Shared features in ear and kidney development - implications for oto-renal syndromes.

The association between ear and kidney anomalies has long been recognized. However, little is known about the underlying mechanisms. In the last two decades, embryonic development of the inner ear and kidney has been studied extensively. Here, we describe the developmental pathways shared between both organs with particular emphasis on the genes that regulate signalling cross talk and the specification of progenitor cells and specialised cell types. We relate this to the clinical features of oto-renal syndromes and explore links to developmental mechanisms.

Evolution of the expression and regulation of the nuclear hormone receptor ERR gene family in the chordate lineage.

The Estrogen Related Receptor (ERR) nuclear hormone receptor genes have a wide diversity of roles in vertebrate development. In embryos, ERR genes are expressed in several tissues, including the central and peripheral nervous systems. Here we seek to establish the evolutionary history of chordate ERR genes, their expression and their regulation. We examine ERR expression in mollusc, amphioxus and sea squirt embryos, finding the single ERR orthologue is expressed in the nervous system in all three, with muscle expression also found in the two chordates. We show that most jawed vertebrates and lampreys have four ERR paralogues, and that vertebrate ERR genes were ancestrally linked to Estrogen Receptor genes. One of the lamprey paralogues shares conserved expression domains with jawed vertebrate ERRγ in the embryonic vestibuloacoustic ganglion, eye, brain and spinal cord. Hypothesising that conserved expression derives from conserved regulation, we identify a suite of pan-vertebrate conserved non-coding sequences in ERR introns. We use transgenesis in lamprey and chicken embryos to show that these sequences are regulatory and drive reporter gene expression in the nervous system. Our data suggest an ancient association between ERR and the nervous system, including expression in cells associated with photosensation and mechanosensation. This includes the origin in the vertebrate common ancestor of a suite of regulatory elements in the 3' introns that drove nervous system expression and have been conserved from this point onwards.

Zbtb16 mediates a switch between Fgf signalling regimes in the developing hindbrain.

Developing tissues are sequentially patterned by extracellular signals that are turned on and off at specific times. In the zebrafish hindbrain, fibroblast growth factor (Fgf) signalling has different roles at different developmental stages: in the early hindbrain, transient Fgf3 and Fgf8 signalling from rhombomere 4 is required for correct segmentation, whereas later, neuronal Fgf20 expression confines neurogenesis to specific spatial domains within each rhombomere. How the switch between these two signalling regimes is coordinated is not known. We present evidence that the Zbtb16 transcription factor is required for this transition to happen in an orderly fashion. Zbtb16 expression is high in the early anterior hindbrain, then gradually upregulated posteriorly and confined to neural progenitors. In mutants lacking functional Zbtb16, fgf3 expression fails to be downregulated and persists until a late stage, resulting in excess and more widespread Fgf signalling during neurogenesis. Accordingly, the spatial pattern of neurogenesis is disrupted in Zbtb16 mutants. Our results reveal how the distinct stage-specific roles of Fgf signalling are coordinated in the zebrafish hindbrain.

scRNA-sequencing in chick suggests a probabilistic model for cell fate allocation at the neural plate border.

The vertebrate 'neural plate border' is a transient territory located at the edge of the neural plate containing precursors for all ectodermal derivatives: the neural plate, neural crest, placodes and epidermis. Elegant functional experiments in a range of vertebrate models have provided an in-depth understanding of gene regulatory interactions within the ectoderm. However, these experiments conducted at tissue level raise seemingly contradictory models for fate allocation of individual cells. Here, we carry out single cell RNA sequencing of chick ectoderm from primitive streak to neurulation stage, to explore cell state diversity and heterogeneity. We characterise the dynamics of gene modules, allowing us to model the order of molecular events which take place as ectodermal fates segregate. Furthermore, we find that genes previously classified as neural plate border 'specifiers' typically exhibit dynamic expression patterns and are enriched in either neural, neural crest or placodal fates, revealing that the neural plate border should be seen as a heterogeneous ectodermal territory and not a discrete transitional transcriptional state. Analysis of neural, neural crest and placodal markers reveals that individual NPB cells co-express competing transcriptional programmes suggesting that their ultimate identify is not yet fixed. This population of 'border located undecided progenitors' (BLUPs) gradually diminishes as cell fate decisions take place. Considering our findings, we propose a probabilistic model for cell fate choice at the neural plate border. Our data suggest that the probability of a progenitor's daughters to contribute to a given ectodermal derivative is related to the balance of competing transcriptional programmes, which in turn are regulated by the spatiotemporal position of a progenitor.

