LGR4 and LGR5 Function Redundantly During Human Endoderm Differentiation

Cell Mol Gastroenterol Hepatol. 2016 Jun 23;2(5):648-662.e8. doi: 10.1016/j.jcmgh.2016.06.002. eCollection 2016 Sep.


Background & aims: 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 of LGR family 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 that LGR5 is part of the human definitive endoderm (DE) gene signature, and LGR5 transcripts are induced robustly when human pluripotent stem cells are differentiated into DE. Our results show that LGR4 and 5 are functionally required for efficient human endoderm induction. Consistent with data in human DE, we observe Lgr5 reporter (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 that LGR5 is induced during DE differentiation, LGR receptors are functionally required for DE induction, and that they function to potentiate WNT signaling during this process.

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.