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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.

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.