Nrg1 Regulates Cardiomyocyte Migration and Cell Cycle in Ventricular Development.

BACKGROUND: Cardiac ventricles provide the contractile force of the beating heart throughout life. How the primitive endocardium-layered myocardial projections called trabeculae form and mature into the adult ventricles is of great interest for biology and regenerative medicine. Trabeculation is dependent on the signaling protein Nrg1 (neuregulin-1). However, the mechanism of action of Nrg1 and its role in ventricular wall maturation are poorly understood. METHODS: We investigated the functions and downstream mechanisms of Nrg1 signaling during ventricular chamber development using confocal imaging, transcriptomics, and biochemical approaches in mice with cardiac-specific inactivation or overexpression of Nrg1. RESULTS: Analysis of cardiac-specific Nrg1 mutant mice showed that the transcriptional program underlying cardiomyocyte-oriented cell division and trabeculae formation depends on endocardial Nrg1 to myocardial ErbB2 (erb-b2 receptor tyrosine kinase 2) signaling and phospho-Erk (phosphorylated extracellular signal-regulated kinase; pErk) activation. Early endothelial loss of Nrg1 and reduced pErk activation diminished cardiomyocyte Pard3 and Crumbs2 (Crumbs Cell Polarity Complex Component 2) protein and altered cytoskeletal gene expression and organization. These alterations are associated with abnormal gene expression related to mitotic spindle organization and a shift in cardiomyocyte division orientation. Nrg1 is crucial for trabecular growth and ventricular wall thickening by regulating an epithelial-to-mesenchymal transition-like process in cardiomyocytes involving migration, adhesion, cytoskeletal actin turnover, and timely progression through the cell cycle G2/M phase. Ectopic cardiac Nrg1 overexpression and high pErk signaling caused S-phase arrest, sustained high epithelial-to-mesenchymal transition-like gene expression, and prolonged trabeculation, blocking compact myocardium maturation. Myocardial trabecular patterning alterations resulting from above- or below-normal Nrg1-dependent pErk activation were concomitant with sarcomere actin cytoskeleton disorganization. The Nrg1 loss- and gain-of-function transcriptomes were enriched for Yap1 (yes-associated protein-1) gene signatures, identifying Yap1 as a potential downstream effector. Furthermore, biochemical and imaging data reveal that Nrg1 influences pErk activation and Yap1 nuclear-cytoplasmic distribution during trabeculation. CONCLUSIONS: These data establish the Nrg1-ErbB2/ErbB4-Erk axis as a crucial regulator of cardiomyocyte cell cycle progression and migration during ventricular development.

Cross-species analysis of LZTR1 loss-of-function mutants demonstrates dependency to RIT1 orthologs.

RAS GTPases are highly conserved proteins involved in the regulation of mitogenic signaling. We have previously described a novel Cullin 3 RING E3 ubiquitin ligase complex formed by the substrate adaptor protein LZTR1 that binds, ubiquitinates, and promotes proteasomal degradation of the RAS GTPase RIT1. In addition, others have described that this complex is also responsible for the ubiquitination of classical RAS GTPases. Here, we have analyzed the phenotypes of Lztr1 loss-of-function mutants in both fruit flies and mice and have demonstrated a biochemical preference for their RIT1 orthologs. Moreover, we show that Lztr1 is haplosufficient in mice and that embryonic lethality of the homozygous null allele can be rescued by deletion of Rit1. Overall, our results indicate that, in model organisms, RIT1 orthologs are the preferred substrates of LZTR1.

MAX mutant small-cell lung cancers exhibit impaired activities of MGA-dependent noncanonical polycomb repressive complex.

