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Redundant and Additive Functions of the Four Lef/Tcf Transcription Factors in Lung Epithelial Progenitors

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Redundant and Additive Functions of the Four Lef/Tcf Transcription Factors in Lung Epithelial Progenitors Redundant and additive functions of the four Lef/Tcf transcription factors in lung epithelial progenitors Kamryn N. Gerner-Mauroa, Haruhiko Akiyamab, and Jichao Chena,1 aDepartment of Pulmonary Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX 77030; and bDepartment of Orthopedics, Gifu University, 501-1194 Gifu, Japan Edited by Brigid L. M. Hogan, Duke University Medical Center, Durham, NC, and approved April 14, 2020 (received for review February 6, 2020) In multicellular organisms, paralogs from gene duplication survive knockout models demonstrate their overlapping but noninter- purifying selection by evolving tissue-specific expression and func- changeable functions, and partially phenocopy deletion of their tion. Whether this genetic redundancy is also selected for within a obligatory transcriptional coactivator CTNNB1 (6). Studying ad- single cell type is unclear for multimember paralogs, as exempli- ditional paralogs involves dauntingly complex genetic crosses and fied by the four obligatory Lef/Tcf transcription factors of canon- is limited to cultured cells and global deletion in early Xenopus ical Wnt signaling, mainly due to the complex genetics involved. embryos (7, 8). As a result, genetic redundancy of Lef/Tcf genes Using the developing mouse lung as a model system, we generate in vivo and within a given cell type, as well as the corresponding two quadruple conditional knockouts, four triple mutants, and requirement of CTNNB1, is unknown. various combinations of double mutants, showing that the four The established requirement of WNT2/2B and CTNNB1 in Lef/Tcf genes function redundantly in the presence of at least two Lef/Tcf paralogs, but additively upon losing additional paral- specifying lung epithelial progenitors from the embryonic fore- ogs to specify and maintain lung epithelial progenitors. Prelung- gut, as well as the continued role of CTNNB1 in maintaining the – specification, pan-epithelial double knockouts have no lung pheno- progenitors (9 11), supports the involvement of canonical Wnt type; triple knockouts have varying phenotypes, including defective signaling and lays the foundation to investigate the identity and branching and tracheoesophageal fistulas; and the quadruple associated genetic redundancy of Lef/Tcf transcription factors. knockout barely forms a lung, resembling the Ctnnb1 mutant. In this study, using pan-lung epithelium and progenitor-specific Postlung-specification deletion of all four Lef/Tcf genes leads to Cre drivers, we first deleted Tcf7l1 and Tcf7l2, the main Lef/Tcf branching defects, down-regulation of progenitor genes, premature paralogs expressed in the lung epithelium, but did not detect any DEVELOPMENTAL BIOLOGY alveolar differentiation, and derepression of gastrointestinal genes, phenotype. Extending our analysis to additional double mutants, again phenocopying the corresponding Ctnnb1 mutant. Our study all possible triple mutants, and the quadruple mutant, we show supports a monotonic, positive signaling relationship between that depending on gene dosage, Lef/Tcf paralogs function ad- CTNNB1 and Lef/Tcf in lung epithelial progenitors as opposed to ditively and redundantly in lung progenitor specification and reported repressor functions of Lef/Tcf, and represents a thorough branching. Single-cell RNA-seq (scRNA-seq) reveals the role of in vivo analysis of cell-type-specific genetic redundancy among the Lef/Tcf paralogs in maintaining lung progenitors by promoting four Lef/Tcf paralogs. progenitor genes and suppressing gastrointestinal genes and pre- lung development | epithelial progenitors | Wnt signaling | Lef/Tcf mature alveolar differentiation, recapitulating the transcriptional transcription factors | gene duplication defects of the progenitor-specific Ctnnb1 mutant. Our study clar- ifies the signaling relationship between CTNNB1 and Lef/Tcf and equenced genomes of key species in the phylogenetic tree, Sespecially the amphioxus, suggest that two rounds of whole Significance genome duplication quadruple ancestral genes in the jawed vertebrates, as exemplified by the single Hox gene cluster in flies Paralogs represent genetic redundancy and survive purifying se- and four in mammals (1–3). This, together with individual gene lection by evolving overlapping and distinct functions. In multi- amplification, generates paralogs that are initially redundant and cellular organisms, such functional diversification can manifest as must evolve unique functions to escape purifying selection. This tissue and cell-type-specific expression, which masks possible se- apparent genetic redundancy is maintained if the surviving