauma Registry or Victorian Orthopaedic Trauma Outcomes Registry 12-24 months post-injury. RESULTS: Key findings of the study were that although out-of-pocket treatment costs were generally low, financial hardship was prevalent after hospitalisation for serious injury, and was predominantly experienced by working age patients due to prolonged absences from paid employment. Where participants were financially pressured prior to injury, injury further exacerbated these financial concerns. Reliance on savings and loans and the need to budget carefully to limit financial burden were discussed. Financial implications of loss of income were generally less for those covered by compensation schemes, with non-compensable participants requiring welfare payments due to an inability to earn an income. Most participants reported that the injury had a negative impact on work. Loss of earnings payments from injury compensation schemes and income protection policies, supportive employers, and return to work programs were perceived as key factors in reducing the financial burden of injured participants. Employer-related barriers to return to work included the employer not listening to the needs of the injured participant, not understanding their physical limitations, and placing unrealistic expectations on the injured person. While the financial benefits of compensation schemes were acknowledged, issues accessing entitlements and delays in receiving benefits were commonly reported by participants, suggesting that improvements in scheme processes could have substantial benefits for injured patients. CONCLUSIONS: Seriously injured patients commonly experienced substantial financial and work-related impacts of injury. Participants of working age who were unemployed prior to injury, did not have extensive leave accrual at their pre-injury employment, and those not covered by injury compensation schemes or income protection insurance clearly represent participants "at risk" for substantial financial hardship post-injury. Early identification of these patients, and improved provision of information about financial support services, budgeting and work retraining could assist in alleviating financial stress after injury.

Patient perspectives of care in a regionalised trauma system: lessons from the Victorian State Trauma System.

OBJECTIVES: To explore injured patients' experiences of trauma care to identify areas for improvement in service delivery. DESIGN, SETTING AND PARTICIPANTS: Qualitative study using in-depth, semi-structured interviews, conducted from 1 April 2011 to 31 January 2012, with 120 trauma patients registered by the Victorian State Trauma Registry and the Victorian Orthopaedic Trauma Outcomes Registry and managed at the major adult trauma services (MTS) in Victoria. MAIN OUTCOME MEASURES: Emergent themes from patients' experiences of acute, rehabilitation and post-discharge care in the Victorian State Trauma System (VSTS). RESULTS: Patients perceived their acute hospital care as high quality, although 3s with communication and surgical management delays were common. Discharge from hospital was perceived as stressful, and many felt ill prepared for discharge. A consistent emerging theme was the sense of a lack of coordination of post-discharge care, and the absence of a consistent point of contact for ongoing management. Most patients' primary point of contact after discharge was outpatient clinics at the MTS, which were widely criticised because of substantial delays in receiving an appointment, prolonged waiting times, limited time with clinicians, lack of continuity of care and inability to see senior clinicians. CONCLUSIONS: This study highlights perceived 3s in the patient care pathway in the VSTS, especially those relating to communication, information provision and post-discharge care. Trauma patients perceived the need for a single point of contact for coordination of post-discharge care.

Evaluating time points for measuring recovery after major trauma in adults.

OBJECTIVE: To evaluate recovery after major trauma over a 24-month time frame. BACKGROUND: Measuring disability after injury is seen as increasingly important but requires knowledge not only of the measures that should be implemented but also of the critical time points for follow-up. METHODS: Six hundred sixty-two adult major trauma patients from 2 level 1 trauma centers (October 2006 to March 2007) were followed up by telephone at 6-, 12-, 18-, and 24 months after injury. SF-12, Glasgow Outcome Scale-Extended (GOS-E), pain scores, and return to work (RTW) were collected. Multilevel mixed-effects regression models were fitted to analyze change in outcomes over time. RESULTS: Six hundred seventeen (93%) were followed up for at least 1 time point. Functional recovery (GOS-E = 8) [odds ratio (OR) 3.1, 95% CI: 1.9, 5.0] and RTW (OR 2.4, 95% CI: 1.4, 4.0) improved, and physical health (PCS-12) scores were better (mean difference 1.9, 95% CI: 0.9, 2.9), from 6 to 12 months after injury, but changed little from 12 months. Pain scores were unchanged from 6 to 12 months but were higher at 18 months than at 12 months (OR 1.8, 95% CI: 1.2, 2.8). SF-12 mental health (MCS-12) scores decreased until 18 months but improved from 18 to 24 months (mean difference 1.5, 95% CI: 0.2, 2.8). The rate of recovery differed by injury group and age. CONCLUSIONS: Different patterns of recovery were evident for each outcome, and there was a variation in the rate of recovery for some subgroups. The selection of time points for follow-up requires consideration of the outcome measurements of interest and the population being studied.

Does recall of preinjury disability change over time?

BACKGROUND: Pre-injury disability must be determined when assessing whether treatment programs return people to pre-injury status, however there is little empirical evidence to support recommendations that this be done as soon as possible after injury to prevent recall bias. OBJECTIVES: To determine disagreement between recall of pre-injury disability at different time points post-injury and bias towards under- or overestimating pre-injury disability. METHODS: Self-reported pre-injury global disability was assessed within days, 6 months and 12 months post-injury in patients admitted to two level 1 adult trauma centres. Kappa statistics and multiple logistic regression models identified predictors of disagreement between time-points. RESULTS: Pre-injury disability was measured at all time-points in 801 patients. Pre-injury disability at baseline was rated as none, mild, moderate, marked and severe in 80%, 12%, 5.1%, 1.9% and 1.0% respectively. Absolute agreement between baseline and 6 and 12 months respectively, was 79% and 80%. Corresponding kappa values (95% confidence intervals) were 0.33 (0.26-0.40) and 0.32 (0-25-0.38). Patients over 65 years or not completing high school were more likely to report less pre-injury disability at 6 and 12 months than at baseline with adjusted odds ratios (95% confidence intervals) for these groups being 8.24 (4.32-15.72) and 1.93 (1.03-3.64) respectively. CONCLUSIONS: There was little evidence of recall bias in an adult trauma population if self-reported global pre-injury disability was assessed 6 months post-injury. The recall of pre-injury disability up to 6 months post-injury can be used to determine return to pre-injury status, if assessment is not feasible shortly after injury.