The MYC axis is disrupted in cancer, predominantly through activation of the MYC family oncogenes but also through inactivation of the MYC partner MAX or of the MAX partner MGA. MGA and MAX are also members of the polycomb repressive complex, ncPRC1.6. Here, we use genetically modified MAX-deficient small-cell lung cancer (SCLC) cells and carry out genome-wide and proteomics analyses to study the tumor suppressor function of MAX. We find that MAX mutant SCLCs have ASCL1 or NEUROD1 or combined ASCL1/NEUROD1 characteristics and lack MYC transcriptional activity. MAX restitution triggers prodifferentiation expression profiles that shift when MAX and oncogenic MYC are coexpressed. Although ncPRC1.6 can be formed, the lack of MAX restricts global MGA occupancy, selectively driving its recruitment toward E2F6-binding motifs. Conversely, MAX restitution enhances MGA occupancy to repress genes involved in different functions, including stem cell and DNA repair/replication. Collectively, these findings reveal that MAX mutant SCLCs have either ASCL1 or NEUROD1 or combined characteristics and are MYC independent and exhibit deficient ncPRC1.6-mediated gene repression.

KrasP34R and KrasT58I mutations induce distinct RASopathy phenotypes in mice.

Somatic KRAS mutations are highly prevalent in many cancers. In addition, a distinct spectrum of germline KRAS mutations causes developmental disorders called RASopathies. The mutant proteins encoded by these germline KRAS mutations are less biochemically and functionally activated than those in cancer. We generated mice harboring conditional KrasLSL-P34Rand KrasLSL-T58I knock-in alleles and characterized the consequences of each mutation in vivo. Embryonic expression of KrasT58I resulted in craniofacial abnormalities reminiscent of those seen in RASopathy disorders, and these mice exhibited hyperplastic growth of multiple organs, modest alterations in cardiac valvulogenesis, myocardial hypertrophy, and myeloproliferation. By contrast, embryonic KrasP34R expression resulted in early perinatal lethality from respiratory failure due to defective lung sacculation, which was associated with aberrant ERK activity in lung epithelial cells. Somatic Mx1-Cre-mediated activation in the hematopoietic compartment showed that KrasP34R and KrasT58I expression had distinct signaling effects, despite causing a similar spectrum of hematologic diseases. These potentially novel strains are robust models for investigating the consequences of expressing endogenous levels of hyperactive K-Ras in different developing and adult tissues, for comparing how oncogenic and germline K-Ras proteins perturb signaling networks and cell fate decisions, and for performing preclinical therapeutic trials.

Coronary arterial development is regulated by a Dll4-Jag1-EphrinB2 signaling cascade.

Coronaries are essential for myocardial growth and heart function. Notch is crucial for mouse embryonic angiogenesis, but its role in coronary development remains uncertain. We show Jag1, Dll4 and activated Notch1 receptor expression in sinus venosus (SV) endocardium. Endocardial Jag1 removal blocks SV capillary sprouting, while Dll4 inactivation stimulates excessive capillary growth, suggesting that ligand antagonism regulates coronary primary plexus formation. Later endothelial ligand removal, or forced expression of Dll4 or the glycosyltransferase Mfng, blocks coronary plexus remodeling, arterial differentiation, and perivascular cell maturation. Endocardial deletion of Efnb2 phenocopies the coronary arterial defects of Notch mutants. Angiogenic rescue experiments in ventricular explants, or in primary human endothelial cells, indicate that EphrinB2 is a critical effector of antagonistic Dll4 and Jag1 functions in arterial morphogenesis. Thus, coronary arterial precursors are specified in the SV prior to primary coronary plexus formation and subsequent arterial differentiation depends on a Dll4-Jag1-EphrinB2 signaling cascade.

In vivo conditional deletion of HDAC7 reveals its requirement to establish proper B lymphocyte identity and development.

