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.

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.

Engrailed controls epaxial-hypaxial muscle innervation and the establishment of vertebrate three-dimensional mobility.

Chordates are characterised by contractile muscle on either side of the body that promotes movement by side-to-side undulation. In the lineage leading to modern jawed vertebrates (crown group gnathostomes), this system was refined: body muscle became segregated into distinct dorsal (epaxial) and ventral (hypaxial) components that are separately innervated by the medial and hypaxial motors column, respectively, via the dorsal and ventral ramus of the spinal nerves. This allows full three-dimensional mobility, which in turn was a key factor in their evolutionary success. How the new gnathostome system is established during embryogenesis and how it may have evolved in the ancestors of modern vertebrates is not known. Vertebrate Engrailed genes have a peculiar expression pattern as they temporarily demarcate a central domain of the developing musculature at the epaxial-hypaxial boundary. Moreover, they are the only genes known with this particular expression pattern. The aim of this study was to investigate whether Engrailed genes control epaxial-hypaxial muscle development and innervation. Investigating chick, mouse and zebrafish as major gnathostome model organisms, we found that the Engrailed expression domain was associated with the establishment of the epaxial-hypaxial boundary of muscle in all three species. Moreover, the outgrowing epaxial and hypaxial nerves orientated themselves with respect to this Engrailed domain. In the chicken, loss and gain of Engrailed function changed epaxial-hypaxial somite patterning. Importantly, in all animals studied, loss and gain of Engrailed function severely disrupted the pathfinding of the spinal motor axons, suggesting that Engrailed plays an evolutionarily conserved role in the separate innervation of vertebrate epaxial-hypaxial muscle.

EYA1 and SIX1 drive the neuronal developmental program in cooperation with the SWI/SNF chromatin-remodeling complex and SOX2 in the mammalian inner ear.

Inner ear neurogenesis depends upon the function of the proneural basic helix-loop-helix (bHLH) transcription factors NEUROG1 and NEUROD1. However, the transcriptional regulation of these factors is unknown. Here, using loss- and gain-of-function models, we show that EYA1 and SIX1 are crucial otic neuronal determination factors upstream of NEUROG1 and NEUROD1. Overexpression of both Eya1 and Six1 is sufficient to convert non-neuronal epithelial cells within the otocyst and cochlea as well as the 3T3 fibroblast cells into neurons. Strikingly, all the ectopic neurons express not only Neurog1 and Neurod1 but also mature neuronal markers such as neurofilament, indicating that Eya1 and Six1 function upstream of, and in the same pathway as, Neurog1 and Neurod1 to not only induce neuronal fate but also regulate their differentiation. We demonstrate that EYA1 and SIX1 interact directly with the SWI/SNF chromatin-remodeling subunits BRG1 and BAF170 to drive neurogenesis cooperatively in 3T3 cells and cochlear nonsensory epithelial cells, and that SOX2 cooperates with these factors to mediate neuronal differentiation. Importantly, we show that the ATPase BRG1 activity is required for not only EYA1- and SIX1-induced ectopic neurogenesis but also normal neurogenesis in the otocyst. These findings indicate that EYA1 and SIX1 are key transcription factors in initiating the neuronal developmental program, probably by recruiting and interacting with the SWI/SNF chromatin-remodeling complex to specifically mediate Neurog1 and Neurod1 transcription.

Awareness and Perceptions of Clinical Trials in Cancer Patients and Their Families in Saudi Arabia.

Despite the increasing number of medical articles being published from the Middle East, clinical research is still lagging behind compared to other regions. Enrolling participants into clinical trials presents an important challenge. We wanted to explore the perception, knowledge, and willingness of cancer patients to participate in oncology clinical trials and to recommend strategies to overcome these challenges. A 31-item questionnaire was administered to cancer patients and their family members in an outpatient clinic. Two hundred four patients and family members were enrolled between December 2011 and February 2013. Fifty-eight percent of the participants were aware of clinical trials. Some misconceptions included the following: 22% believed that no clinical trials were conducted in the Arab world, 19% believed that clinical trials in the Arab world were not under any regulatory authority supervision, and 15% believed that local clinical trials are conducted on subjects without their consent. One third of patients assumed that clinical trials are executed on animals instead of humans, and greater than 40% believed that clinical trials are performed for new medications only. Finally, 61% of the survey participants who were aware of clinical trials expressed their willingness to participate in trials. This large cohort survey demonstrated that a relatively significant number of Saudi cancer patients and their families are aware of clinical trials and a similarly high number of participants are willing to participate in clinical trials. This leads us to believe that patients' awareness and perception of clinical trials are not a significant limiting factor in clinical trial recruitment in our region.

RGMa and RGMb expression pattern during chicken development suggest unexpected roles for these repulsive guidance molecules in notochord formation, somitogenesis, and myogenesis.