A gene regulatory network for neural induction.

During early vertebrate development, signals from a special region of the embryo, the organizer, can redirect the fate of non-neural ectoderm cells to form a complete, patterned nervous system. This is called neural induction and has generally been imagined as a single signalling event, causing a switch of fate. Here, we undertake a comprehensive analysis, in very fine time course, of the events following exposure of competent ectoderm of the chick to the organizer (the tip of the primitive streak, Hensen's node). Using transcriptomics and epigenomics we generate a gene regulatory network comprising 175 transcriptional regulators and 5614 predicted interactions between them, with fine temporal dynamics from initial exposure to the signals to expression of mature neural plate markers. Using in situ hybridization, single-cell RNA-sequencing, and reporter assays, we show that the gene regulatory hierarchy of responses to a grafted organizer closely resembles the events of normal neural plate development. The study is accompanied by an extensive resource, including information about conservation of the predicted enhancers in other vertebrates.

Sox8 remodels the cranial ectoderm to generate the ear.

The vertebrate inner ear arises from a pool of progenitors with the potential to contribute to all the sense organs and cranial ganglia in the head. Here, we explore the molecular mechanisms that control ear specification from these precursors. Using a multiomics approach combined with loss-of-function experiments, we identify a core transcriptional circuit that imparts ear identity, along with a genome-wide characterization of noncoding elements that integrate this information. This analysis places the transcription factor Sox8 at the top of the ear determination network. Introducing Sox8 into the cranial ectoderm not only converts non-ear cells into ear progenitors but also activates the cellular programs for ear morphogenesis and neurogenesis. Thus, Sox8 has the unique ability to remodel transcriptional networks in the cranial ectoderm toward ear identity.

The chromatin remodelling factor Chd7 protects auditory neurons and sensory hair cells from stress-induced degeneration.

Neurons and sensory cells are particularly vulnerable to oxidative stress due to their high oxygen demand during stimulus perception and transmission. The mechanisms that protect them from stress-induced death and degeneration remain elusive. Here we show that embryonic deletion of the chromodomain helicase DNA-binding protein 7 (CHD7) in auditory neurons or hair cells leads to sensorineural hearing loss due to postnatal degeneration of both cell types. Mechanistically, we demonstrate that CHD7 controls the expression of major stress pathway components. In its absence, hair cells are hypersensitive, dying rapidly after brief exposure to stress inducers, suggesting that sound at the onset of hearing triggers their degeneration. In humans, CHD7 haploinsufficiency causes CHARGE syndrome, a disorder affecting multiple organs including the ear. Our findings suggest that CHD7 mutations cause developmentally silent phenotypes that predispose cells to postnatal degeneration due to a failure of protective mechanisms.

Fibroscan® probe selection for lean adults.

BACKGROUND AND AIM: Fibroscan® is used to assess fibrosis and steatosis of the liver noninvasively. The company suggests to use the S+-probe in people <18 years with a thoracic circumference (TC) between 45 and 75 cm and the M+-probe in children with a TC >75 cm and adults with a skin-liver capsule distance <2.5 cm. For lean adults with a TC ≤75 cm, no comparative studies have been performed. Furthermore, it is unclear whether lean adults need to be fasted before assessment. METHODS: We compared liver stiffness (LS) using Fibroscan® S+- and M+-probes and controlled attenuation parameter (CAP; only available for M+-probe) in healthy volunteers with a TC ≤75 cm compared with those with a TC >75 cm in fasting state and after intake of a standardized light meal (300 kcal). RESULTS: We examined 50 volunteers (26 female, 24 ± 3 years). Twenty-two participants were in the TC ≤75 cm group and 28 in TC >75 cm group. LS values with the S+-probe were 15% higher than with the M+-probe in both groups (median difference 0.6 kPa, P < 0.001). Both probes showed good agreement with minimal bias (Spearman correlation r = 0.754, P < 0.001; Interclass Correlation Coefficient 0.843, P < 0.001; Bland-Altman bias 0.6 ± 0.9 kPa, linear regression r 2 = 0.557, P < 0.001). Intake of a light meal had no relevant influence on LS (S+- and M+-probes) or CAP measurements (M+-probe) in both groups. CONCLUSION: Lean adults with a TC below 75 cm can be assessed with either the S+-probe or the M+-probe and may take a light meal before assessment.

