Notch and retinoic acid signals regulate macrophage formation from endocardium downstream of Nkx2-5.

Hematopoietic progenitors are enriched in the endocardial cushion and contribute, in a Nkx2-5-dependent manner, to tissue macrophages required for the remodeling of cardiac valves and septa. However, little is known about the molecular mechanism of endocardial-hematopoietic transition. In the current study, we identified the regulatory network of endocardial hematopoiesis. Signal network analysis from scRNA-seq datasets revealed that genes in Notch and retinoic acid (RA) signaling are significantly downregulated in Nkx2-5-null endocardial cells. In vivo and ex vivo analyses validate that the Nkx2-5-Notch axis is essential for the generation of both hemogenic and cushion endocardial cells, and the suppression of RA signaling via Dhrs3 expression plays important roles in further differentiation into macrophages. Genetic ablation study revealed that these macrophages are essential in cardiac valve remodeling. In summary, the study demonstrates that the Nkx2-5/Notch/RA signaling plays a pivotal role in macrophage differentiation from hematopoietic progenitors.

Cell disorientation by loss of SHH-dependent mechanosensation causes cyclopia.

The physical causes of organ malformation remain largely unclear in most cases due to a lack of information on tissue/cell dynamics. Here, we address this issue by considering onset of cyclopia in sonic hedgehog (SHH)-inhibited chick embryos. We show that ventral forebrain-specific self-organization ability driven by SHH-dependent polarized patterns in cell shape, phosphorylated myosin localization, and collective cell motion promotes optic vesicle elongation during normal development. Stress loading tests revealed that these polarized dynamics result from mechanical responses. In particular, stress and active tissue deformation satisfy orthogonality, defining an SHH-regulated morphogenetic law. Without SHH signaling, cells cannot detect the direction of stress and move randomly, leading to insufficient optic vesicle elongation and consequently a cyclopia phenotype. Since polarized tissue/cell dynamics are common in organogenesis, cell disorientation caused by loss of mechanosensation could be a pathogenic mechanism for other malformations.

Nucleome programming is required for the foundation of totipotency in mammalian germline development.

Germ cells are unique in engendering totipotency, yet the mechanisms underlying this capacity remain elusive. Here, we perform comprehensive and in-depth nucleome analysis of mouse germ-cell development in vitro, encompassing pluripotent precursors, primordial germ cells (PGCs) before and after epigenetic reprogramming, and spermatogonia/spermatogonial stem cells (SSCs). Although epigenetic reprogramming, including genome-wide DNA de-methylation, creates broadly open chromatin with abundant enhancer-like signatures, the augmented chromatin insulation safeguards transcriptional fidelity. These insulatory constraints are then erased en masse for spermatogonial development. Notably, despite distinguishing epigenetic programming, including global DNA re-methylation, the PGCs-to-spermatogonia/SSCs development entails further euchromatization. This accompanies substantial erasure of lamina-associated domains, generating spermatogonia/SSCs with a minimal peripheral attachment of chromatin except for pericentromeres-an architecture conserved in primates. Accordingly, faulty nucleome maturation, including persistent insulation and improper euchromatization, leads to impaired spermatogenic potential. Given that PGCs after epigenetic reprogramming serve as oogenic progenitors as well, our findings elucidate a principle for the nucleome programming that creates gametogenic progenitors in both sexes, defining a basis for nuclear totipotency.

Light-induced local gene expression in primary chick cell culture system.

The ability to manipulate gene expression at a specific region in a tissue or cell culture system is critical for analysis of target gene function. For chick embryos/cells, several gene introduction/induction methods have been established such as those involving retrovirus, electroporation, sonoporation, and lipofection. However, these methods have limitations in the accurate induction of localized gene expression. Here we demonstrate the effective application of a recently developed light-dependent gene expression induction system (LightOn system) using the Neurospora crassa photoreceptor Vivid fused with a Gal4 DNA binding domain and p65 activation domain (GAVPO) that alters its activity in response to light stimulus in a primary chicken cell culture system. We show that the gene expression level and induction specificity in this system are strongly dependent on the light irradiation conditions. Especially, the irradiation interval is an important parameter for modulating gene expression; for shorter time intervals, higher induction specificity can be achieved. Further, by adjusting light irradiation conditions, the expression level in primary chicken cells can be regulated in a multiple step manner, in contrast to the binary expression seen for gene disruption or introduction (i.e., null or overexpression). This result indicates that the light-dependent expression control method can be a useful technique in chick models to examine how gene function is affected by gradual changes in gene expression levels. We applied this light induction system to regulate Sox9 expression in cultures of chick limb mesenchyme cells and showed that induced SOX9 protein could modulate expression of downstream genes.

Quantitative Analysis of 3D Tissue Deformation Reveals Key Cellular Mechanism Associated with Initial Heart Looping.

Despite extensive study, the morphogenetic mechanisms of heart looping remain controversial because of a lack of information concerning precise tissue-level deformation and the quantitative relationship between tissue and cellular dynamics; this lack of information causes difficulties in evaluating previously proposed models. To overcome these limitations, we perform four-dimensional (4D) high-resolution imaging to reconstruct a tissue deformation map, which reveals that, at the tissue scale, initial heart looping is achieved by left-right (LR) asymmetry in the direction of deformation within the myocardial tube. We further identify F-actin-dependent directional cell rearrangement in the right myocardium as a major contributor to LR asymmetric tissue deformation. Our findings demonstrate that heart looping involves dynamic and intrinsic cellular behaviors within the tubular tissue and provide a significantly different viewpoint from current models that are based on LR asymmetry of growth and/or stress at the tube boundaries. Finally, we propose a minimally sufficient model for initial heart looping that is also supported by mechanical simulations.