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Bud tip progenitors (BTPs) in the developing lung give rise to all epithelial cell types found in the airways and alveoli. This work aimed to develop an iPSC organoid model enriched with NKX2-1+ BTP-like cells. Building on previous studies, we optimized a directed differentiation paradigm to generate spheroids with more robust NKX2-1 expression. Spheroids were expanded into organoids that possessed NKX2-1+/CPM+ BTP-like cells, which increased in number over time. Single cell RNA-sequencing analysis revealed a high degree of transcriptional similarity between induced BTPs (iBTPs) and in vivo BTPs. Using FACS, iBTPs were purified and expanded as induced bud tip progenitor organoids (iBTOs), which maintained an enriched population of bud tip progenitors. When iBTOs were directed to differentiate into airway or alveolar cell types using well-established methods, they gave rise to organoids composed of organized airway or alveolar epithelium, respectively. Collectively, iBTOs are transcriptionally and functionally similar to in vivo BTPs, providing an important model for studying human lung development and differentiation.

Recent advances using single cell genomic approaches have identified new epithelial cell types and uncovered cellular heterogeneity in the murine and human lung (1). Here, using scRNA-seq and microscopy we identify and describe a secretory-like cell that is enriched in the small airways of the developing human lung and identified by the unique co-expression of SCGB3A2/SFTPB/CFTR. To place these cells in the hierarchy of airway development, we apply a single cell barcode-based lineage tracing method track the fate of SCGB3A2/SFTPB/CFTR cells during airway organoid differentiation in vitro (2). Lineage tracing revealed that these cells have distinct developmental potential from basal cells, giving rise predominantly to pulmonary neuroendocrine cells (PNECs) and a subset of multiciliated cells distinguished by high C6 and low MUC16 expression. We conclude that SCGB3A2/SFTPB/CFTR cells act as a progenitor cell contributing to the cellular diversity and heterogeneity in the developing human airway.SIGNIFICANCE STATEMENTThe current study identifies a novel secretory cell type that is present predominantly in the small airway of the developing human lung. These secretory cells are defined by co-expression of SCGB3A2/SFTPB/CFTR, and functional studies show that this cell gives rise to pulmonary neuroendocrine cells and a sub-population of multiciliated cells, thereby leading to cellular heterogeneity.

Bud tip progenitors (BTPs) in the developing lung give rise to all epithelial cell types found in the airways and alveoli. The current work aimed to develop an iPSC organoid model enriched with stable NKX2-1+ BTP-like cells. Building on prior work, we optimized a directed differentiation paradigm to generate spheroids with robust NKX2-1 expression. Spheroids were expanded into organoids that possessed NKX2-1+/CPM+ BTP-like cells, which increased in number over time. Single cell RNA-sequencing analysis revealed a high degree of transcriptional similarity between induced BTPs (iBTPs) and in vivo BTPs. Using FACS, iBTPs can be purified and expanded as induced bud tip organoids (iBTO), which maintain an enriched population of bud tip progenitors. When iBTOs are directed to differentiate into airway or alveolar cell types using well-established methods, they give rise to organoids composed of organized airway or alveolar epithelium, respectively. Collectively, iBTOs are transcriptionally and functionally similar to in vivo BTPs, providing an important model to study human lung development and differentiation.SUMMARY STATEMENTiPSC-derived lung bud tip progenitors emerge in organoid culture, can be isolated and expanded, are transcriptionally similar to primary bud tip progenitors, and can differentiate into airway or alveolar organoids.

SUMMARYMammalian respiratory system development is regulated by complex reciprocal signaling events that take place between epithelial cells and the surrounding mesenchymal cells; however, mesenchymal heterogeneity and function in the developing human lung is poorly understood. We interrogated single cell RNA sequencing data from multiple human lung specimens and identified a mesenchymal cell population present during development that is highly enriched for expression of the WNT agonistR-SPONDIN2(RSPO2), and we found that adjacent epithelial bud tip progenitors are enriched for the RSPO2 receptorLGR5. By carrying out functional experiments using organoid models, lung explant cultures, and FACS-isolatedRSPO2+mesenchyme, we show that RSPO2 is a critical niche cue that potentiates WNT signaling in human lung progenitors to maintain their multipotency.