Direct acting antiviral therapy rescues neutrophil dysfunction and reduces hemolysis in hepatitis C infection.

Chronic hepatitis C virus infection is characterized by multiple extra-hepatic manifestations. Innate immune dysfunction and hemolysis are symptoms which might be associated with each other. We investigated the impact of direct acting antivirals on neutrophil function and its connection to hemolysis. In this prospective study, 85 patients with or without cirrhosis and 21 healthy controls were included. Patients' blood samples were taken at baseline, at the end of therapy and at follow-up 12 weeks after end of therapy. Neutrophil phagocytosis, oxidative burst, and hemolysis parameters were studied. Multivariate analysis was performed to decipher the relationship between hemolysis and neutrophil function. Ex vivo cross-incubation experiments with neutrophils and serum fractions were done. Impaired neutrophil phagocytosis and mild hemolysis were observed in patients with and without cirrhosis. A proteome approach revealed different expression of hemolysis-related serum proteins in patients and controls. Direct acting antiviral therapy restored neutrophil function irrespective of severity of liver disease, achievement of sustained virologic response or type of drug and reduced hemolysis. Treatment with ribavirin delayed the improvement of neutrophil function. Statistical analysis revealed associations of haptoglobin with neutrophil phagocytic capacity. Neutrophil dysfunction could be transferred to healthy cells by incubation with patients' serum fractions (>30 kDa) ex vivo. Neutrophil dysfunction and hemolysis represent extrahepatic manifestations of chronic hepatitis C virus infection and simultaneously improve during direct acting antiviral therapy independently of therapy-related liver function recovery. Therefore, large-scale treatment would not only drive viral eradication but also improve patients' immune system and may reduce susceptibility to infections.

Cell fate decisions during the development of the peripheral nervous system in the vertebrate head.

Sensory placodes and neural crest cells are among the key cell populations that facilitated the emergence and diversification of vertebrates throughout evolution. Together, they generate the sensory nervous system in the head: both form the cranial sensory ganglia, while placodal cells make major contributions to the sense organs-the eye, ear and olfactory epithelium. Both are instrumental for integrating craniofacial organs and have been key to drive the concentration of sensory structures in the vertebrate head allowing the emergence of active and predatory life forms. Whereas the gene regulatory networks that control neural crest cell development have been studied extensively, the signals and downstream transcriptional events that regulate placode formation and diversity are only beginning to be uncovered. Both cell populations are derived from the embryonic ectoderm, which also generates the central nervous system and the epidermis, and recent evidence suggests that their initial specification involves a common molecular mechanism before definitive neural, neural crest and placodal lineages are established. In this review, we will first discuss the transcriptional networks that pattern the embryonic ectoderm and establish these three cell fates with emphasis on sensory placodes. Second, we will focus on how sensory placode precursors diversify using the specification of otic-epibranchial progenitors and their segregation as an example.

Transplantation of Neural Tissue: Quail-Chick Chimeras.

Tissue transplantation is an important approach in developmental neurobiology to determine cell fate, to uncover inductive interactions required for tissue specification and patterning as well as to establish tissue competence and commitment. Combined with state-of-the-art molecular approaches, transplantation assays have been instrumental for the discovery of gene regulatory networks controlling cell fate choices and how such networks change over time. Avian species are among the favorite model systems for these approaches because of their accessibility and relatively large size. Here we describe two culture techniques used to generate quail-chick chimeras at different embryonic stages and methods to distinguish graft and donor tissue.

Enhancer activation by FGF signalling during otic induction.