Improved functional outcomes for major trauma patients in a regionalized, inclusive trauma system.

OBJECTIVE: To describe outcomes of major trauma survivors managed in an organized trauma system, including the association between levels of care and outcomes over time. BACKGROUND: Trauma care systems aim to reduce deaths and disability. Studies have found that regionalization of trauma care reduces mortality but the impact on quality of survival is unknown. Evaluation of a trauma system should include mortality and morbidity. METHODS: Predictors of 12-month functional (Glasgow Outcome Scale-Extended) outcomes after blunt major trauma (Injury Severity Score >15) in an organized trauma system were explored using ordered logistic regression for the period October 2006 to June 2009. Data from the population-based Victorian State Trauma Registry were used. RESULTS: There were 4986 patients older than 18 years. In-hospital mortality decreased from 11.9% in 2006-2007 to 9.9% in 2008-2009. The follow-up rate at 12 months was 86% (n = 3824). Eighty percent reported functional limitations. Odds of better functional outcome increased in the 2007-2008 [adjusted odds ratio (AOR): 1.22; 95% CI: 1.05, 1.41] and 2008-2009 (AOR: 1.16; 95% CI: 1.01, 1.34) years compared with 2006-2007. Cases managed at major trauma services (MTS) achieved better functional outcome (AOR: 1.22; 95% CI: 1.03, 1.45). Female gender, older age, and lower levels of education demonstrated lower adjusted odds of better outcome. CONCLUSIONS: Despite an annual decline in mortality, risk-adjusted functional outcomes improved over time, and cases managed at MTS (level-1 trauma centers) demonstrated better functional outcomes. The findings provide early evidence that this inclusive, regionalized trauma system is achieving its aims.

Level of agreement between patient and proxy responses to the EQ-5D health questionnaire 12 months after injury.

BACKGROUND: Health-related quality of life represents a patient's experiences and expectations and should be collected from the patient. In trauma, collection of information from the patient can be challenging, particularly for subgroups where cognitive impairment is prevalent, increasing reliance on proxy reporting. This study assessed the agreement between patient and proxy reporting of health-related quality of life 12 months after injury. METHODS: The Victorian State Trauma Registry and Victorian Orthopaedic Trauma Outcomes Registry collect EQ-5D data at 12 months after injury. Cases where data were collected from the patient and proxy were extracted. Agreement between patient and proxy responses was compared using kappa (K) coefficients for the individual EQ-5D items, and Bland-Altman plots and Wilcoxon signed-rank tests for the EQ-5D summary score and visual analog scale (VAS). RESULTS: Agreement between patient and proxy respondents was substantial for the mobility (K = 0.61) and personal care items (K = 0.67) and moderate for the usual activities (K = 0.50), pain/discomfort (K = 0.42), and anxiety/depression items (K = 0.47). The mean difference between proxy and patient-reported scores for the VAS (0.74, 95% confidence interval: -2.73, 4.21) and the EQ-5D summary score (-0.02, 95% confidence interval: -0.07, 0.03) was small, but the limits of agreement were wide (-34.22 to 35.71 for VAS and -0.55 to 0.51 for summary score), suggesting no systematic bias. CONCLUSIONS: Although proxy and patient responses for the EQ-5D VAS may differ, the differences show random variability rather than systematic bias. Group comparisons using proxy responses are unlikely to be biased, but proxy responses should be used with caution when assessing individual patient recovery.

Loss of Tgif function causes holoprosencephaly by disrupting the SHH signaling pathway.

Holoprosencephaly (HPE) is a severe human genetic disease affecting craniofacial development, with an incidence of up to 1/250 human conceptions and 1.3 per 10,000 live births. Mutations in the Sonic Hedgehog (SHH) gene result in HPE in humans and mice, and the Shh pathway is targeted by other mutations that cause HPE. However, at least 12 loci are associated with HPE in humans, suggesting that defects in other pathways contribute to this disease. Although the TGIF1 (TG-interacting factor) gene maps to the HPE4 locus, and heterozygous loss of function TGIF1 mutations are associated with HPE, mouse models have not yet explained how loss of Tgif1 causes HPE. Using a conditional Tgif1 allele, we show that mouse embryos lacking both Tgif1 and the related Tgif2 have HPE-like phenotypes reminiscent of Shh null embryos. Eye and nasal field separation is defective, and forebrain patterning is disrupted in embryos lacking both Tgifs. Early anterior patterning is relatively normal, but expression of Shh is reduced in the forebrain, and Gli3 expression is up-regulated throughout the neural tube. Gli3 acts primarily as an antagonist of Shh function, and the introduction of a heterozygous Gli3 mutation into embryos lacking both Tgif genes partially rescues Shh signaling, nasal field separation, and HPE. Tgif1 and Tgif2 are transcriptional repressors that limit Transforming Growth Factor ß/Nodal signaling, and we show that reducing Nodal signaling in embryos lacking both Tgifs reduces the severity of HPE and partially restores the output of Shh signaling. Together, these results support a model in which Tgif function limits Nodal signaling to maintain the appropriate output of the Shh pathway in the forebrain. These data show for the first time that Tgif1 mutation in mouse contributes to HPE pathogenesis and provide evidence that this is due to disruption of the Shh pathway.