Proneural gene requirements and progenitor dynamics in sensory organ development

The inner ear is the sensory organ for hearing and balance. Its functional unit is the sensory patch that comprises: i) hair cells, which are the mechano- transducers sensing the stimuli and are embedded in the supporting cell layer, and ii) sensory neurons, which conduct these stimuli to the hindbrain. The generation of hair cells and neurons occurs in the otic placode early during embryonic development. Cell fate specification relies on expression of proneural genes and is concomitant with organ growth and morphogenesis. We used zebrafish embryos and combined live imaging and genetic tools to investigate: i) the location of the different progenitor pools, ii) the potentialities they exhibit, and iii) the dynamic behavior of these cells in generating the different fates. We have generated progenitor maps for the different cell fates by lineage tracing and focused our analysis on the behavioral changes of progenitors upon depletion of a proneural gene and the spatial and temporal aspects of cell fate specification.L'oïda interna és l'òrgan sensorial responsable de l'audició i l'equilibri. La seva unitat funcional és el parxe sensorial que contèn: i) les cèl.lules ciliades, que són els mecano-transductors que detecten, i ii) les neurones sensorials, que envien aquests estímuls al cervell posterior. La generació de cèl.lules ciliades i de neurones te lloc a la placoda òtica molt aviat durant el desenvolupament embrionari . L'especificació del destí cel.lular es basa en l'expressió dels gens proneurals i és concomitant amb el creixement de l’òrgan i la seva morfogènesi. Hem utilitzat embrions de peix zebra i combinat imatges en viu amb eines genètiques per investigar: i) la ubicació dels diferents grups de progenitors, ii) les potencialitats que presenten, i iii) el comportament dinàmic d'aquestes cèl.lules en la generació dels diferents destins. Hem generat mapes progenitors pels diferents destins cel.lulars a partir d’experiments de llinatge i hem centrat la nostra anàlisi en els canvis de comportament dels progenitors després de la inactivació d'un gen proneural i els aspectes espacials i temporals de l'especificació de destí cel.lular

Proneural gene requirements and progenitor dynamics in sensory organ development

The inner ear is the sensory organ for hearing and balance. Its functional unit is the sensory patch that comprises: i) hair cells, which are the mechano- transducers sensing the stimuli and are embedded in the supporting cell layer, and ii) sensory neurons, which conduct these stimuli to the hindbrain. The generation of hair cells and neurons occurs in the otic placode early during embryonic development. Cell fate specification relies on expression of proneural genes and is concomitant with organ growth and morphogenesis. We used zebrafish embryos and combined live imaging and genetic tools to investigate: i) the location of the different progenitor pools, ii) the potentialities they exhibit, and iii) the dynamic behavior of these cells in generating the different fates. We have generated progenitor maps for the different cell fates by lineage tracing and focused our analysis on the behavioral changes of progenitors upon depletion of a proneural gene and the spatial and temporal aspects of cell fate specification.L'oïda interna és l'òrgan sensorial responsable de l'audició i l'equilibri. La seva unitat funcional és el parxe sensorial que contèn: i) les cèl.lules ciliades, que són els mecano-transductors que detecten, i ii) les neurones sensorials, que envien aquests estímuls al cervell posterior. La generació de cèl.lules ciliades i de neurones te lloc a la placoda òtica molt aviat durant el desenvolupament embrionari . L'especificació del destí cel.lular es basa en l'expressió dels gens proneurals i és concomitant amb el creixement de l’òrgan i la seva morfogènesi. Hem utilitzat embrions de peix zebra i combinat imatges en viu amb eines genètiques per investigar: i) la ubicació dels diferents grups de progenitors, ii) les potencialitats que presenten, i iii) el comportament dinàmic d'aquestes cèl.lules en la generació dels diferents destins. Hem generat mapes progenitors pels diferents destins cel.lulars a partir d’experiments de llinatge i hem centrat la nostra anàlisi en els canvis de comportament dels progenitors després de la inactivació d'un gen proneural i els aspectes espacials i temporals de l'especificació de destí cel.lular

Cell lineage analysis reveals three different progenitor pools for neurosensory elements in the otic vesicle