Patients with coronavirus disease 2019 (COVID-19) present a wide range of acute clinical manifestations affecting the lungs, liver, kidneys and gut. Angiotensin converting enzyme (ACE) 2, the best-characterized entry receptor for the disease-causing virus SARS-CoV-2, is highly expressed in the aforementioned tissues. However, the pathways that underlie the disease are still poorly understood. Here, we unexpectedly found that the complement system was one of the intracellular pathways most highly induced by SARS-CoV-2 infection in lung epithelial cells. Infection of respiratory epithelial cells with SARS-CoV-2 generated activated complement component C3a and could be blocked by a cell-permeable inhibitor of complement factor B (CFBi), indicating the presence of an inducible cell-intrinsic C3 convertase in respiratory epithelial cells. Within cells of the bronchoalveolar lavage of patients, distinct signatures of complement activation in myeloid, lymphoid and epithelial cells tracked with disease severity. Genes induced by SARS-CoV-2 and the drugs that could normalize these genes both implicated the interferon-JAK1/2-STAT1 signaling system and NF-κB as the main drivers of their expression. Ruxolitinib, a JAK1/2 inhibitor, normalized interferon signature genes and all complement gene transcripts induced by SARS-CoV-2 in lung epithelial cell lines, but did not affect NF-κB-regulated genes. Ruxolitinib, alone or in combination with the antiviral remdesivir, inhibited C3a protein produced by infected cells. Together, we postulate that combination therapy with JAK inhibitors and drugs that normalize NF-κB-signaling could potentially have clinical application for severe COVID-19.

The human respiratory epithelium is derived from a progenitor cell in the distal buds of the developing lung. These "bud tip progenitors" are regulated by reciprocal signaling with surrounding mesenchyme; however, mesenchymal heterogeneity and function in the developing human lung are poorly understood. We interrogated single-cell RNA sequencing data from multiple human lung specimens and identified a mesenchymal cell population present during development that is highly enriched for expression of the WNT agonist RSPO2, and we found that the adjacent bud tip progenitors are enriched for the RSPO2 receptor LGR5. Functional experiments using organoid models, explant cultures, and FACS-isolated RSPO2

The human respiratory epithelium is derived from a progenitor cell in the distal buds of the developing lung. These ""bud tip progenitors'' are regulated by reciprocal signaling with surrounding mesenchyme; however, mesenchymal heterogeneity and function in the developing human lung are poorly understood. We interrogated single-cell RNA sequencing data from multiple human lung specimens and identified a mesenchymal cell population present during development that is highly enriched for expression of theWNT agonist RSPO2, and we found that the adjacent bud tip progenitors are enriched for the RSPO2 receptor LGR5. Functional experiments using organoid models, explant cultures, and FACS-isolated RSPO2(+) mesenchyme show that RSPO2 is a critical niche cue that potentiates WNT signaling in bud tip progenitors to support their maintenance and multipotency., This project has been made possible in part by grant number CZF2019-002440 from the Chan Zuckerberg Initiative DAF, an advised fund from the Silicon Valley Community Foundation, and in part by the NIH-NHLBI (R01HL119215) funding to J.R.S.; R.F.C.H. was supported by a NIH Tissue En- gineering and Regenerative Medicine Training Grant (NIH-NIDCR T32DE 007057) and by a Ruth L. Kirschstein Predoctoral Individual National Research Service Award (NIH-NHLBI F31HL152531); A.J.M. was supported by a Ruth L. Kirschstein Predoctoral Individual National Research Service Award (NIH- NHLBI F31HL142197); E.M.H. was supported by a Ruth L. Kirschstein Predoc- toral Individual National Research Service Award (NIH-NHBLI F31HL146162); T.F. was supported by a NIH Tissue Engineering and Regenerative Medicine Training Grant (NIH-NIDCR T32DE007057); A.S.C. was supported by the T32 Michigan Medical Scientist Training Program (5T32GM007863-40); I.G. and the University of Washington Laboratory of Developmental Biology was supported by NIH award number 5R24HD000836 from the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD). B.R. is a fellow of the Branco Weiss Foundation (Switzerland) and the National Research Foundation (Singapore), and an A*STAR and EMBO Young Investi- gator. This work was supported by an inaugural Use-Inspired Basic Research (UIBR) central fund from the Agency for Science, Technology and Research (A*STAR) and National Medical Research Council Open Fund– Individual Research Grants (OF-YIRG,#OFYIRG18May-0053; and OF-IRG, #OFIRG20- Nov-0057), to E.S.-R and B.R., respectively. We would like to thank Judy Opp and the University of Michigan Advanced Genomics core for the operation of the 103 chromium single-cell capture plat- form, the University of Michigan Microscopy core for providing access to confocal microscopes, the Flow Cytometry core for providing access to flow cytometers, and the Vector core for providing lentivirus production and adeno- virus purification and expansion services. We would also like to thank the Uni- versity of Washington Laboratory of Developmental Biology. Lastly, we would like to thank Michael Dame and the Translational Tissue Modeling Laboratory (TTML) for providing helpful suggestions regarding the use of WNT3a afamin conditioned media as well as Lindy K. Brastrom for her careful technical editing of the manuscript.

