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Adult-onset hearing loss is very common, but we know little about the underlying molecular pathogenesis impeding the development of therapies. We took a genetic approach to identify new molecules involved in hearing loss by screening a large cohort of newly generated mouse mutants using a sensitive electrophysiological test, the auditory brainstem response (ABR). We review here the findings from this screen. Thirty-eight unexpected genes associated with raised thresholds were detected from our unbiased sample of 1,211 genes tested, suggesting extreme genetic heterogeneity. A wide range of auditory pathophysiologies was found, and some mutant lines showed normal development followed by deterioration of responses, revealing new molecular pathways involved in progressive hearing loss. Several of the genes were associated with the range of hearing thresholds in the human population and one, SPNS2, was involved in childhood deafness. The new pathways required for maintenance of hearing discovered by this screen present new therapeutic opportunities.

The developing cerebellum of amniotes is characterised by a unique, transient, secondary proliferation zone: the external germinal layer (EGL). The EGL is comprised solely of granule cell precursors, whose progeny migrate inwardly to form the internal granule cell layer. While a range of cell morphologies in the EGL has long been known, how they reflect the cells’ differentiation status has previously only been inferred. Observations have suggested a deterministic maturation from outer to inner EGL that we wished to test experimentally. To do this, we electroporated granule cell precursors in chick with plasmids encoding fluorescent proteins and probed labelled cells with markers of both proliferation (phosphohistone H3) and differentiation (Axonin1/TAG1 and NeuroD1). We show that granule cell precursors can display a range of complex forms throughout the EGL while mitotically active. Overexpression of full length NeuroD1 within granule cell precursors does not abolish proliferation, but biases granule cells towards precocious differentiation, alters their migration path and results in a smaller and less foliated cerebellum. Our results show that granule cells show a greater flexibility in differentiation than previously assumed. We speculate that this allows the EGL to regulate its proliferative activity in response to overall patterns of cerebellar growth.

Background Mice carrying targeted mutations are important for investigating gene function and the role of genes in disease, but off-target mutagenic effects associated with the processes of generating targeted alleles, for instance using Crispr, and culturing embryonic stem cells, offer opportunities for spontaneous mutations to arise. Identifying spontaneous mutations relies on the detection of phenotypes segregating independently of targeted alleles, and having a broad estimate of the level of mutations generated by intensive breeding programmes is difficult given that many phenotypes are easy to miss if not specifically looked for. Here we present data from a large, targeted knockout programme in which mice were analysed through a phenotyping pipeline. Such spontaneous mutations segregating within mutant lines may confound phenotypic analyses, highlighting the importance of record-keeping and maintaining correct pedigrees. Results Twenty-five lines out of 1311 displayed different deafness phenotypes that did not segregate with the targeted allele. We observed a variety of phenotypes by Auditory Brainstem Response (ABR) and behavioural assessment and isolated eight lines showing early-onset severe progressive hearing loss, later-onset progressive hearing loss, low frequency hearing loss, or complete deafness, with vestibular dysfunction. The causative mutations identified include deletions, insertions, and point mutations, some of which involve new genes not previously associated with deafness while others are new alleles of genes known to underlie hearing loss. Two of the latter show a phenotype much reduced in severity compared to other mutant alleles of the same gene. We investigated the ES cells from which these lines were derived and determined that only one of the 8 mutations could have arisen in the ES cell, and in that case, only after targeting. Instead, most of the non-segregating mutations appear to have occurred during breeding of mutant mice. In one case, the mutation arose within the wildtype colony used for expanding mutant lines. Conclusions Our data show that spontaneous mutations with observable effects on phenotype are a common side effect of intensive breeding programmes, including those underlying targeted mutation programmes. Such spontaneous mutations segregating within mutant lines may confound phenotypic analyses, highlighting the importance of record-keeping and maintaining correct pedigrees.

