Inhibitory G proteins play multiple roles to polarize sensory hair cell morphogenesis

Reviewer #1 (Public Review):

A subclass of inhibitory heterotrimeric guanine nucleotide-binding protein subunits, GNAI, has been implicated in sensory hair cell formation, namely the establishment of hair bundle (stereocilia) orientation and staircase formation. However, the former role of hair bundle orientation has only been demonstrated in mutants expressing pertussis toxin, which blocks all GNAI subunits, but not in mutants with a single knockout of any of the Gnai genes, suggesting that there is a redundancy among various GNAI proteins in this role. Using various conditional mutants, the authors concluded that GNAI3 is the primary GNAI proteins required for hair bundle morphogenesis, whereas hair bundle orientation requires both GNAI2 and GNAI3.

Strength
Various compound mutants were generated to decipher the contribution of individual GNAI1, GNAI2, GNAI3 and GNAIO in the establishment of hair bundle orientation and morphogenesis. The study is thorough with detailed quantification of hair bundle orientation and morphogenesis, as well as auditory functions.

Weakness
While the hair bundle orientation phenotype in the Foxg1-cre; Gnai2-/-; Gnai3 lox/lox (double mutants) appear more severe than those observed in Ptx cKO mutants, it may be an oversimplification to attribute the differences to more GNAI function in the Ptx cko mutants. The phenotypes between the double mutants and Ptx cko mutants appear qualitatively different. For example, assuming the milder phenotypes in the Ptx cKO is due to incomplete loss of GNAI function, one would expect the Ptx phenotype would be reproducible by some combination of compound mutants among various Gnai genes. Such information was not provided. Furthermore, of all the double mutant specimens analyzed for hair bundle orientation (Fig. 8), the hair bundle/kinocilium position started out normally in the lateral quadrant at E17.5 but failed to be maintained by P0. This does not appear to be the case for Ptx cKO, in which all affected hair cells showed inverted orientation by E17.5. It is not clear whether this is the end-stage of bundle orientation in Ptx cKO, and the kinocilium position started out normal, similar to the double mutants before the age of analysis at E17.5. Understanding these differences may reveal specific requirements of individual GNAI subunits or other factors are being affected in the Ptx mutants.

Reviewer #2 (Public Review):

Jarysta and colleagues set out to define how similar GNAI/O family members contribute to the shape and orientation of stereocilia bundles on auditory hair cells. Previous work demonstrated that loss of particular GNAI proteins, or inhibition of GNAIs by pertussis toxin, caused several defects in hair bundle morphogenesis, but open questions remained which the authors sought to address. Some of these questions include whether all phenotypes resulting from expression of pertussis toxin stemmed from GNAI inhibition; which GNAI family members are most critical for directing bundle development; whether GNAI proteins are needed for basal body movements that contribute to bundle patterning. These questions are important for understanding how tissue is patterned in response to planar cell polarity cues.

To address questions related to the GNAI family in auditory hair cell development, the authors assembled an impressive and nearly comprehensive collection of mouse models. This approach allowed for each Gnai and Gnao gene to be knocked out individually or in combination with each other. Notably, a new floxed allele was generated for Gnai3 because loss of this gene in combination with Gnai2 deletion was known to be embryonic lethal. Besides these lines, a new knockin mouse was made to conditionally express untagged pertussis toxin following cre induction from a strong promoter. The breadth and complexity involved in generating and collecting these strains makes this study unique, and likely the authoritative last word on which GNAI proteins are needed for which aspect of auditory hair bundle development.

Appropriate methods were employed by the authors to characterize auditory hair bundle morphology in each mouse line. Conclusions were carefully drawn from the data and largely based on excellent quantitative analysis. The main conclusions are that GNAI3 has the largest effect on hair bundle development. GNAI2 can compensate for GNAI3 loss in early development but incompletely in late development. The Gnai2 Gnai3 double mutant recapitulates nearly all the phenotypic effects associated with pertussis toxin expression and also reveals a role for GNAIs in early movement of the basal body. Although these results are not entirely unexpected based on earlier reports, the current results both uncover new functions and put putative functions on more solid ground.

Based on this study, loss of GNAI1 and GNAO show a slight shortening of the tallest row of stereocilia but no other significant changes to bundle shape. Antibody staining shows no change in GNAI localization in the Gnai1 knockout, suggesting that little to no protein is found in hair cells. One caveat to this interpretation is that the antibody, while proposed to cross-react with GNAI1, is not clearly shown to immunolabel GNAI1. More than anything, this reservation mostly serves to illustrate how challenging it is to nail down every last detail. In turn, the comprehensive nature of the current study seems all the more impressive.