Developmental gene expression profile of Vmo1 in the mouse auditory system

Hearing loss (HL) is a sensory disorder that affects an estimated 250 million people worldwide and can greatly affect quality of life. In New Zealand, more than 10% of the population is affected by HL with the Māori population being overrepresented among all age groups. Therefore, understanding the mechanism of HL is extremely important for the development of new pharmaceuticals for the prevention or treatment of HL disorders. The main aim of the research undertaken in this thesis was to characterise the function of the Mus musculus (mouse) vitelline membrane outer layer one (Vmo1) gene. This gene is considered an excellent candidate for being involved in human HL and/or balance disorders. Our hypothesis is based on its restricted gene localisation within the mouse inner ear and the postulated function of Reissner’s membrane. Two methods were used to address this aim. Firstly, comparative genomics was used to determine the level of nucleotide and amino acid conservation of VMO1 across mammalian species, and to search for DNA motifs that may imply a biological function. Secondly, molecular biology and histochemical techniques were used to DNA sequence the Vmo1 gene, detect the expression of 22 kDa VMO1 protein within mouse tissues, and to localise the expression of VMO1 protein within the mouse inner ear. Comparative genomics results showed VMO1 to be highly conserved across 36 species. An in-depth analysis of the differences and similiarites between the mouse, human and chicken indicated a high level of gene conservation with an even greater degree of identity and similarity seen at a proteomic level. In addition, a high level of conservation across amino acids involved in the formation and stabilisation of the three dimensional structure. Thus, results suggest an important function for the VMO1 protein. Two commerical VMO1 antibodies were purchased to determine the localisation of the mouse VMO1 protein. They were validated for specificity using western blot analysis of protein lysates dissected from postnatal day 28 mice (P28). VMO1 was identified within the inner ear protein lysate and tear gland protein lysate of an expected molecular weight size of 20-37kDa with additional binding observed in the ear sample at 250kDa. Immunohistochemistry detected high concentrations of VMO1 protein within the tectorial membrane (TM) and inner pillar cells (IPC) in inner ear sections from the mouse at P5. In agreement with the comparative genomics analysis, VMO1 is a secreted protein. The movement of the hair cells (HC) relative to the TM is is essential for the transduction of sound into electrical signals. The IPC act as supporting cells for the hair cells, and help to couple movement of the basilar membrane to the HC. In conclusion, the importance of the TM and IPC in hearing function, and the localisation of the VMO1 protein within these structures implies an important role for VMO1 in hearing function. We recommend further studies to examine the specificity of the VMO1 antibody, and the development of a Vmo1 knockout mouse to support the functional analysis of Vmo1 in the auditory system

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