Cerebellar granule cell precursors (GCP) are the most abundant neuronal progenitor population in the brain, which, given the structural simplicity of the cerebellum make them a good model for investigating mechanisms regulating neurogenesis. GCPs are born at the rhombic lip; the germinal zone of glutamatergic neurons in the cerebellar anlage from E6 in the chick. From the rhombic lip GCPs undergo subpial, tangential migration to form a transiently proliferative layer; the external granule layer (EGL), followed by inward radial migration towards the inner granule cell layer (IGL) once they have undergone neurogenic differentiation. GCPs undergo massive proliferation within the EGL, influenced by Sonic Hedgehog (SHH) from underlying Purkinje neurons, however triggers for termination of GCP proliferation are poorly understood. Through spatiotemporal characterisation and in ovo targeted genetic dysregulation of the bone morphogenetic protein (BMP) signalling pathway, a known inhibitor of SHH, in the embryonic chicken hindbrain, I have shown similarities in expression of BMP activity between chick and human cerebellar development and suggest a novel role for BMP signalling during GCP development in the chick. In vivo experimental data shows that inhibition of BMP signalling affects the recruitment to and subpial migration of granule cell precursors along the EGL, however not their specification. GCPs can be recruited to the EGL following upregulation of BMP signalling, however, following the onset of SHH signalling at E8 GCPs in the EGL exit the cell cycle, differentiate, and radially migrate inwards along the Bergmann glia fibres, which are shown here to mature at E8 in the chick. This novel role is temporally intermediate to those previously described during early rhombic lip specification and later in the induction of differentiation and suggest that BMP signalling is involved in regulating the migration of granule cells from their specification at the rhombic lip to their final destination in the granule cell layer.

The external granule layer (EGL) is a transient proliferative layer that gives rise to cerebellar granule cell neurons. Extensive EGL proliferation characterises the foliated structure of amniote cerebella, but the factors that regulate EGL formation, amplification within it, and differentiation from it, are incompletely understood. Here, we characterise bone morphogenic protein (BMP) signalling during cerebellar development in chick and human and show that while in chick BMP signalling correlates with external granule layer formation, in humans BMP signalling is maintained throughout the external granule layer after the onset of foliation. We also show via Immunohistochemical labelling of phosphorylated Smad1/5/9, that the spatiotemporal activity of BMP signalling is conserved between chick and human. Using in ovo electroporation in chick, we demonstrate that BMP signalling is necessary for subpial migration of granule cell precursors and hence the formation of the external granule layer (EGL) prior to transit amplification. However, altering BMP signalling does not block the formation of mature granule neurons but significantly disrupts that pattern of morphological transitions that accompany transit amplification. Our results elucidate two key, temporally distinct roles for BMP signalling in vivo in organising first the assembly of the EGL from the rhombic lip and subsequently the tempo of granule neuron production within the EGL.Competing Interest StatementThe authors have declared no competing interest.Footnotes* We have revised our manuscript significantly in response to reviewers' comments and in particular made significant additions comprising: - a new comparative timeline for cerebellar development across amniote species (human, mouse, chick). - quantification of cell numbers in folia tips vs troughs for both chick and human. - quantification of pSmad expression at e5 following Smad expression manipulation at e3 in chick. - quantification of cellular morphology at e7 following Smad expression manipulation at e4 in chick. - quantification of sub-pial layer thickness at e7 and e8 in chick. - new data illustrating cellular morphology following double electroporation at e2 (with tol2-GFP) and e4 (with induction of Smad6) that distinguishes wild type from Smad6-expressing cellular morphologies within the same cerebellum. We have also edited and re-written the manuscript to describe, explain, and interpret these new findings and to clarify the text in response to reviewers' comments.

Mice carrying targeted mutations are important for investigating gene function and the role of genes in disease, but the process of culturing embryonic stem cells during the making of a targeted allele offers opportunities for spontaneous mutations to arise. Identifying spontaneous mutations relies on the detection of phenotypes segregating independently of targeted alleles, and many phenotypes are easy to miss if not specifically looked for. Here we present data from a large, targeted knockout programme in which mice were analysed through a phenotyping pipeline. Twenty-five lines out of 1311 displayed different deafness phenotypes that did not segregate with the targeted allele. We have identified 8 different mutations causing deafness in 16 of these 25 lines and characterised the resulting phenotypes. Our data show that spontaneous mutations with observable effects on phenotype are a common side effect of intensive breeding programmes, including those underlying targeted mutation programmes.

The yolk sac (YS) represents an evolutionarily-conserved extraembryonic structure that ensures timely delivery of nutritional support and oxygen to the developing embryo. However, the YS remains ill-defined in humans. We therefore assemble a complete single cell 3D map of human YS from 3-8 post conception weeks by integrating multiomic protein and gene expression data. We reveal the YS as a site of primitive and definitive haematopoiesis including a YS-specific accelerated route to macrophage production, a source of nutritional/metabolic support and a regulator of oxygen-carrying capacity. We reconstruct the emergence of primitive haematopoietic stem and progenitor cells from YS hemogenic endothelium and their decline upon stromal support modulation as intraembryonic organs specialise to assume these functions. The YS therefore functions as ‘three organs in one’ revealing a multifaceted relay of vital organismal functions as pregnancy proceeds. Human yolk sac is a key staging post in a relay of vital organismal functions during human pregnancy.