Vertebrate ear progenitors are induced by fibroblast growth factor signalling, however the molecular mechanisms leading to the coordinate activation of downstream targets are yet to be discovered. The ear, like other sensory placodes, arises from the pre-placodal region at the border of the neural plate. Using a multiplex NanoString approach, we determined the response of these progenitors to FGF signalling by examining the changes of more than 200 transcripts that define the otic and other placodes, neural crest and neural plate territories. This analysis identifies new direct and indirect FGF targets during otic induction. Investigating changes in histone marks by ChIP-seq reveals that FGF exposure of pre-placodal cells leads to rapid deposition of active chromatin marks H3K27ac near FGF-response genes, while H3K27ac is depleted in the vicinity of non-otic genes. Genomic regions that gain H3K27ac act as cis-regulatory elements controlling otic gene expression in time and space and define a unique transcription factor signature likely to control their activity. Finally, we show that in response to FGF signalling the transcription factor dimer AP1 recruits the histone acetyl transferase p300 to selected otic enhancers. Thus, during ear induction FGF signalling modifies the chromatin landscape to promote enhancer activation and chromatin accessibility.

PRDM1 controls the sequential activation of neural, neural crest and sensory progenitor determinants.

During early embryogenesis, the ectoderm is rapidly subdivided into neural, neural crest and sensory progenitors. How the onset of lineage determinants and the loss of pluripotency markers are temporally and spatially coordinated in vivo is still debated. Here, we identify a crucial role for the transcription factor PRDM1 in the orderly transition from epiblast to defined neural lineages in chick. PRDM1 is initially expressed broadly in the entire epiblast, but becomes gradually restricted as cell fates are specified. We find that PRDM1 is required for the loss of some pluripotency markers and the onset of neural, neural crest and sensory progenitor specifier genes. PRDM1 directly activates their expression by binding to their promoter regions and recruiting the histone demethylase Kdm4a to remove repressive histone marks. However, once neural lineage determinants become expressed, they in turn repress PRDM1, whereas prolonged PRDM1 expression inhibits neural, neural crest and sensory progenitor genes, suggesting that its downregulation is necessary for cells to maintain their identity. Therefore, PRDM1 plays multiple roles during ectodermal cell fate allocation.

Specification of sensory placode progenitors: signals and transcription factor networks.

Sensory placodes contribute to much of the sensory nervous system in the vertebrate head. They give rise to parts of the eye, ear and nose, as well as to the sensory ganglia that innervate the face, tongue, oesophagus and visceral tissues. Despite their diversity, during development placodes arise from a population of common progenitor cells, which are first specified at the border of the neural plate. The chick has been particularly instrumental in dissecting the timing of these events, and recent evidence has highlighted the close relationship of placode progenitors and precursors for neural crest cells and the central nervous system. This review focuses on the induction of placode progenitors by localised signalling events, and the transcriptional networks that lead to their specification.

Lsd1 interacts with cMyb to demethylate repressive histone marks and maintain inner ear progenitor identity.

During development, multipotent progenitor cells must maintain their identity while retaining the competence to respond to new signalling cues that drive cell fate decisions. This depends on both DNA-bound transcription factors and surrounding histone modifications. Here, we identify the histone demethylase Lsd1 as a crucial component of the molecular machinery that preserves progenitor identity in the developing ear prior to lineage commitment. Although Lsd1 is mainly associated with repressive complexes, we show that, in ear precursors, it is required to maintain active transcription of otic genes. We reveal a novel interaction between Lsd1 and the transcription factor cMyb, which in turn recruits Lsd1 to the promoters of key ear transcription factors. Here, Lsd1 prevents the accumulation of repressive H3K9me2, while allowing H3K9 acetylation. Loss of Lsd1 function causes rapid silencing of active promoters and loss of ear progenitor genes, and shuts down the entire ear developmental programme. Our data suggest that Lsd1-cMyb acts as a co-activator complex that maintains a regulatory module at the top of the inner ear gene network.

Neural induction by the node and placode induction by head mesoderm share an initial state resembling neural plate border and ES cells.

Around the time of gastrulation in higher vertebrate embryos, inductive interactions direct cells to form central nervous system (neural plate) or sensory placodes. Grafts of different tissues into the periphery of a chicken embryo elicit different responses: Hensen's node induces a neural plate whereas the head mesoderm induces placodes. How different are these processes? Transcriptome analysis in time course reveals that both processes start by induction of a common set of genes, which later diverge. These genes are remarkably similar to those induced by an extraembryonic tissue, the hypoblast, and are normally expressed in the pregastrulation stage epiblast. Explants of this epiblast grown in the absence of further signals develop as neural plate border derivatives and eventually express lens markers. We designate this state as "preborder"; its transcriptome resembles embryonic stem cells. Finally, using sequential transplantation experiments, we show that the node, head mesoderm, and hypoblast are interchangeable to begin any of these inductions while the final outcome depends on the tissue emitting the later signals.

