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

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

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

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

Production of light-coloured, low heat-absorbing Holstein Friesian cattle by precise embryo-mediated genome editing

Context. Genome editing enables the introduction of beneficial sequence variants into the genomes of animals with high genetic merit in a single generation. This can be achieved by introducing variants into primary cells followed by producing a live animal from these cells by somatic cell nuclear transfer cloning. The latter step is associated with low efficiencies and developmental problems due to incorrect reprogramming of the donor cells, causing animal welfare concerns. Direct editing of fertilised one-cell embryos could circumvent this issue and might better integrate with genetic improvement strategies implemented by the industry. Methods. In vitro fertilised zygotes were injected with TALEN editors and repair template to introduce a known coat colour dilution mutation in the PMEL gene. Embryo biopsies of injected embryos were screened by polymerase chain reaction and sequencing for intended biallelic edits before transferring verified embryos into recipients for development to term. Calves were genotyped and their coats scanned with visible and hyperspectral cameras to assess thermal energy absorption. Key results. Multiple non-mosaic calves with precision edited genotypes were produced, including calves from high genetic merit parents. Compared to controls, the edited calves showed a strong coat colour dilution which was associated with lower thermal energy absorbance. Conclusions. Although biopsy screening was not absolutely accurate, non-mosaic, precisely edited calves can be readily produced by embryo-mediated editing. The lighter coat colouring caused by the PMEL mutation can lower radiative heat gain which might help to reduce heat stress. Implications. The study validates putative causative sequence variants to rapidly adapt grazing cattle to changing environmental conditions

Image5_Double cytoplast embryonic cloning improves in vitro but not in vivo development from mitotic pluripotent cells in cattle.tif

Cloning multiple animals from genomically selected donor embryos is inefficient but would accelerate genetic gain in dairy cattle breeding. To improve embryo cloning efficiency, we explored the idea that epigenetic reprogramming improves when donor cells are in mitosis. We derived primary cultures from bovine inner cell mass (ICM) cells of in vitro fertilized (IVF) embryos. Cells were grown feeder-free in a chemically defined medium with increased double kinase inhibition (2i+). Adding recombinant bovine interleukin 6 to 2i+ medium improved plating efficiency, outgrowth expansion, and expression of pluripotency-associated epiblast marker genes (NANOG, FGF4, SOX2, and DPPA3). For genotype multiplication by embryonic cell transfer (ECT) cloning, primary colonies were treated with nocodazole, and single mitotic donors were harvested by mechanical shake-off. Immunofluorescence against phosphorylated histone 3 (P-H3) showed 37% of nocodazole-treated cells in metaphase compared to 6% in DMSO controls (P −5), with an average of 53% of P-H3-positive cells expressing the pluripotency marker SOX2. We optimized several parameters (fusion buffer, pronase treatment, and activation timing) for ECT with mitotic embryonic donors. Sequential double cytoplast ECT, whereby another cytoplast was fused to the first cloned reconstruct, doubled cloned blastocyst development and improved morphological embryo quality. However, in situ karyotyping revealed that over 90% of mitotic ECT-derived blastocysts were tetraploid or aneuploid with extra chromosomes, compared to less than 2% in the original ICM donor cells. Following the transfer of single vs. double cytoplast embryos, there was no difference between the two methods in pregnancy establishment at D35 (1/22 = 5% vs. 4/53 = 8% for single vs. double ECT, respectively). Overall, post-implantation development was drastically reduced from embryonic mitotic clones when compared to somatic interphase clones and IVF controls. We conclude that mitotic donors cause ploidy errors during in vitro development that cannot be rescued by enhanced epigenetic reprogramming through double cytoplast cloning.</p

Image2_Double cytoplast embryonic cloning improves in vitro but not in vivo development from mitotic pluripotent cells in cattle.tif

Cloning multiple animals from genomically selected donor embryos is inefficient but would accelerate genetic gain in dairy cattle breeding. To improve embryo cloning efficiency, we explored the idea that epigenetic reprogramming improves when donor cells are in mitosis. We derived primary cultures from bovine inner cell mass (ICM) cells of in vitro fertilized (IVF) embryos. Cells were grown feeder-free in a chemically defined medium with increased double kinase inhibition (2i+). Adding recombinant bovine interleukin 6 to 2i+ medium improved plating efficiency, outgrowth expansion, and expression of pluripotency-associated epiblast marker genes (NANOG, FGF4, SOX2, and DPPA3). For genotype multiplication by embryonic cell transfer (ECT) cloning, primary colonies were treated with nocodazole, and single mitotic donors were harvested by mechanical shake-off. Immunofluorescence against phosphorylated histone 3 (P-H3) showed 37% of nocodazole-treated cells in metaphase compared to 6% in DMSO controls (P −5), with an average of 53% of P-H3-positive cells expressing the pluripotency marker SOX2. We optimized several parameters (fusion buffer, pronase treatment, and activation timing) for ECT with mitotic embryonic donors. Sequential double cytoplast ECT, whereby another cytoplast was fused to the first cloned reconstruct, doubled cloned blastocyst development and improved morphological embryo quality. However, in situ karyotyping revealed that over 90% of mitotic ECT-derived blastocysts were tetraploid or aneuploid with extra chromosomes, compared to less than 2% in the original ICM donor cells. Following the transfer of single vs. double cytoplast embryos, there was no difference between the two methods in pregnancy establishment at D35 (1/22 = 5% vs. 4/53 = 8% for single vs. double ECT, respectively). Overall, post-implantation development was drastically reduced from embryonic mitotic clones when compared to somatic interphase clones and IVF controls. We conclude that mitotic donors cause ploidy errors during in vitro development that cannot be rescued by enhanced epigenetic reprogramming through double cytoplast cloning.</p