lective pressure for genetic redundancy within a single cell type. paralogs provide 1) an extra amount of the same function, 2) a Using in vivo genetic and genomic analyses, we show that al- back-up to buffer environmental fluctuations including somatic though the four mammalian Lef/Tcf transcription factors have mutations, or 3) a new function such as alternative enzymatic evolved organ-specific functions, they function additively and re- substrates (4). In multicellular organisms, when paralogs differ in dundantly, depending on gene dosage, to promote lung epithelial their tissue and cell type distributions, such differential expres- progenitors and do so in a monotonic, positive manner with beta- sion even in the absence of differential protein activity provides catenin in the canonical Wnt signaling pathway. unique functions and allows their existence in the genome. This Author contributions: K.N.G.-M. and J.C. designed research; K.N.G.-M. and J.C. performed organism-level genetic redundancy, however, masks possible re- research; H.A. contributed new reagents/analytic tools; K.N.G.-M. and J.C. analyzed data; dundancy within individual cell types, which is only revealed by and K.N.G.-M. and J.C. wrote the paper. studying cell-type-specific mutants. The authors declare no competing interest. The canonical Wnt signaling pathway culminates in beta-catenin This article is a PNAS Direct Submission. (CTNNB1) binding to and activating the Lef/Tcf transcription Published under the PNAS license. factors, which include a single member in flies (Pangolin or dTCF) Data deposition: Raw data for scRNA-seq data have been deposited in GEO under the and worms (POP-1) but four in mice and humans (LEF1, TCF7, accession number GSE144170.(https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi? TCF7L1, and TCF7L2) (5). Global, single knockout of individual acc=GSE144170). Lef/Tcf genes in mice results in defects in skin appendages (Lef1), 1To whom correspondence may be addressed. Email: [email protected]. Tcells(Tcf7), embryonic axis induction (Tcf7l1), and intestinal This article contains supporting information online at https://www.pnas.org/lookup/suppl/ epithelial stem cells (Tcf7l2), highlighting the aforementioned doi:10.1073/pnas.2002082117/-/DCSupplemental. tissue-specific functions of paralogs. Several subsequent double www.pnas.org/cgi/doi/10.1073/pnas.2002082117 PNAS Latest Articles | 1of10 Downloaded by guest on September 27, 2021 provides an in vivo demonstration of genetic redundancy within a branch tip, suggesting thwarted SOX9 progenitor expansion and/or single cell type. their accelerated differentiation into SOX2 progeny (Fig. 2B and SI Appendix,Fig.S1B). The Lef1L/+ triple mutant additionally had a Results fused trachea and esophagus—recapitulating the tracheoesophageal TCF7L1 and TCF7L2, the Main LEF/TCF Factors in the Lung Epithelium, fistula phenotype in the pan-epithelial Ctnnb1 mutant (9–11) Are Not Required for Epithelial Progenitor Specification and Maintenance. (Fig. 2B and SI Appendix,Fig.S1C). Intriguingly, whereas no vi- The lung epithelial primordium originates fromtheanteriorforegut able pups were obtained for the three triple mutants carrying as a group of NKX2-1/SOX9 double positive progenitors, which re- Tcf7l2 deletion presumably due to the reported intestinal defects main distally during subsequent branching morphogenesis, leaving (15), the Tcf7l2L/+ triple mutant—despite defective branching— behind SOX2-expressing progeny for the airways early in develop- formed apparently normal alveoli and survived postnatally, albeit ment and alveolar cells late in development (12). The established role with no hair as expected from Lef1 deletion (16) (SI Appendix, Fig. of canonical Wnt signaling components, CTNNB1 and WNT2/2B, S1D), suggesting that the lung normally has sufficient functional in specifying and maintaining these lung progenitors (9–11) pro- reserve to tolerate to some extent early branching defects. These vides an in vivo system to investigate the four corresponding data suggest that when only one of the four Lef/Tcf paralogs re- transcription factor paralogs: LEF1, TCF7, TCF7L1, and TCF7L2 mains in the lung progenitors, individual paralogs are no longer (6). We set out to identify which LEF/TCF was present in the redundant nor equivalent: TCF7 or TCF7L1 supports sufficient epithelium using immunostaining, and found that while all four Wnt signaling, whereas LEF1 or TCF7L2 is partially functional at factors were expressed in the surrounding mesenchyme, only different levels. TCF7L1 and TCF7L2 were readily detectible in the epithelium Finally, the ShhCre quadruple mutant lung consisted of two (Fig. 1A). Accordingly, we deleted both Tcf7l1 and Tcf7l2 using Cre small sacs that fused with the esophagus and expressed SOX2 Shh (13) throughout the epithelium and before lung specifica- except for occasional clusters of SOX9
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