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Mouse models of lineage tracing have helped to describe the important subpopulations of hepatocytes responsible for liver regeneration. However, conflicting results have been obtained from different models. Herein, we aimed to reconcile these conflicting reports by repeating a key lineage-tracing study from pericentral hepatocytes and characterising this Axin2CreERT2 model in detail. We performed detailed characterisation of the labelled population in the Axin2CreERT2 model. We lineage traced this cell population, quantifying the labelled population over 1 year and performed in-depth phenotypic comparisons, including transcriptomics, metabolomics and analysis of proteins through immunohistochemistry, of Axin2CreERT2 mice to WT counterparts. We found that after careful definition of a baseline population, there are marked differences in labelling between male and female mice. Upon induced lineage tracing there was no expansion of the labelled hepatocyte population in Axin2CreERT2 mice. We found substantial evidence of disrupted homeostasis in Axin2CreERT2 mice. Offspring are born with sub-Mendelian ratios and adult mice have perturbations of hepatic Wnt/β-catenin signalling and related metabolomic disturbance. We find no evidence of predominant expansion of the pericentral hepatocyte population during liver homeostatic regeneration. Our data highlight the importance of detailed preclinical model characterisation and the pitfalls which may occur when comparing across sexes and backgrounds of mice and the effects of genetic insertion into native loci. Understanding the source of cells which regenerate the liver is crucial to harness their potential to regrow injured livers. Herein, we show that cells which were previously thought to repopulate the liver play only a limited role in physiological regeneration. Our data helps to reconcile differing conclusions drawn from results from a number of prior studies and highlights methodological challenges which are relevant to preclinical models more generally. [Display omitted] • Pericentral hepatocytes do not play a dominant role in physiological hepatocyte regeneration. • The Wnt pathway is suppressed by Axin2 haploinsufficiency. • Mouse genetics and environmental conditions should be rigorously controlled in preclinical models. [ABSTRACT FROM AUTHOR]

Precisely deciphering the cellular plasticity in vivo is essential in understanding many key biological processes. Site-specific recombinases are genetic tools used for in vivo lineage tracing and gene manipulation. Conventional Cre- loxP , Dre- rox , and Flp- frt technologies form the orthogonal recombination systems that can also be used in combination to increase the precision. As such, more than one marker gene can be targeted for lineage tracing, studying cellular heterogeneity, recording cellular activities, or even genome editing. Their combinatory use has recently resolved some controversies in defining cellular fate plasticity. Focusing on cell fate studies, we introduce the design principles of orthogonal recombinases-based strategies, describe some working examples in resolving cell fate-related controversies, and discuss some of their technical strengths and limits. Orthogonal recombinase systems are superior to conventional single-recombinase for studying in vivo cell fate, as they employ multiple markers to increase the resolution and precision of cell fate mapping results. Orthogonal recombinase systems enable genetic labeling of the intersect or subtractive parts of multiple cell populations, which helps clarify some controversial issues in cell origins and fate plasticity. Transitional transcriptional activation could be genetically recorded by orthogonal recombinase systems that could continuously capture some brief yet biologically important cellular events or states. Orthogonal recombinase systems have broad applications in cell lineage tracing and gene manipulation in fields of developmental biology, neuroscience, cardiovascular biology, oncology, and regenerative medicine. [ABSTRACT FROM AUTHOR]

Cardiac resident macrophages play vital roles in heart development, homeostasis, repair, and regeneration. Recent studies documented the hematopoietic potential of cardiac endothelium that supports the generation of cardiac macrophages and peripheral blood cells in mice. However, the conclusion was not strongly supported by previous genetic tracing studies, given the non-specific nature of conventional Cre- loxP tracing tools. Here, we develop an intercellular genetic labeling system that can permanently trace heart-specific endothelial cells based on cell-cell interaction in mice. Results from cell-cell contact-mediated genetic fate mapping demonstrate that cardiac endothelial cells do not exhibit hemogenic potential and do not contribute to cardiac macrophages or other circulating blood cells. This Matters Arising paper is in response to Shigeta et al. (2019), published in Developmental Cell. See also the response by Liu and Nakano (2023), published in this issue. [Display omitted] • Generation of intercellular genetic recording system to label neighboring cells • Genetic tracing of endocardium and coronary endothelial cells by cell-cell contact • Cardiac endothelium does not have hematopoietic potential in development • Cell-cell contact-mediated labeling enhances the specificity of genetic tracing In this Matters Arising article, Liu and Jin et al. re-evaluate the model that the developing endocardium is hemogenic and generates cardiac macrophages and circulating blood cells, which was proposed by Shigeta et al. By using an intercellular genetic tracing system that enables specific labeling of cardiac endothelium, they find that the cardiac endothelium does not have hematopoietic activity during heart development. [ABSTRACT FROM AUTHOR]