An abundance of information about lung development in animal models exists; however, comparatively little is known about lung development in humans. Recent advances using primary human lung tissue combined with the use of human in vitro model systems, such as human pluripotent stem cell-derived tissue, have led to a growing understanding of the mechanisms governing human lung development. They have illuminated key differences between animal models and humans, underscoring the need for continued advancements in modeling human lung development and utilizing human tissue. This review discusses the use of human tissue and the use of human in vitro model systems that have been leveraged to better understand key regulators of human lung development and that have identified uniquely human features of development. This review also examines the implementation and challenges of human model systems and discusses how they can be applied to address knowledge gaps.

CRISPR technologies used for mammalian embryology have wide implications from basic research to applications in agriculture and biomedicine. Confirmation of successful gene editing following CRISPR/Cas9 delivery is often limited to either protein expression or sequencing analyses of embryos but not both, due to technical challenges. Herein we report an integrative approach for evaluating both protein expression and genotype of single embryos from fixed bovine embryos previously subjected to CRISPR/Cas9 microinjection. The techniques described facilitate investigation of functional genomics in bovine embryos compatible with gene editing in livestock after zygotic CRISPR microinjection. These methods avoid traditional avenues that necessitate the use of gene-edited cell lines followed by nuclear transfer that hinder efficiency, limit physiological relevance and contribute to technical challenges.

The HIPPO signaling pathway plays an early and essential role in mammalian embryogenesis. The earliest known roles for HIPPO signaling during mouse development include segregating fetal and extraembryonic lineages and establishing the pluripotent progenitors of embryonic stem (ES) cells. In the mouse early embryo, HIPPO signaling responds to multiple cell biological inputs, including cell polarization, cytoskeleton, and cell environment, to influence gene expression and the first cell fate decisions in development. Methods to monitor and manipulate HIPPO signaling in the mouse early embryo are fundamental to discovering mechanisms regulating pluripotency in vivo, but properties of the early embryo, such as small cell number and spherical architecture, pose unique challenges for signaling pathway analysis. Here, we share approaches for visualizing HIPPO signaling in mouse early embryos. In addition, these methods can be applied to visualize HIPPO signaling in other spherical or cystic structures comprised of relatively few cells, such as organoids, or for the examination of other signaling pathways in these contexts.