Class IIa histone deacetylase (HDAC) subfamily members are tissue-specific gene repressors with crucial roles in development and differentiation processes. A prominent example is HDAC7, a class IIa HDAC that shows a lymphoid-specific expression pattern within the hematopoietic system. In this study, we explored its potential role in B cell development by generating a conditional knockout mouse model. Our study demonstrates for the first time that HDAC7 deletion dramatically blocks early B cell development and gives rise to a severe lymphopenia in peripheral organs, while also leading to pro-B cell lineage promiscuity. We find that HDAC7 represses myeloid and T lymphocyte genes in B cell progenitors through interaction with myocyte enhancer factor 2C (MEFC2). In B cell progenitors, HDAC7 is recruited to promoters and enhancers of target genes, and its absence leads to increased enrichment of histone active marks. Our results prove that HDAC7 is a bona fide transcriptional repressor essential for B cell development.

The tumor suppressor PTEN and the PDK1 kinase regulate formation of the columnar neural epithelium.

Epithelial morphogenesis and stability are essential for normal development and organ homeostasis. The mouse neural plate is a cuboidal epithelium that remodels into a columnar pseudostratified epithelium over the course of 24 hr. Here we show that the transition to a columnar epithelium fails in mutant embryos that lack the tumor suppressor PTEN, although proliferation, patterning and apical-basal polarity markers are normal in the mutants. The Pten phenotype is mimicked by constitutive activation of PI3 kinase and is rescued by the removal of PDK1 (PDPK1), but does not depend on the downstream kinases AKT and mTORC1. High resolution imaging shows that PTEN is required for stabilization of planar cell packing in the neural plate and for the formation of stable apical-basal microtubule arrays. The data suggest that appropriate levels of membrane-associated PDPK1 are required for stabilization of apical junctions, which promotes cell elongation, during epithelial morphogenesis.

Morphogenesis of the mouse neural plate depends on distinct roles of cofilin 1 in apical and basal epithelial domains.

The genetic control of mammalian epithelial polarity and dynamics can be studied in vivo at cellular resolution during morphogenesis of the mouse neural tube. The mouse neural plate is a simple epithelium that is transformed into a columnar pseudostratified tube over the course of ∼ 24 h. Apical F-actin is known to be important for neural tube closure, but the precise roles of actin dynamics in the neural epithelium are not known. To determine how the organization of the neural epithelium and neural tube closure are affected when actin dynamics are blocked, we examined the cellular basis of the neural tube closure defect in mouse mutants that lack the actin-severing protein cofilin 1 (CFL1). Although apical localization of the adherens junctions, the Par complex, the Crumbs complex and SHROOM3 is normal in the mutants, CFL1 has at least two distinct functions in the apical and basal domains of the neural plate. Apically, in the absence of CFL1 myosin light chain does not become phosphorylated, indicating that CFL1 is required for the activation of apical actomyosin required for neural tube closure. On the basal side of the neural plate, loss of CFL1 has the opposite effect on myosin: excess F-actin and myosin accumulate and the ectopic myosin light chain is phosphorylated. The basal accumulation of F-actin is associated with the assembly of ectopic basal tight junctions and focal disruptions of the basement membrane, which eventually lead to a breakdown of epithelial organization.

ß-Pix directs collective migration of anterior visceral endoderm cells in the early mouse embryo.

Collective epithelial migration is important throughout embryonic development. The underlying mechanisms are poorly understood but likely involve spatially localized activation of Rho GTPases. We previously reported that Rac1 is essential for generating the protrusive activity that drives the collective migration of anterior visceral endoderm (AVE) cells in the early mouse embryo. To identify potential regulators of Rac1, we first performed an RNAi screen of Rho family exchange factors (guanine nucleotide exchange factor [GEF]) in an in vitro collective epithelial migration assay and identified ß-Pix. Genetic deletion of ß-Pix in mice disrupts collective AVE migration, while high-resolution live imaging revealed that this is associated with randomly directed protrusive activity. We conclude that ß-Pix controls the spatial localization of Rac1 activity to drive collective AVE migration at a critical stage in mouse development.

Hand2 is an essential regulator for two Notch-dependent functions within the embryonic endocardium.