Endocardial development involves a complex orchestration of cell fate decisions that coordinate with endoderm formation and other mesodermal cell lineages. Historically, investigations into the contribution of endocardium in the developing embryo was constrained to the heart where these cells give rise to the inner lining of the myocardium and are a major contributor to valve formation. In recent years, studies have continued to elucidate the complexities of endocardial fate commitment revealing a much broader scope of lineage potential from developing endocardium. These studies cover a wide range of species and model systems and show direct contribution or fate potential of endocardium giving rise to cardiac vasculature, blood, fibroblast, and cardiomyocyte lineages. This review focuses on the marked expansion of knowledge in the area of endocardial fate potential. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Integration of Developmental and Environmental Cues in the Heart edited by Marcus Schaub and Hughes Abriel. [ABSTRACT FROM AUTHOR]

Abstract: The origins of liver progenitor cells have been extensively studied, but evidence on the origin of pancreatic precursor cells is currently limited. Pancreatic and duodenal homeobox gene 1 (Pdx1) is one of the earliest known markers for the pancreas. A transgenic mouse line expressing green fluorescent protein (GFP) under the control of the Pdx1 promoter showed that Pdx1/GFP expression was first observed in the mid-region of the anterior intestinal portal (AIP) lip at embryonic day (E) 8.5 at the 5–6 somite stage (ss). The liver progenitors were confirmed to originate from separate domains at the lateral endoderm and the inner part of the medial AIP as previously reported (), which turned out to lie caudally to the Pdx1/GFP-expressing domain. To confirm if the early Pdx1/GFP-positive cells give rise to the pancreatic bud, we labeled the cells on the lip of the AIP using the carbocyanine dye CM-DiI and traced their fates in 1–4 ss, 5–6 ss and 7–9 ss E8.5 embryos using an ex utero whole embryo culture method. At 1 ss, the ventral pancreas progenitors were observed in the lateral endoderm, not yet being segregated from the liver or gut progenitors. Cells that contributed solely to the ventral pancreas first appeared at the AIP lip from 5 ss. At 5–6 ss, cells from the medial of the AIP lip contributed to the ventral pancreas. The pancreas fate region become narrower as development progresses. At 7–9 ss, the cells contributing to the ventral pancreas resided in a narrow region of the AIP lip. From 5 ss, the right flanking region contributes to the posterior gut, and the left flanking region contributes to the anterior gut. Dorsal pancreatic progenitors originate from the dorsal endoderm at the 3–6 somite level at 7–9 ss, though they have not yet diverged from the dorsal gut progenitors at this stage. [Copyright &y& Elsevier]

Genetic lineage tracing unravels cell fate and plasticity in development, tissue homeostasis, and diseases. However, it remains technically challenging to trace temporary or transient cell fate, such as epithelial-to-mesenchymal transition (EMT) in tumor metastasis. Here, we generated a genetic fate-mapping system for temporally seamless tracing of transient cell fate. Highlighting its immediate application, we used it to study EMT gene activity from the local primary tumor to a distant metastatic site in vivo. In a spontaneous breast-to-lung metastasis model, we found that primary tumor cells activated vimentin and N-cadherin in situ , but only N-cadherin was activated and functionally required during metastasis. Tumor cells that have ever expressed N-cadherin constituted the majority of metastases in lungs, and functional deletion of N-cad significantly reduced metastasis. The seamless genetic recording system described here provides an alternative way for understanding transient cell fate and plasticity in biological processes. • Generation of a genetic system for recording transient cell fate in vivo • Genetic tracing of EMT gene activity during breast-to-lung tumor metastasis • Vimentin is not involved during mammary tumor metastasis in MMTV-PyMT model • N-cadherin is activated and functionally required during tumor metastasis Li, Lv et al. establish a genetic system to seamlessly record transient cell fate in vivo. Using this approach, they study epithelial-to-mesenchymal transition (EMT) gene activity from the local primary tumor to a distant metastatic site showing that N-cadherin but not vimentin is activated and functionally required during breast-to-lung tumor metastasis. [ABSTRACT FROM AUTHOR]