Characterisation of cardiomyocyte plasticity and the role of fibroblast during zebrafish heart regeneration

The zebrafish is an established model organism to study heart regeneration, in which pre-existing cardiomyocytes (CMs) proliferate to replace the lost myocardium. During development, mesodermal progenitors from the first heart field (FHF) form a primitive cardiac tube, to which cells from the second heart field (SHF) are added. Here we investigated whether FHF and SHF derivatives in the zebrafish give rise to distinct CM populations, and examined the degree of cell fate plasticity of SHF derivatives during heart regeneration. Using tbx5a-lineage tracing we found that the adult zebrafish heart is also composed of CM populations from the FHF and SHF. Furthermore, ablation of FHF-derived CMs in the embryo is compensated by expansion of SHF derived cells. tbx5a lineage-tracing was also employed to investigate the fate of trabecular CMs during adult heart regeneration. While previous clonal analysis suggested that the different myocardial layers are rebuilt by CMs within each layers, we describe that trabecular CMs can switch their fate and differentiate into cortical myocardium. Heart regeneration is preceded by a fibrotic response. Thus, fibrosis and regeneration are not mutually exclusive responses. Upon cardiac cryoinjury, collagen and other extracellular matrix (ECM) components accumulate at the injury site. Unlike the situation in mammals, fibrosis in zebrafish is transient and its regression is concomitant with regrowth of the myocardial wall. We describe that during fibrosis regression, fibroblasts are not fully eliminated and become inactivated. Unexpectedly, limiting the fibrotic response by genetic ablation of col1a2-expressing cells not only failed to enhance regeneration but also impaired CMs proliferation. We conclude that zebrafish regeneration is a process that requires CM plasticity, and involves ECM-producing cells that become inactive and promote CMs proliferation

Adult sox10+ Cardiomyocytes Contribute to Myocardial Regeneration in the Zebrafish

During heart regeneration in the zebrafish, fibrotic tissue is replaced by newly formed cardiomyocytes derived from preexisting ones. It is unclear whether the heart is composed of several cardiomyocyte populations bearing different capacity to replace lost myocardium. Here, using sox10 genetic fate mapping, we identify a subset of preexistent cardiomyocytes in the adult zebrafish heart with a distinct gene expression profile that expanded after cryoinjury. Genetic ablation of sox10+ cardiomyocytes impairs cardiac regeneration, revealing that these cells play a role in heart regeneration.This work has been funded by the Spanish Ministry of Economy and Competitiveness (BFU2014–56970–P), the Swiss National Science Foundation (grant 31003A_159721), the ERC (starting grant 337703–zebra–Heart), Comunidad de Madrid (FIBROTEAM S2010/BMD-2321), and co-funding by Fondo Europeo de Desarrollo Regional (FEDER). I.J.M. was supported by Marie Curie Slodowska fellowship (PIEF-A-2012-330728). D.F. was supported by BtRAIN (European Brain Barriers Training Network) (H2020-MSCA-ITN-2015, n 675619). The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia, Innovacion y Universidades (MCNU), and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015-0505).S

Nanog regulates Pou3f1 expression at the exit from pluripotency during gastrulation.

Pluripotency is regulated by a network of transcription factors that maintain early embryonic cells in an undifferentiated state while allowing them to proliferate. NANOG is a critical factor for maintaining pluripotency and its role in primordial germ cell differentiation has been well described. However, Nanog is expressed during gastrulation across all the posterior epiblast, and only later in development is its expression restricted to primordial germ cells. In this work, we unveiled a previously unknown mechanism by which Nanog specifically represses genes involved in anterior epiblast lineage. Analysis of transcriptional data from both embryonic stem cells and gastrulating mouse embryos revealed Pou3f1 expression to be negatively correlated with that of Nanog during the early stages of differentiation. We have functionally demonstrated Pou3f1 to be a direct target of NANOG by using a dual transgene system for the controlled expression of Nanog Use of Nanog null ES cells further demonstrated a role for Nanog in repressing a subset of anterior neural genes. Deletion of a NANOG binding site (BS) located nine kilobases downstream of the transcription start site of Pou3f1 revealed this BS to have a specific role in the regionalization of the expression of this gene in the embryo. Our results indicate an active role of Nanog inhibiting neural regulatory networks by repressing Pou3f1 at the onset of gastrulation.This article has an associated First Person interview with the joint first authors of the paper.This work was funded by the Spanish government [grant BFU2017-84914-P to M.M.]. The Gottgens laboratory is supported by core funding from the Wellcome Trust and Medical Research Council to the Wellcome and Medical Research Council Cambridge Stem Cell Institute. The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia, Innovación y Universidades (MCNU) and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence [SEV-2015-0505]

Pluripotency factors regulate the onset of Hox cluster activation in the early embryo

Pluripotent cells are a transient population of the mammalian embryo dependent on transcription factors, such as OCT4 and NANOG, which maintain pluripotency while suppressing lineage specification. However, these factors are also expressed during early phases of differentiation, and their role in the transition from pluripotency to lineage specification is largely unknown. We found that pluripotency factors play a dual role in regulating key lineage specifiers, initially repressing their expression and later being required for their proper activation. We show that Oct4 is necessary for activation of HoxB genes during differentiation of embryonic stem cells and in the embryo. In addition, we show that the HoxB cluster is coordinately regulated by OCT4 binding sites located at the 3′ end of the cluster. Our results show that core pluripotency factors are not limited to maintaining the precommitted epiblast but are also necessary for the proper deployment of subsequent developmental programs

Nanog regulates Pou3f1 expression at the exit from pluripotency during gastrulation

Pluripotency is regulated by a network of transcription factors that maintain early embryonic cells in an undifferentiated state while allowing them to proliferate. NANOG is a critical factor for maintaining pluripotency and its role in primordial germ cell differentiation has been well described. However, Nanog is expressed during gastrulation across all the posterior epiblast, and only later in development is its expression restricted to primordial germ cells. In this work, we unveiled a previously unknown mechanism by which Nanog specifically represses genes involved in anterior epiblast lineage. Analysis of transcriptional data from both embryonic stem cells and gastrulating mouse embryos revealed Pou3f1 expression to be negatively correlated with that of Nanog during the early stages of differentiation. We have functionally demonstrated Pou3f1 to be a direct target of NANOG by using a dual transgene system for the controlled expression of Nanog Use of Nanog null ES cells further demonstrated a role for Nanog in repressing a subset of anterior neural genes. Deletion of a NANOG binding site (BS) located nine kilobases downstream of the transcription start site of Pou3f1 revealed this BS to have a specific role in the regionalization of the expression of this gene in the embryo. Our results indicate an active role of Nanog inhibiting neural regulatory networks by repressing Pou3f1 at the onset of gastrulation.This article has an associated First Person interview with the joint first authors of the paper.This work was funded by the Spanish government [grant BFU2017-84914-P to M.M.]. The Gottgens laboratory is supported by core funding from the Wellcome Trust and Medical Research Council to theWellcome and Medical Research Council Cambridge Stem Cell Institute. The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia, Innovación y Universidades MCNU) and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence [SEV-2015-0505].S