Grainyhead-like 2 is required for morphological integrity of mouse embryonic stem cells and orderly formation of inner ear-like organoids

Abstract

Mutations in the transcription factor gene grainyhead-like 2 (GRHL2) are associated with progressive non-syndromic sensorineural deafness autosomal dominant type 28 (DFNA28) in humans. Since complete loss of Grhl2 is lethal in mouse embryos, we studied its role during inner ear pathology and hearing loss in vitro. To this end, we generated different homozygous deletions to knockout Grhl2 in mouse embryonic stem cells (Grhl2-KO ESCs), including some mimicking naturally occurring truncations in the dimerisation domain related to human DFNA28. Under naïve culture conditions, Grhl2-KO cells in suspension were more heterogenous in size and larger than wild-type controls. Adherent Grhl2-KO cells were also larger, with a less uniform shape, flattened, less circular morphology, forming loose monolayer colonies with poorly defined edges. These changes correlated with lower expression of epithelial cadherin Cdh1 but no changes in tight junction markers (Ocln, Tjp2) or other Grhl isoforms (Grhl1, Grhl3). Clonogenicity from single cells, proliferation rates of cell populations and proliferation markers were reduced in Grhl2-KO ESCs. We next induced stepwise directed differentiation of Grhl2-KO ESCs along an otic pathway, giving rise to three-dimensional inner ear-like organoids (IELOs). Quantitative morphometry revealed that Grhl2-KO cells initially formed larger IELOs with a less compacted structure, more eccentric shape and increased surface area. These morphological changes persisted for up to one week. They were partially rescued by forced cell aggregation and fully restored by stably overexpressing exogenous Grhl2 in Grhl2-KO ESCs, indicating that Grhl2 alters cell-cell interactions. On day 8, aggregates were transferred into minimal maturation medium to allow self-guided organogenesis for another two weeks. During this period, Grhl2-KO cells and wild-type controls developed similarly, expressing neural, neuronal and sensory hair cell markers, while maintaining their initial differences in size and shape. In summary, Grhl2 is required for morphological maintenance of ESCs and orderly formation of IELOs, consistent with an essential role in organising epithelial integrity during inner ear development. Our findings validate quantitative morphometry as a useful, non-invasive screening method for molecular phenotyping of candidate mutations during organoid development.

Keywords: autosomal-dominant hearing loss; cadherin; cell adhesion; embryonic stem cells; grainyhead; inner ear; organoids; quantitative morphometry.

FIGURE 1

gRNA/Cas9 genome editing of murine Grhl2. (A) Murine Grhl2 wild-type (WT) gene structure with untranslated regions (black), numbered exons of coding sequence (CDS, grey), including functional domains for transactivation (blue), DNA-binding (yellow) and dimerisation (green). Mapped to the sequence are the gRNA targets (red arrows) and the c.1609dupC mutation in human DFNA28 (black arrow). (B) Predicted amino acid (aa) sequences for murine WT and human DFNA28 heterozygous genotype. (C) Predicted amino acid sequences for gene edited ESC strains with loss of three or two functional domains after homology-directed insertion of red fluorescent protein (RFP) and puromycin (Puro) transgene (3KO or 2KO-1, -2, respectively). Compound heterozygous DFNA28-like frameshift mutations after non-homologous end joining only affect the dimerisation domain (1KO-1, -2, -3).

FIGURE 2

Loss of Grhl2 alters ESC morphology. (A) Qualitative morphology of representative Grhl2-KO grown in group cultures, compared to wild-type (WT) ESCs. Alkaline phosphatase (AP) activity was stained with a colorimetric assay and visualised with brightfield (AP-BF), phase contrast (Phase) or epifluorescence in the FITC channel (AP-Fluo). DNA was visualised with Hoechst 33,342. Scale bar = 400 µm. (B) Quantitative morphometry of single ESCs and ESC colonies. Colony formation assay from single cells (clonogenicity) was determined in several strains for each genotype (N = 3, N = 3 and N = 2 for WT, 2KO/3KO and 1KO, respectively). Single cells were sized in suspension with several strains analysed for each genotype (N = 6, N = 4 and N = 3 for WT, 2KO/3KO and 1KO, respectively). To determine cell size variability, each strain was analysed in 3 replicates (N = 1,122, N = 708 and N = 568 cells, respectively). Colony morphology and composition (cell size within colonies, density and circularity) was calculated using colonies from the colony formation assay (N = 49, N = 14, N = 13 colonies for WT, 2KO/3KO and 1KO, respectively). All measurements were taken 12 days after plating single ESCs. (C–E) Gene expression changes for Grhl transcription factors (C), cell-cell junctional markers (D) and pluripotency markers (E). Expression was normalised on the geometric mean of three reference genes. *, **** = p < 0.05, p < 0.0001, respectively. (F) Representative images of EB-like aggregates formed during hanging drop differentiation for several days. Scale bar = 400 µm.

FIGURE 3

Loss of Grhl2 disrupts early IELO integrity. (A) SFEBq-spin partially restores IELO morphology; scale bar = 400 μm (B) Quantitative morphometry of Grhl2-KO IELOs. Morphology (area, perimeter, circularity and compactness) was determined after 1 day in culture. ns = non-significant. *, **, *** = p < 0.05, p < 0.01 and p < 0.001, respectively. (C) Gene expression changes for cell-cell junctional markers, normalised on the geometric mean of four reference genes. (D) Qualitative and (E) quantitative restoration of area by overexpressing Grhl1, or -2 transgenes (Grhl-TG). ab = letters differ by p < 0.05; scale bar = 400 μm. (F) Complementation of 3KO-ESCs carrying a red fluorescent protein (RFP) transgene with WT-ESC (1:1). Non-aggregated 3KO and WT ESCs provide negative and positive controls, respectively. Scale bars = 200 µm.

FIGURE 4

Grhl2 is not required for late IELO differentiation. (A) Brightfield images of IELOs showing presumptive otic vesicles on day 17–21 (red triangles). (B) Gene expression changes for neural (Cdh2, Nes, Otx2, Sox2), neuronal (Tubb3) and hair cell specific (Myo7a) markers in day 26 IELOs. Gene expression relative to Gapdh. abcde = samples with different letters differ by p < 0.05. (C) IELOs at day 21 contained neural rosettes (red box) with cells expressing CDH2 (green). Negative control was only incubated with the secondary antibody (2° Ab). DNA was counterstained with Hoechst 33,342 (blue). Scale bars = 400 µm.