Image1_Double cytoplast embryonic cloning improves in vitro but not in vivo development from mitotic pluripotent cells in cattle.tif

Cloning multiple animals from genomically selected donor embryos is inefficient but would accelerate genetic gain in dairy cattle breeding. To improve embryo cloning efficiency, we explored the idea that epigenetic reprogramming improves when donor cells are in mitosis. We derived primary cultures from bovine inner cell mass (ICM) cells of in vitro fertilized (IVF) embryos. Cells were grown feeder-free in a chemically defined medium with increased double kinase inhibition (2i+). Adding recombinant bovine interleukin 6 to 2i+ medium improved plating efficiency, outgrowth expansion, and expression of pluripotency-associated epiblast marker genes (NANOG, FGF4, SOX2, and DPPA3). For genotype multiplication by embryonic cell transfer (ECT) cloning, primary colonies were treated with nocodazole, and single mitotic donors were harvested by mechanical shake-off. Immunofluorescence against phosphorylated histone 3 (P-H3) showed 37% of nocodazole-treated cells in metaphase compared to 6% in DMSO controls (P −5), with an average of 53% of P-H3-positive cells expressing the pluripotency marker SOX2. We optimized several parameters (fusion buffer, pronase treatment, and activation timing) for ECT with mitotic embryonic donors. Sequential double cytoplast ECT, whereby another cytoplast was fused to the first cloned reconstruct, doubled cloned blastocyst development and improved morphological embryo quality. However, in situ karyotyping revealed that over 90% of mitotic ECT-derived blastocysts were tetraploid or aneuploid with extra chromosomes, compared to less than 2% in the original ICM donor cells. Following the transfer of single vs. double cytoplast embryos, there was no difference between the two methods in pregnancy establishment at D35 (1/22 = 5% vs. 4/53 = 8% for single vs. double ECT, respectively). Overall, post-implantation development was drastically reduced from embryonic mitotic clones when compared to somatic interphase clones and IVF controls. We conclude that mitotic donors cause ploidy errors during in vitro development that cannot be rescued by enhanced epigenetic reprogramming through double cytoplast cloning.</p

Image3_Double cytoplast embryonic cloning improves in vitro but not in vivo development from mitotic pluripotent cells in cattle.tif

Cloning multiple animals from genomically selected donor embryos is inefficient but would accelerate genetic gain in dairy cattle breeding. To improve embryo cloning efficiency, we explored the idea that epigenetic reprogramming improves when donor cells are in mitosis. We derived primary cultures from bovine inner cell mass (ICM) cells of in vitro fertilized (IVF) embryos. Cells were grown feeder-free in a chemically defined medium with increased double kinase inhibition (2i+). Adding recombinant bovine interleukin 6 to 2i+ medium improved plating efficiency, outgrowth expansion, and expression of pluripotency-associated epiblast marker genes (NANOG, FGF4, SOX2, and DPPA3). For genotype multiplication by embryonic cell transfer (ECT) cloning, primary colonies were treated with nocodazole, and single mitotic donors were harvested by mechanical shake-off. Immunofluorescence against phosphorylated histone 3 (P-H3) showed 37% of nocodazole-treated cells in metaphase compared to 6% in DMSO controls (P −5), with an average of 53% of P-H3-positive cells expressing the pluripotency marker SOX2. We optimized several parameters (fusion buffer, pronase treatment, and activation timing) for ECT with mitotic embryonic donors. Sequential double cytoplast ECT, whereby another cytoplast was fused to the first cloned reconstruct, doubled cloned blastocyst development and improved morphological embryo quality. However, in situ karyotyping revealed that over 90% of mitotic ECT-derived blastocysts were tetraploid or aneuploid with extra chromosomes, compared to less than 2% in the original ICM donor cells. Following the transfer of single vs. double cytoplast embryos, there was no difference between the two methods in pregnancy establishment at D35 (1/22 = 5% vs. 4/53 = 8% for single vs. double ECT, respectively). Overall, post-implantation development was drastically reduced from embryonic mitotic clones when compared to somatic interphase clones and IVF controls. We conclude that mitotic donors cause ploidy errors during in vitro development that cannot be rescued by enhanced epigenetic reprogramming through double cytoplast cloning.</p