title: LGR4andLGR5Function Redundantly During Human Endoderm Differentiation.
abstract: OBJECTIVE
  The Lgr family of transmembrane proteins (Lgr4, 5, 6) act as functional receptors for R-spondin proteins (Rspo 1, 2, 3, 4), and potentiate Wnt signaling in different contexts. Lgr5 is arguably the best characterized of the Lgr family members in a number of adult and embryonic contexts in mice. However, the function ofLGRfamily members in early embryonic development is unclear, and has not been explored during human development or tissue differentiation in detail.
  
  
  METHODS
  We interrogated the function and expression of LGR family members using human pluripotent stem cell-derived tissues including definitive endoderm, mid/hindgut, and intestinal organoids. We performed embryonic lineage tracing in Lgr5-GFP-IRES-CreERT2 mice.
  
  
  RESULTS
  We show thatLGR5is part of the human definitive endoderm (DE) gene signature, andLGR5transcripts are induced robustly when human pluripotent stem cells are differentiated into DE. Our results show thatLGR4and5are functionally required for efficient human endoderm induction. Consistent with data in human DE, we observeLgr5reporter (eGFP) activity in the embryonic day 8.5 mouse endoderm, and show the ability to lineage trace these cells into the adult intestine. However, gene expression data also suggest that there are human-mouse species-specific differences at later time points of embryonic development.
  
  
  CONCLUSIONS
  Our results show thatLGR5is induced during DE differentiation, LGR receptors are functionally required for DE induction, and that they function to potentiate WNT signaling during this process.
authors.affiliation: Division of Gastroenterology, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan.
authors.forename: Yu-Hwai
authors.initials: YH
authors.lastname: Tsai
authors.affiliation: Division of Gastroenterology, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan.
authors.forename: David R
authors.initials: DR
authors.lastname: Hill
authors.affiliation: Department of Genetics, Rutgers, The State University of New Jersey, Piscataway, New Jersey.
authors.forename: Namit
authors.initials: N
authors.lastname: Kumar
authors.affiliation: Division of Gastroenterology, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan.
authors.forename: Sha
authors.initials: S
authors.lastname: Huang
authors.affiliation: Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, Michigan.
authors.forename: Alana M
authors.initials: AM
authors.lastname: Chin
authors.affiliation: Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, Michigan.
authors.forename: Briana R
authors.initials: BR
authors.lastname: Dye
authors.affiliation: Division of Gastroenterology, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan.
authors.forename: Melinda S
authors.initials: MS
authors.lastname: Nagy
authors.affiliation: Department of Genetics, Rutgers, The State University of New Jersey, Piscataway, New Jersey.
authors.forename: Michael P
authors.initials: MP
authors.lastname: Verzi
authors.affiliation: Division of Gastroenterology, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan; Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, Michigan; Center for Organogenesis, University of Michigan Medical School, Ann Arbor, Michigan.
authors.forename: Jason R
authors.initials: JR
authors.lastname: Spence
keywords: CDX2, caudal type homeobox2; ChIPseq, chromatin immunoprecipitation sequencing; Ct, cycle threshold; DE, definitive endoderm; E, embryonic day; Endoderm; GFP, green fluorescent protein; Intestine; LGR5; Organoid; Pluripotent Stem Cells; Rspo, R-spondin protein; WNT; creER, cre recombinase protein fused to estrogen receptor; hESC, human embryonic stem cell; mRNA, messenger RNA; qRT-PCR, quantitative reverse-transcription polymerase chain reaction; shRNA, short hairpin RNA