The HIPPO signaling pathway plays an early and essential role in mammalian embryogenesis. The earliest known roles for HIPPO signaling during mouse development include segregating fetal and extraembryonic lineages and establishing the pluripotent progenitors of embryonic stem (ES) cells. In the mouse early embryo, HIPPO signaling responds to multiple cell biological inputs, including cell polarization, cytoskeleton, and cell environment, to influence gene expression and the first cell fate decisions in development. Methods to monitor and manipulate HIPPO signaling in the mouse early embryo are fundamental to discovering mechanisms regulating pluripotency in vivo, but properties of the early embryo, such as small cell number and spherical architecture, pose unique challenges for signaling pathway analysis. Here, we share approaches for visualizing HIPPO signaling in mouse early embryos. In addition, these methods can be applied to visualize HIPPO signaling in other spherical or cystic structures comprised of relatively few cells, such as organoids, or for the examination of other signaling pathways in these contexts.

During mammalian development, the challenge for the embryo is to override intrinsic cellular plasticity to drive cells to distinct fates. Here, we unveil novel roles for the HIPPO signaling pathway in controlling cell positioning and expression of Sox2, the first marker of pluripotency in the mouse early embryo. We show that maternal and zygotic YAP1 and WWTR1 repress Sox2 while promoting expression of the trophectoderm gene Cdx2 in parallel. Yet, Sox2 is more sensitive than Cdx2 to Yap1/Wwtr1 dosage, leading cells to a state of conflicted cell fate when YAP1/WWTR1 activity is moderate. Remarkably, HIPPO signaling activity resolves conflicted cell fate by repositioning cells to the interior of the embryo, independent of its role in regulating Sox2 expression. Rather, HIPPO antagonizes apical localization of Par complex components PARD6B and aPKC. Thus, negative feedback between HIPPO and Par complex components ensure robust lineage segregation., eLife digest As an embryo develops, its cells divide, grow and migrate in specific patterns to build an organized collection of cells that go on to form our tissues and organs. One of the first steps – well before the embryo has implanted into the womb – is to allocate cells to make part of the placenta. Once this process is complete, the remaining cells continue building the organism. These cells are pluripotent, meaning they can develop into any part of the body. Scientists think that the embryo manages to sort ‘placenta cells’ from pluripotent ones with the help of certain proteins, which the mother has packaged into her eggs. To investigate this further, Frum et al. used genetic tools to track a specific gene called Sox2 that identifies pluripotent cells as soon as they are formed in mouse embryos. The experiments revealed that the mother places two closely related proteins known as YAP1 and WWTR1 within each egg, which help to make placenta cells different from pluripotent cells. Moreover, both proteins enable the embryo to segregate these two cell types to two different locations: placenta cells are moved to the outer layer of the embryo, while pluripotent cells are moved to the inside. Current technologies allow researchers to create pluripotent cells in the laboratory. But these approaches often result in error, failing to replicate the embryo’s natural ability. By studying how embryos form and arrange pluripotent cells, scientists hope to advance stem cell technology (which emerge from pluripotent cells). This may help to find new ways to heal damaged tissues and organs, or to treat or even prevent many diseases.

A new study by Zenker et al. uses time-lapse imaging to discover how dynamic actin movements contribute to epithelialization of living mouse embryos. Together with work from other labs, this study presents exciting new ways to think about the emergence of cell fates during mammalian development.

The first cell fate decisions during mammalian development establish tissues essential for healthy pregnancy. The mouse has served as a valuable model for discovering pathways regulating the first cell fate decisions because of the ease with which early embryos can be recovered and the availability of an arsenal of classical and emerging methods for manipulating gene expression. We summarize the major pathways that govern the first cell fate decisions in mouse development. This knowledge serves as a paradigm for exploring how emergent properties of a self-organizing system can dynamically regulate gene expression and cell fate plasticity. Moreover, it brings to light the processes that establish healthy pregnancy and ES cells. We also describe unsolved mysteries and new technologies that could help to overcome experimental challenges in the field.

