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

Cloned cattle derived from a novel zona-free embryo reconstruction system

As the demand for cloned embryos and offspring increases, the need arises for the development of nuclear transfer procedures that are improved in both efficiency and ease of operation. Here, we describe a novel zona-free cloning method that doubles the throughput in cloned bovine embryo production over current procedures and generates viable offspring with the same efficiency. Elements of the procedure include zona-free enucleation without a holding pipette, automated fusion of 5-10 oocyte-donor cell pairs and microdrop in vitro culture. Using this system, zona-free embryos were reconstructed from five independent primary cell lines and cultured either singularly (single-IVC) or as aggregates of three (triple-IVC). Blastocysts of transferable quality were obtained at similar rates from zona-free single-IVC, triple-IVC, and control zona-intact embryos (33%, 25%, and 29%, respectively). In a direct comparison, there was no significant difference in development to live calves at term between single-IVC, triple-IVC, and zona-intact embryos derived from the same adult fibroblast line (10%, 13%, and 15%, respectively). This zona-free cloning method could be straightforward for users of conventional cloning procedures to adopt and may prove a simple, fast, and efficient alternative for nuclear cloning of other species as well

Improved in vitro development of cloned bovine embryos using S-adenosylhomocysteine, a non-toxic epigenetic modifying reagent.

In this study, fibroblast cells were stably transfected with mouse POU5F1 promoter-driven enhanced green fluorescent protein (EGFP) to investigate the effect of S-adenosylhomocysteine (SAH), the reversible non-toxic inhibitor of DNA-methyltransferases (DNMTs), at different intervals post-fusion on in vitro development of cloned bovine embryos. Treatment with SAH for 12 hr resulted in 54.6 ± 7.7% blastocyst production, which was significantly greater than in vitro fertilized embryos (IVF: 37.2 ± 2.7%), cloned embryos treated with SAH for 72 hr (31.0 ± 7.6%), and control cloned embryos (34.6 ± 3.6%). The fluorescence intensities of the EGFP-POU5F1 reporter gene at all intervals of SAH treatment, except of 72 hr, were significantly higher than control somatic cell nuclear transfers (SCNT) embryos. The intensity of DNA-methylation in cloned embryos treated with SAH for 48 hr was similar to that of IVF embryos, and was significantly lower than the other SCNT groups. The levels of H3K9 acetylation in all SCNT groups were significantly lower than IVF embryos. Real-time PCR analysis of gene expression revealed significantly higher expression of POU5F1 in cloned versus IVF blastocysts. Neither embryo production method (SCNT vs. IVF) nor the SAH treatment interval affected expression of the BCL2 gene. Cloned embryos at all intervals of SAH treatment, except for 24 hr, had significantly increased VEGF transcript compared to IVF and control SCNT embryos. It was suggested that the time interval of DNMT inhibition may have important consequences on different in vitro features of bovine SCNT, and the improving effects of DNMT inhibition on developmental competency of cloned embryos are restricted to a specific period of time preceding de novo methylation. Mol. Reprod. Dev. 78:576–584, 2011

Primary Transgenic Bovine Cells and Their Rejuvenated Cloned Equivalents Show Transgene-Specific Epigenetic Differences

Cell-mediated transgenesis, based on somatic cell nuclear transfer (SCNT), provides the opportunity to shape the genetic make-up of cattle. Bovine primary fetal fibroblasts, commonly used cells for SCNT, have a limited lifespan, and complex genetic modifications that require sequential transfections can be challenging time and cost-wise. To overcome these limitations, SCNT is frequently used to rejuvenate the cell lines and restore exhausted growth potential. We have designed a construct to be used in a 2-step cassette exchange experiment. Our transgene contains a puromycin resistance marker gene and an enhanced green fluorescence protein (EGFP) expression cassette, both driven by a strong mammalian promoter, and flanked by loxP sites and sequences from the bovine β-casein locus. Several transgenic cell lines were generated by random insertion into primary bovine cell lines. Two of these original cell lines were rederived by SCNT and new primary cells, with the same genetic makeup as the original donors, were established. While the original cell lines were puromycin-resistant and had a characteristic EGFP expression profile, all rejuvenated cell lines were sensitive to puromycin, and displayed varied EGFP expression, indicative of various degrees of silencing. When the methylation states of individual CpG sites within the transgene were analyzed, a striking increase in transgene-specific methylation was observed in all rederived cell lines. The results indicate that original transgenic donor cells and their rejuvenated derivatives may not be equivalent and differ in the functionality of their transgene sequences

Cytokine Gene Expression in the Maternal-Fetal Interface in Somatic Cell Nuclear Transfer Pregnancies in Small Ruminants

The present retrospective study investigates pregnancy rates, incidence of pregnancy losses and large offspring syndrome (LOS), and immune-related gene expression of sheep and goat somatic cell nuclear transfer (SCNT) pregnancies. We hypothesized that significantly higher pregnancy losses observed in sheep SCNT pregnancies compared to goats are due to the increased amounts of T-helper 1 cytokines and pro-inflammatory mediators at the maternal-fetal interface. Sheep and goat SCNT pregnancies were generated using the same procedure. Control pregnancies were established by natural breeding. Although SCNT pregnancy rates at 45 days were similar in both species, pregnancy losses between 45 and 60 days and incidence of LOS were significantly increased in sheep compared with goats. At term, the expression of pro-inflammatory genes in sheep SCNT placentas was increased while the one of goat SCNT was similar to the control animals. Among the genes that had altered expression in sheep SCNT placentas are CTLA4, IL2RA, CD28, IFNG, IL6, IL10, TGFB1, TNF, IL1A and CXCL8. MHC-I protein expression was greater in sheep and goat SCNT placentas at term compared with control pregnancies. An unfavorable immune environment is present at the maternal-fetal interface in sheep SCNT pregnancies