Inducible and reversible inhibition of miRNA-mediated gene repression in vivo

Essential revisions:

1. An overall histopathologic analysis of P0 T6B mice exposed to dox between E13.5-E18.5 could be interesting to see if the development of any tissues are particularly sensitive to inhibition of miRNA-mediated silencing during this window. This would be straightforward if fixed P0 mice are available, although this analysis could be the subject of a future study if tissues are not readily available.

We agree with the Reviewer that a detailed characterization of the consequences of acute miRNA inhibition on mouse development is of great interest. We have asked the MSKCC mouse pathology service to carry out a pathologic analysis of the embryos described in Figure 3B and Figure 3—figure supplement 1. The results of this analysis confirmed the observed general delay in development of the T6B mice compared to wild type controls, but did not highlight any specific developmental defect that can be detected by histologic analysis of H and E stained sections.

We have initiated a series of more detailed studies, comparing different timing of T6B induction during embryonic development, and examining the effect of miRNA inhibition on in vitro differentiation trajectories by using single cell RNAseq analysis. We are thrilled about these studies, but they will require considerable time and effort and therefore, if the Editor and the Reviewers agree, we would prefer to present them in a follow-up manuscript.

2. Figure 2 – supplement 2 shows T6B activation in lung in CAG-T6B but not R26-T6B animals. Does activation of the transgene in CAG-T6B mice result in any noticeable abnormalities in adult lung?

We thank the Editor and Reviewers for the opportunity to clarify this aspect of the phenotypic analysis of the CAG-T6B strain that had been superficially described in our previous version of the manuscript. To address this point, we asked an experienced mouse pathologist from the MSKCC animal core to review the lung histology of adult CAG-T6B mice. As described in the revised manuscript, this analysis revealed marked vasculitis of the pulmonary veins (Figure 6—figure supplement 2). The most likely explanation for this phenotype is that these lesions are secondary to the increased pulmonary vein pressure caused by congestive heart failure, but we cannot exclude that a primary effect caused by T6B expression in the lung contributed to this phenotype. Follow-up studies using transgenic animals harboring tissue-restricted expression of rtTA will be needed to conclusively address this important point.