BACKGROUND: Repulsive guidance molecules (RGM) are high-affinity ligands for the Netrin receptor Neogenin, and they are crucial for nervous system development including neural tube closure; neuronal and neural crest cell differentiation and axon guidance. Recent studies implicated RGM molecules in bone morphogenetic protein signaling, which regulates a variety of developmental processes. Moreover, a role for RGMc in iron metabolism has been established. This suggests that RGM molecules may play important roles in non-neural tissues. RESULTS: To explore which tissues and processed may be regulated by RGM molecules, we systematically investigated the expression of RGMa and RGMb, the only RGM molecules currently known for avians, in the chicken embryo. CONCLUSIONS: Our study suggests so far unknown roles of RGM molecules in notochord, somite and skeletal muscle development.

Gradients of glucose metabolism regulate morphogen signalling required for specifying tonotopic organisation in the chicken cochlea.

In vertebrates with elongated auditory organs, mechanosensory hair cells (HCs) are organised such that complex sounds are broken down into their component frequencies along a proximal-to-distal long (tonotopic) axis. Acquisition of unique morphologies at the appropriate position along the chick cochlea, the basilar papilla, requires that nascent HCs determine their tonotopic positions during development. The complex signalling within the auditory organ between a developing HC and its local niche along the cochlea is poorly understood. Using a combination of live imaging and NAD(P)H fluorescence lifetime imaging microscopy, we reveal that there is a gradient in the cellular balance between glycolysis and the pentose phosphate pathway in developing HCs along the tonotopic axis. Perturbing this balance by inhibiting different branches of cytosolic glucose catabolism disrupts developmental morphogen signalling and abolishes the normal tonotopic gradient in HC morphology. These findings highlight a causal link between graded morphogen signalling and metabolic reprogramming in specifying the tonotopic identity of developing HCs.

Telocytes regulate macrophages in periodontal disease.

Telocytes (TCs) or interstitial cells are characterised in vivo by their long projections that contact other cell types. Although telocytes can be found in many different tissues including the heart, lung, and intestine, their tissue-specific roles are poorly understood. Here we identify a specific cell signalling role for telocytes in the periodontium whereby telocytes regulate macrophage activity. We performed scRNA-seq and lineage tracing to identify telocytes and macrophages in mouse periodontium in homeostasis and periodontitis and carried out hepatocyte growth factor (HGF) signalling inhibition experiments using tivantinib. We show that telocytes are quiescent in homeostasis; however, they proliferate and serve as a major source of HGF in periodontitis. Macrophages receive telocyte-derived HGF signals and shift from an M1 to an M1/M2 state. Our results reveal the source of HGF signals in periodontal tissue and provide new insights into the function of telocytes in regulating macrophage behaviour in periodontitis through HGF/Met cell signalling, which may provide a novel approach in periodontitis treatment.

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.

MRI-guided histology of TDP-43 knock-in mice implicates parvalbumin interneuron loss, impaired neurogenesis and aberrant neurodevelopment in amyotrophic lateral sclerosis-frontotemporal dementia.

Amyotrophic lateral sclerosis and frontotemporal dementia are overlapping diseases in which MRI reveals brain structural changes in advance of symptom onset. Recapitulating these changes in preclinical models would help to improve our understanding of the molecular causes underlying regionally selective brain atrophy in early disease. We therefore investigated the translational potential of the TDP-43Q331K knock-in mouse model of amyotrophic lateral sclerosis-frontotemporal dementia using MRI. We performed in vivo MRI of TDP-43Q331K knock-in mice. Regions of significant volume change were chosen for post-mortem brain tissue analyses. Ex vivo computed tomography was performed to investigate skull shape. Parvalbumin neuron density was quantified in post-mortem amyotrophic lateral sclerosis frontal cortex. Adult mutants demonstrated parenchymal volume reductions affecting the frontal lobe and entorhinal cortex in a manner reminiscent of amyotrophic lateral sclerosis-frontotemporal dementia. Subcortical, cerebellar and brain stem regions were also affected in line with observations in pre-symptomatic carriers of mutations in C9orf72, the commonest genetic cause of both amyotrophic lateral sclerosis and frontotemporal dementia. Volume loss was also observed in the dentate gyrus of the hippocampus, along with ventricular enlargement. Immunohistochemistry revealed reduced parvalbumin interneurons as a potential cellular correlate of MRI changes in mutant mice. By contrast, microglia was in a disease activated state even in the absence of brain volume loss. A reduction in immature neurons was found in the dentate gyrus, indicative of impaired adult neurogenesis, while a paucity of parvalbumin interneurons in P14 mutant mice suggests that TDP-43Q331K disrupts neurodevelopment. Computerized tomography imaging showed altered skull morphology in mutants, further suggesting a role for TDP-43Q331K in development. Finally, analysis of human post-mortem brains confirmed a paucity of parvalbumin interneurons in the prefrontal cortex in sporadic amyotrophic lateral sclerosis and amyotrophic lateral sclerosis linked to C9orf72 mutations. Regional brain MRI changes seen in human amyotrophic lateral sclerosis-frontotemporal dementia are recapitulated in TDP-43Q331K knock-in mice. By marrying in vivo imaging with targeted histology, we can unravel cellular and molecular processes underlying selective brain vulnerability in human disease. As well as helping to understand the earliest causes of disease, our MRI and histological markers will be valuable in assessing the efficacy of putative therapeutics in TDP-43Q331K knock-in mice.