Anti-HCV treatment with ombitasvir/paritaprevir/ritonavir ± dasabuvir is associated with increased bile acid levels and pruritus.

BACKGROUND: Direct acting antiviral (DAA)-based treatment with ombitasvir/paritaprevir/ritonavir ± dasabuvir (OBV/PTV/r ± DSV) is highly effective in HCV genotype 1 or 4 infection and well-tolerated with only few side effects. However, pruritus has been observed in several trials in up to 20% of patients and seems to be unique for this DAA combination. OBJECTIVES: The aim of this preliminary study was to investigate the effect of OBV/PTV/r ± DSV on bile acid levels and to correlate them to the emergence of pruritus during treatment. METHODS: Twenty patients with chronic hepatitis C genotype 1 or 4 were treated for 12 or 24 weeks with OBV/PTV/r ± DSV with or without ribavirin. Side effects including pruritus were assessed every 4 weeks during treatment or on demand. Blood was collected in fasting state at baseline and at treatment week 4 for determination of bile acid concentrations by high-resolution mass spectrometry. RESULTS: Pruritus developed in 5 out of 20 patients during the first 4 weeks of DAA treatment. Pruritus was self-limiting during DAA treatment in 4 patients while one patient required cholestyramine treatment and responded well. Total bile acid levels increased approximately 4­fold by treatment week 4. CONCLUSIONS: Pruritus observed during OBV/PTV/r ± DSV treatment of chronic hepatitis C is associated with increased on-treatment serum bile acid levels, possibly due to ritonavir-induced alterations of bile acid transport.

A gene network regulated by FGF signalling during ear development.

During development cell commitment is regulated by inductive signals that are tightly controlled in time and space. In response, cells activate specific programmes, but the transcriptional circuits that maintain cell identity in a changing signalling environment are often poorly understood. Specification of inner ear progenitors is initiated by FGF signalling. Here, we establish the genetic hierarchy downstream of FGF by systematic analysis of many ear factors combined with a network inference approach. We show that FGF rapidly activates a small circuit of transcription factors forming positive feedback loops to stabilise otic progenitor identity. Our predictive network suggests that subsequently, transcriptional repressors ensure the transition of progenitors to mature otic cells, while simultaneously repressing alternative fates. Thus, we reveal the regulatory logic that initiates ear formation and highlight the hierarchical organisation of the otic gene network.

Cell interactions, signals and transcriptional hierarchy governing placode progenitor induction.

In vertebrates, cranial placodes contribute to all sense organs and sensory ganglia and arise from a common pool of Six1/Eya2+ progenitors. Here we dissect the events that specify ectodermal cells as placode progenitors using newly identified genes upstream of the Six/Eya complex. We show in chick that two different tissues, namely the lateral head mesoderm and the prechordal mesendoderm, gradually induce placode progenitors: cells pass through successive transcriptional states, each identified by distinct factors and controlled by different signals. Both tissues initiate a common transcriptional state but over time impart regional character, with the acquisition of anterior identity dependent on Shh signalling. Using a network inference approach we predict the regulatory relationships among newly identified transcription factors and verify predicted links in knockdown experiments. Based on this analysis we propose a new model for placode progenitor induction, in which the initial induction of a generic transcriptional state precedes regional divergence.

A systems-level approach reveals new gene regulatory modules in the developing ear.

The inner ear is a complex vertebrate sense organ, yet it arises from a simple epithelium, the otic placode. Specification towards otic fate requires diverse signals and transcriptional inputs that act sequentially and/or in parallel. Using the chick embryo, we uncover novel genes in the gene regulatory network underlying otic commitment and reveal dynamic changes in gene expression. Functional analysis of selected transcription factors reveals the genetic hierarchy underlying the transition from progenitor to committed precursor, integrating known and novel molecular players. Our results not only characterize the otic transcriptome in unprecedented detail, but also identify new gene interactions responsible for inner ear development and for the segregation of the otic lineage from epibranchial progenitors. By recapitulating the embryonic programme, the genes and genetic sub-circuits discovered here might be useful for reprogramming naïve cells towards otic identity to restore hearing loss.