The basic-helix-loop-helix (bHLH) transcription factor Hand2 plays critical roles during cardiac morphogenesis via expression and function within myocardial, neural crest, and epicardial cell populations. Here, we show that Hand2 plays two essential Notch-dependent roles within the endocardium. Endocardial ablation of Hand2 results in failure to develop a patent tricuspid valve, intraventricular septum defects, and hypotrabeculated ventricles, which collectively resemble the human congenital defect tricuspid atresia. We show endocardial Hand2 to be an integral downstream component of a Notch endocardium-to-myocardium signaling pathway and a direct transcriptional regulator of Neuregulin1. Additionally, Hand2 participates in endocardium-to-endocardium-based cell signaling, with Hand2 mutant hearts displaying an increased density of coronary lumens. Molecular analyses further reveal dysregulation of several crucial components of Vegf signaling, including VegfA, VegfR2, Nrp1, and VegfR3. Thus, Hand2 functions as a crucial downstream transcriptional effector of endocardial Notch signaling during both cardiogenesis and coronary vasculogenesis.

Pten regulates collective cell migration during specification of the anterior-posterior axis of the mouse embryo.

Pten, the potent tumor suppressor, is a lipid phosphatase that is best known as a regulator of cell proliferation and cell survival. Here we show that mouse embryos that lack Pten have a striking set of morphogenetic defects, including the failure to correctly specify the anterior-posterior body axis, that are not caused by changes in proliferation or cell death. The majority of Pten null embryos express markers of the primitive streak at ectopic locations around the embryonic circumference, rather than at a single site at the posterior of the embryo. Epiblast-specific deletion shows that Pten is not required in the cells of the primitive streak; instead, Pten is required for normal migration of cells of the Anterior Visceral Endoderm (AVE), an extraembryonic organizer that controls the position of the streak. Cells of the wild-type AVE migrate within the visceral endoderm epithelium from the distal tip of the embryo to a position adjacent to the extraembryonic region. In all Pten null mutants, AVE cells move a reduced distance and disperse in random directions, instead of moving as a coordinated group to the anterior of the embryo. Aberrant AVE migration is associated with the formation of ectopic F-actin foci, which indicates that absence of Pten disrupts the actin-based migration of these cells. After the initiation of gastrulation, embryos that lack Pten in the epiblast show defects in the migration of mesoderm and/or endoderm. The findings suggest that Pten has an essential and general role in the control of mammalian collective cell migration.

Integration of a Notch-dependent mesenchymal gene program and Bmp2-driven cell invasiveness regulates murine cardiac valve formation.

Cardiac valve formation is crucial for embryonic and adult heart function. Valve malformations constitute the most common congenital cardiac defect, but little is known about the molecular mechanisms regulating valve formation and homeostasis. Here, we show that endocardial Notch1 and myocardial Bmp2 signal integration establish a valve-forming field between 2 chamber developmental domains. Patterning occurs through the activation of endocardial epithelial-to-mesenchymal transition (EMT) exclusively in prospective valve territories. Mice with constitutive endocardial Notch1 activity ectopically express Hey1 and Heyl. They also display an activated mesenchymal gene program in ventricles and a partial (noninvasive) EMT in vitro that becomes invasive upon BMP2 treatment. Snail1, TGF-ß2, or Notch1 inhibition reduces BMP2-induced ventricular transformation and invasion, whereas BMP2 treatment inhibits endothelial Gsk3ß, stabilizing Snail1 and promoting invasiveness. Integration of Notch and Bmp2 signals is consistent with Notch1 signaling being attenuated after myocardial Bmp2 deletion. Notch1 activation in myocardium extends Hey1 expression to nonchamber myocardium, represses Bmp2, and impairs EMT. In contrast, Notch deletion abrogates endocardial Hey gene transcription and extends Bmp2 expression to the ventricular endocardium. This embryonic Notch1-Bmp2-Snail1 relationship may be relevant in adult valve disease, in which decreased NOTCH signaling causes valve mesenchyme cell formation, fibrosis, and calcification.