Protein and genomic organisation of vertebrate MyoR and Capsulin genes and their expression during avian development.

The related bHLH transcription factors MyoR and Capsulin control craniofacial myogenesis and the development of a number of mesoderm-derived organs in the mouse. However, their molecular function as regulators of differentiation processes is conversely debated. One approach to clarify the roles of these genes is to comparatively analyse their biological and molecular function in various vertebrate models. For this, a prerequisite is the determination of their similarity and their expression patterns. Here we show that vertebrate MyoR and Capsulin are paralogous genes with a high level of conservation regarding their protein sequence, their cDNA sequence and their chromosomal organisation. In the chick, both genes are co-expressed in the developing branchiomeric muscles, the anterior heart field and the splanchnopleura lining the foregut. However, both genes show unique expression domains in trunk skeletal muscle precursors, in the lateral and intermediate mesoderm.

Auditory hair cell defects as potential cause for sensorineural deafness in Wolf-Hirschhorn syndrome.

WHSC1 is a histone methyltransferase (HMT) that catalyses the addition of methyl groups to lysine 36 on histone 3. In humans, WHSC1 haploinsufficiency is associated with all known cases of Wolf-Hirschhorn syndrome (WHS). The cardinal feature of WHS is a craniofacial dysmorphism, which is accompanied by sensorineural hearing loss in 15% of individuals with WHS. Here, we show that WHSC1-deficient mice display craniofacial defects that overlap with WHS, including cochlea anomalies. Although auditory hair cells are specified normally, their stereocilia hair bundles required for sound perception fail to develop the appropriate morphology. Furthermore, the orientation and cellular organisation of cochlear hair cells and their innervation are defective. These findings identify, for the first time, the likely cause of sensorineural hearing loss in individuals with WHS.

EYA1-SIX1 complex in neurosensory cell fate induction in the mammalian inner ear.

The phosphatase-transactivator EYA1 interacts with the homeodomain protein SIX1 to form transcriptional activation complexes, which play essential roles in regulating cell proliferation, survival and induction of sensory and neuronal differentiation programs during inner ear development. Mutations of the Eya1 and Six1 genes cause profound developmental auditory defects in mice and humans. The molecular mechanisms and developmental processes controlled by the EYA1 and SIX1 complex in inner ear development and neurosensory fate induction are the focus of this review.

The phosphatase-transcription activator EYA1 is targeted by anaphase-promoting complex/Cdh1 for degradation at M-to-G1 transition.

The phosphatase and transactivator EYA family proteins are overexpressed in many cancer cell lines and are abundantly distributed in undifferentiated cells during development. Loss-of-function studies have shown that EYA1 is required for cell proliferation and survival during mammalian organogenesis. However, how EYA1 is regulated during development is unknown. Here, we report that EYA1 is regulated throughout the cell cycle via ubiquitin-mediated proteolysis. The level of EYA1 protein fluctuates in the cell cycle, peaking during mitosis and dropping drastically as cells exit into G(1). We found that EYA1 is efficiently degraded during mitotic exit in a Cdh1-dependent manner and that these two proteins physically interact. Overexpression of Cdh1 reduces the protein levels of ectopically expressed or endogenous EYA1, whereas depletion of Cdh1 by RNA interference stabilizes the EYA1 protein. Together, our results indicate that anaphase-promoting complex/cyclosome (APC/C)-Cdh1 specifically targets EYA1 for degradation during M-to-G(1) transition, failure of which may compromise cell proliferation and survival.

Eya1-Six1 interaction is sufficient to induce hair cell fate in the cochlea by activating Atoh1 expression in cooperation with Sox2.