In the inner ear, sensory versus neuronal specification is achieved through few well-defined bHLH transcription factors. However, the molecular mechanisms regulating the generation of the appropriate cell type in the correct place and at the correct time are not completely understood yet. Various studies have shown that hair cell- and neuron-specifying genes partially overlap in the otic territory, suggesting that mutual interactions among these bHLH factors could direct the generation of the two cell types from a common neurosensory progenitor. Although there is little evidence for a clonal relationship between macular hair cells and sensory neurons, the existence of a single progenitor able to give both sensory and neuronal cell types remains an open question. Here, we identified a population of common neurosensory progenitors in the zebrafish inner ear and studied the proneural requirement for cell fate decision within this population. Expression analysis reveals that proneural genes for hair cells and neurons overlap within the posteromedial otic epithelium. Combined results from single-cell lineage and functional studies on neurog1 and neuroD1 further demonstrate the following: (1) in the anterior region of the ear, neuronal and sensory lineages have already segregated at the onset of proneural gene expression and are committed to a given fate very early; (2) in contrast, the posteromedial part of the ear harbors a population of common progenitors giving both neurons and hair cells until late stages; and finally (3) neuroD1 is required within this pool of bipotent progenitors to generate the hair cell fate.The work was funded by Ministry of Science and Innovation Grant BFU2009-07010 (C.P.). D.S. was a recipient of a postdoctoral Juan de la Cierva contract (Ministry of Science and Innovation) and S.D. of a predoctoral FI fellowship (Agency for Management of University and Research Grants, Government of Catalonia

Distribution of neurosensory progenitor pools during inner ear morphogenesis unveiled by cell lineage reconstruction

Reconstructing the lineage of cells is central to understanding how the wide diversity of cell types develops. Here, we provide the neurosensory lineage reconstruction of a complex sensory organ, the inner ear, by imaging zebrafish embryos in vivo over an extended timespan, combining cell tracing and cell fate marker expression over time. We deliver the first dynamic map of early neuronal and sensory progenitor pools in the whole otic vesicle. It highlights the remodeling of the neuronal progenitor domain upon neuroblast delamination, and reveals that the order and place of neuroblasts' delamination from the otic epithelium prefigure their position within the SAG. Sensory and non-sensory domains harbor different proliferative activity contributing distinctly to the overall growth of the structure. Therefore, the otic vesicle case exemplifies a generic morphogenetic process where spatial and temporal cues regulate cell fate and functional organization of the rudiment of the definitive organ.This work was supported from Spanish Ministry of Economy and Competitiveness (MINECO) by Grant BFU2012-31994 to CP, Unidad de Excelencia María de Maetzu 2015–19 MDM-2014–0370 to DCEXS-UPF, Centro de Excelencia Severo Ochoa 2013–17, SEV-2012–0208 and Swiss National Science Foundation (SINERGIA CRSII3 141918) to CRG. BioEmergences services were funded by ANR-10-INBS-04 and ANR-11-EQPX-0029. SD and AZ were recipients of predoctoral FI-fellowships from AGAUR (Generalitat de Catalunya). CP is recipient of an ICREA Academia award (Generalitat de Catalunya

Comparison of zebrafish larvae and hiPSC cardiomyocytes for predicting drug-induced cardiotoxicity in humans

Cardiovascular drug toxicity is responsible for 17% of drug withdrawals in clinical phases, half of post-marketed drug withdrawals and remains an important adverse effect of several marketed drugs. Early assessment of drug-induced cardiovascular toxicity is mandatory and typically done in cellular systems and mammals. Current in vitro screening methods allow high-throughput but are biologically reductionist. The use of mammal models, which allow a better translatability for predicting clinical outputs, is low-throughput, highly expensive, and ethically controversial. Given the analogies between the human and the zebrafish cardiovascular systems, we propose the use of zebrafish larvae during early drug discovery phases as a balanced model between biological translatability and screening throughput for addressing potential liabilities. To this end, we have developed a high-throughput screening platform that enables fully automatized in vivo image acquisition and analysis to extract a plethora of relevant cardiovascular parameters: heart rate, arrhythmia, AV blockage, ejection fraction, and blood flow, among others. We have used this platform to address the predictive power of zebrafish larvae for detecting potential cardiovascular liabilities in humans. We tested a chemical library of 92 compounds with known clinical cardiotoxicity profiles. The cross-comparison with clinical data and data acquired from human induced pluripotent stem cell cardiomyocytes calcium imaging showed that zebrafish larvae allow a more reliable prediction of cardiotoxicity than cellular systems. Interestingly, our analysis with zebrafish yields similar predictive performance as previous validation meta-studies performed with dogs, the standard regulatory preclinical model for predicting cardiotoxic liabilities prior to clinical phases