Polarization is a mechanism commonly used by cells to establish asymmetries and create specialized subcellular functional domains. The process of cell polarization is especially important during mammalian development, when pluripotent cells are first established. Pluripotent cells are, by definition, capable of giving rise to the entire fetus. In addition, pluripotent stem cell lines can be derived from mammalian embryos and can be created by reprogramming differentiated cells. Understanding the molecular and cell biological differences between pluripotent and nonpluripotent cells is an important research direction in regenerative medicine. This chapter focuses on examining the role that cell polarization plays in establishing and maintaining pluripotency in the mouse embryo and in pluripotent stem cell lines. The literature shows that, in the early embryo, cell polarization is repressed in emerging pluripotent epiblast cells, while apicobasal polarization is established in multipotent cells of the extraembryonic lineages. Since polarization of extraembryonic cells is essential for defining which cells become pluripotent, cell polarization is indirectly responsible for establishing pluripotency in the embryo. Later, in the established pluripotent epiblast, cells polarize just before they exit pluripotency and commence differentiation. Just as multiple stages of pluripotency exist in the embryo, multiple pluripotent states exist among stem cell lines, ranging from naive to primed pluripotency. Mirroring the embryonic stages of pluripotency, polarity is associated with primed, and not naive, pluripotent stem cell lines. Therefore, in both embryos and in stem cell lines, cell polarization is a hallmark of decreased developmental potential, suggesting that the purpose of pluripotent cell polarization is to prepare cells for the next developmental goal: differentiation.

SummaryIn embryonic stem (ES) cells and in early mouse embryos, the transcription factor Oct4 is an essential regulator of pluripotency. Oct4 transcriptional targets have been described in ES cell lines; however, the molecular mechanisms by which Oct4 regulates establishment of pluripotency in the epiblast (EPI) have not been fully elucidated. Here, we show that neither maternal nor zygotic Oct4 is required for the formation of EPI cells in the blastocyst. Rather, Oct4 is first required for development of the primitive endoderm (PE), an extraembryonic lineage. EPI cells promote PE fate in neighboring cells by secreting Fgf4, and Oct4 is required for expression of Fgf4, but we show that Oct4 promotes PE development cell-autonomously, downstream of Fgf4 and Mapk. Finally, we show that Oct4 is required for the expression of multiple EPI and PE genes as well as multiple metabolic pathways essential for the continued growth of the preimplantation embryo.

In many invertebrate and vertebrate species, cell fates are assigned through the cellular inheritance of differentially localized maternal determinants. Whether mammalian embryogenesis is also regulated by deterministic mechanisms is highly controversial. The caudal domain transcription factor CDX2 has been reported to act as a maternal determinant regulating cell fate decisions in mouse development. However, this finding is contentious because of reports that maternal Cdx2 is not essential for development. Notably, all of the previously published studies of maternal Cdx2 relied on injected RNA interference constructs, which could introduce experimental variation. Only deletion of the maternal gene can unambiguously resolve its requirement in mouse development. Here, we genetically ablated maternal Cdx2 using a Cre/lox strategy, and we definitively establish that maternal Cdx2 is not essential for mouse development.