Objective Public Involvement (PI) in applied health and social care research has grown exponentially in the UK. This review aims to synthesise published UK evidence that evaluates the process and/or outcome(s) of PI in applied health and social care research to identify key contextual factors, effective strategies, outcomes and public partner experiences underpinning meaningful PI in research. Methods Following a pre‐registered protocol, we systematically searched four databases and two key journals for studies conducted within the UK between January 2006 and July 2024. A team of public partners and researchers carried out independent dual screening and data extraction. Included studies were narratively synthesised via Framework Synthesis. Results Nineteen studies evaluated the PI process with a range of populations including National Health Service (NHS) users, carers, and low‐income communities. No specific outcome evaluations were identified. Through their experience, public partners described important components of meaningful PI such as mutual respect and seeing and contributing to change, as well as some unintended harms of involvement. Harms related to ‘experiencing negative attitudes’, ‘emotional burden of involvement’, ‘frustration and disappointment’ and ‘further marginalisation’. Meaningful PI was underpinned by structural, organisational, interpersonal and individual factors; as well as practical and principle‐based strategies of involvement. Both public partners and researchers reflected on a range of outcomes of meaningful PI including changes to the research process and longer term impacts on organisations, researchers and public partners. Conclusions PI in research must be facilitated at multiple levels to reduce unintended harm and encourage meaningful and impactful outcomes. Findings are summarised within a model which gives an overview of priorities for individual researchers, organisations and funders to ensure best practice is achievable. From a methodological perspective, researchers should prioritise robust, transparent and co‐produced approaches to evaluating PI to increase knowledge in the field. Patient and Public Involvement A regional public advisory network provided insight on the relevance and acceptability of the review concept. Our core research team included three public partners. Public partners contributed to the development of the initial review protocol, and full‐text screening, reviewing findings and their interpretation and writing the final report.

BackgroundTesting for germline pathogenic variants (GPVs) in cancer predisposition genes is increasingly offered as part of routine care for patients with cancer. This is often urgent in oncology clinics due to potential implications on treatment and surgical decisions. This also allows identification of family members who should be offered predictive genetic testing. In the UK, it is common practice for healthcare professionals to provide a patient information leaflet (PIL) at point of care for diagnostic genetic testing in patients with cancer, after results disclosure when a GPV is identified, and for predictive testing of at-risk relatives. Services usually create their own PIL, resulting in duplication of effort and wide variability regarding format, content, signposting and patient input in co-design and evaluation.MethodsRepresentatives from UK Cancer Genetics Group (UKCGG), Cancer Research UK (CRUK) funded CanGene-CanVar programme and Association of Genetic Nurse Counsellors (AGNC) held a 2-day meeting with the aim of making recommendations for clinical practice regarding co-design of PIL for germline cancer susceptibility genetic testing. Lynch syndrome and haematological malignancies were chosen as exemplar conditions.ResultsMeeting participants included patient representatives including as co-chair, multidisciplinary clinicians and other experts from across the UK. High-level consensus for UK recommendations for clinical practice was reached on several aspects of PIL using digital polling, including that PIL should be offered, accessible, co-designed and evaluated with patients.ConclusionsRecommendations from the meeting are likely to be applicable for PIL co-design for a wide range of germline genetic testing scenarios.

Backgound: Survivorship clinics aim to identify and treat late-effects of cancer and subsequent treatment. Purpose/Objective: The purpose of this study is to assess the feasibility of using objective cognitive, social, physical, and dietary screening tools in the oncology survivorship clinic to identify intervention needs and referral rates among survivors of pediatric cancer when compared to traditional physician examination. Methods: Survivors of pediatric cancer attending the Pennsylvania State Hershey Long-Term Follow-up Clinic (n=12, age 12-27) participated in this study. Participants completed 4 objective screening tools: the Functional Mobility Assessment (FMA), Body Mass Index (BMI), modified Youth Risk Behavior Surveillance System (YRBSS), and Mini Mental State Exam (MMSE). A retrospective chart review of patients from this same clinic was completed for comparison of referral rates based on traditional physician exam. Results: The findings indicated that the use of these objective screening tools identified more participants (83.3%) as needing referral to specialists than traditional physician exam (26.7%). More than half (58.3%) of the participants had deficits in more than one area. No correlations were found between the objective measures. This small pilot study was limited by the small sample size and inability for comparisons to be made between referrals to specific specialists. Conclusion: Use of the FMA, BMI, modified YRBSS, and MMSE are feasible in the survivorship clinic setting. Additional investigation is needed to assess the YRBSS, MMSE, and alternative measures of social and cognitive functioning. Further assessment of the role of a physical therapist in the survivorship clinic is needed.