Inner-ear hair cell differentiation requires Atoh1 function, while Eya1, Six1, and Sox2 are coexpressed in sensory progenitors and mutations in these genes cause sensorineural hearing loss. However, how these genes are linked functionally and the transcriptional networks controlling hair cell induction remain unclear. Here, we show (1) that Eya1/Six1 are necessary for hair cell development, and their coexpression in mouse cochlear explants is sufficient to induce hair cell fate in the nonsensory epithelium expressing low-level Sox2 by activating not only Atoh1-dependent but also Atoh1-independent pathways and (2) that both pathways induce Pou4f3 to promote hair cell differentiation. Sox2 cooperates with Eya1/Six1 to synergistically activate Atoh1 transcription via direct binding to the conserved Sox- and Six-binding sites in Atoh1 enhancers, and these proteins physically interact. Our findings demonstrate that direct and cooperative interactions between the Sox2, Six1, and Eya1 proteins coordinate Atoh1 expression to specify hair cell fate.

Fibroblast growth factor (FGF) gene expression in the developing cerebellum suggests multiple roles for FGF signaling during cerebellar morphogenesis and development.

The cerebellum is derived from the anterior-most segment of the embryonic hindbrain, rhombomere 1 (r1). Previous studies have shown that the early development and patterning of r1 requires fibroblast growth factor (FGF) signaling. However, many of the developmental processes that shape cerebellar morphogenesis take place later in embryonic development and during the first 2 weeks of postnatal life in the mouse. Here, we present a more comprehensive analysis of the expression patterns of genes encoding FGF receptors and secreted FGF ligands during these later stages of cerebellar development. We show that these genes are expressed in multiple cell types in the developing cerebellum, in an astonishing array of distinct patterns. These data suggest that FGF signaling functions throughout cerebellar development to regulate many processes that shape the formation of a functional cerebellum.

Extrinsic versus intrinsic cues in avian paraxial mesoderm patterning and differentiation.

Somitic and head mesoderm contribute to cartilage and bone and deliver the entire skeletal musculature. Studies on avian somite patterning and cell differentiation led to the view that these processes depend solely on cues from surrounding tissues. However, evidence is accumulating that some developmental decisions depend on information within the somitic tissue itself. Moreover, recent studies established that head and somitic mesoderm, though delivering the same tissue types, are set up to follow their own, distinct developmental programmes. With a particular focus on the chicken embryo, we review the current understanding of how extrinsic signalling, operating in a framework of intrinsically regulated constraints, controls paraxial mesoderm patterning and cell differentiation.

Establishment of the epaxial-hypaxial boundary in the avian myotome.

Trunk skeletal muscles are segregated into dorsomedial epaxial and ventrolateral hypaxial muscles, separated by a myoseptum. In amniotes, they are generated from a transient structure, the dermomyotome, which lays down muscle, namely the myotome underneath. However, the dermomyotome and myotome are dorsoventrally continuous, with no morphologically defined epaxial-hypaxial boundary. The transcription factors En1 and Sim1 have been shown to molecularly subdivide the amniote dermomyotome, with En1 labeling the epaxial dermomyotome and Sim1 the hypaxial counterpart. Here, we demonstrate that En1 and Sim1 expression persists in cells leaving the dermomyotome, superimposing the expression boundary onto muscle and skin. En1-expressing cells colonize the myotome initially from the rostral and caudal lips, and slightly later, directly from the de-epithelializing dermomyotomal center. En1 expression in the myotome is concomitant with the appearance of Fgfr4/Pax7-expressing mitotically active myoblasts. This finding suggests that Fgfr4+/Pax7+/En1+ cells carry their expression with them when entering the myotome. Furthermore, it suggests that the epaxial-hypaxial boundary of the myotome is established through the late arising, mitotically active myoblasts.

The epaxial-hypaxial subdivision of the avian somite.

In all jaw-bearing vertebrates, three-dimensional mobility relies on segregated, separately innervated epaxial and hypaxial skeletal muscles. In amniotes, these muscles form from the morphologically continuous dermomyotome and myotome, whose epaxial-hypaxial subdivision and hence the formation of distinct epaxial-hypaxial muscles is not understood. Here we show that En1 expression labels a central subdomain of the avian dermomyotome, medially abutting the expression domain of the lead-lateral or hypaxial marker Sim1. En1 expression is maintained when cells from the En1-positive dermomyotome enter the myotome and dermatome, thereby superimposing the En1-Sim1 expression boundary onto the developing musculature and dermis. En1 cells originate from the dorsomedial edge of the somite. Their development is under positive control by notochord and floor plate (Shh), dorsal neural tube (Wnt1) and surface ectoderm (Wnt1-like signalling activity) but negatively regulated by the lateral plate mesoderm (BMP4). This dependence on epaxial signals and suppression by hypaxial signals places En1 into the epaxial somitic programme. Consequently, the En1-Sim1 expression boundary marks the epaxial-hypaxial dermomyotomal or myotomal boundary. In cell aggregation assays, En1- and Sim1-expressing cells sort out, suggesting that the En1-Sim1 expression boundary may represent a true compartment boundary, foreshadowing the epaxial-hypaxial segregation of muscle.