Confirmation of gene editing in livestock following CRISPR/Cas9 zygotic microinjection is often limited to either protein expression or gene sequencing analyses of unfixed embryos, but not both, due to the challenges of extracting DNA from fixed samples suitable for genotyping. Bovine embryos have been derived from gene-edited cell lines to screen for mutations followed by nuclear transfer, but these techniques limit embryo production efficiency, include technical challenges, and alter physiological relevance. Herein we report an integrative approach to evaluate both protein expression and the genotype of individual, fixed bovine embryos subjected to CRISPR/Cas9 microinjection. Bovine zygotes were derived from IVF and parthenogenetic activation of in vitro-matured oocytes followed by intracytoplasmic injection of CRISPR/Cas9 targeting embryonic POU5F1. Embryos were cultured for 7 days and fixed in 4% paraformaldehyde and stored at 5°C for up to 4 weeks. Fixed embryos were first subjected to immunohistochemistry to determine POU5F1 mutation success by protein expression. Binder reinforcement labels were affixed to glass slides as a substitute for adhesive spacers. Single embryos were added to 9µL of PBS with a coverslip for imaging. Individual embryos were then recovered and placed in 10µL of QuickExtract (Lucigen, Middleton, WI, USA) for DNA extraction by heating samples to 65°C for 6min and 95°C for 2min. Two rounds of PCR were applied to templates in 20-µL reactions consisting of 10µL of GoTaq Hot Start Green Master Mix (2×; Promega, Madison, WI, USA) with the addition of 0.4µL (10µM) of forward and reverse primer each and 9.2µL of template. The second PCR reaction contained 5µL of PCR product from the first reaction, nested primers, and 4.2µL of H2O. The PCR conditions were |95°C, 3 min| and 35 cycles of |95°C, 30s |56°C, 30 s|72°C, 30 s|72°C, 7 min|. The PCR product was run on an agarose gel to confirm DNA amplification of a single band, and the remainder was purified (QIAquick PCR Purification Kit, Qiagen, Hilden, Germany) and submitted (100ng) for Sanger sequencing. The CRISPR-injected embryos were aligned to wild-type embryo sequences using SnapGene (http://www.snapgene.com/) and TIDE software (https://tide.nki.nl/). Both DNA extraction (n=18/19) and Sanger sequencing (n=19/23) proved highly efficient and repeatable (83 and 95%, respectively). These methods provide an efficient, cost effective, and reproducible approach for confirmation of CRISPR gene editing in individual and fixed bovine embryos by both protein expression and gene sequencing. In addition, these techniques complement the production of gene editing in livestock after zygotic CRISPR microinjection by conclusively determining mutation efficiency through both proteomic and genomic analyses. This research was supported by a USDA NIFA AFRI ELI Postdoctoral Fellowship 2016-67012-25254 to BWD and NIFA multistate research project W3171 to PJR. BD is currently supported by a grant from the Eunice Kennedy Shiver National Institute of Child Health and Human Development of the National Institutes of Health under the award numbers T32HD087166.

Pluripotent epiblast (EPI) cells, present in the inner cell mass (ICM) of the mouse blastocyst, are progenitors of both embryonic stem (ES) cells and the fetus. Discovering how pluripotency genes regulate cell fate decisions in the blastocyst provides a valuable way to understand how pluripotency is normally established. EPI cells are specified by two consecutive cell fate decisions. The first decision segregates ICM from trophectoderm (TE), an extraembryonic cell type. The second decision subdivides ICM into EPI and primitive endoderm (PE), another extraembryonic cell type. Here, we investigate the roles and regulation of the pluripotency gene Sox2 during blastocyst formation. First, we investigate the regulation of Sox2 patterning and show that SOX2 is restricted to ICM progenitors prior to blastocyst formation by members of the HIPPO pathway, independent of CDX2, the TE transcription factor that restricts Oct4 and Nanog to the ICM. Second, we investigate the requirement for Sox2 in cell fate specification during blastocyst formation. We show that neither maternal (M) nor zygotic (Z) Sox2 is required for blastocyst formation, nor for initial expression of the pluripotency genes Oct4 or Nanog in the ICM. Rather, Z Sox2 initially promotes development of the primitive endoderm (PE) non cell-autonomously via FGF4, and then later maintains expression of pluripotency genes in the ICM. The significance of these observations is that 1) ICM and TE genes are spatially patterned in parallel prior to blastocyst formation and 2) both the roles and regulation of Sox2 in the blastocyst are unique compared to other pluripotency factors such as Oct4 or Nanog., Author Summary Pluripotent stem cells can give rise to any cell type in the body, making them an attractive tool for regenerative medicine. Pluripotent stem cells can be derived from the mammalian embryo at the blastocyst stage or they can be created from mature adult cells by reprogramming. During reprogramming, SOX2 helps establish pluripotency, but it is not clear how SOX2 establishes pluripotency in the blastocyst. We evaluated where SOX2 is present, how SOX2 is regulated, and where SOX2 is active during blastocyst formation. Our data show that the roles and the regulation of SOX2 are unique compared to other pluripotency/reprogramming factors, such as OCT4 and NANOG. SOX2 marks pluripotent cells earlier than do other factors, but does not regulate pluripotency until several days later. Rather, the earlier role of SOX2 is to help establish the yolk sac lineage, which is essential for gestation.