Brain atlas for glycoprotein hormone receptors at single-transcript level

Essential revisions:

1. As the authors did not stain tanycytes using tanycyte-specific antibodies, I would suggest co-labelling tanycyte and TSHR in Figure 1, or removing the word "tanycyte" in the text but with an alternative statement "ependymal layer of the third ventricle".

As we do not currently have Tshr^+/- mice and it would take a while to grow these colonies, we have chosen to replace the word “tanycyte” with an “ependymal layer of the third ventricle or cells” in the text.

2. In this study, the authors used young adult animals. Are there any differences in those gene and receptor expressions in young or old mouse brains? It would gain more information to discuss.

We fully concur with the review comment. As for the receptor, Kerp et al., showed no difference in pituitary Tshr expression in young vs. old mice. For the ligand, while Wang et al., did not detect alterations in TSH levels in 6, 15 and 22–month–old mice, Miler et al., reported an increase in pituitary TSH by 44% in old Wistar rats compared with young rats. These references have been quoted and discussed in lines 226 to 229 in the revised manuscript.

3. Figure 1C- based on the figure legend, n=4-5 mice were used for each group. The bar diagram should be changed into a scatter dot plot format.

Individual data points have been added to the bar graph in Figure 1C.

4. Figure 4A- the scale bars of the 2 images in the upper panel are not clear.

The scale bar has been updated to provide more clarity.

5. Supplemental Figure 2- the resolution of some higher power images needs to be improved.

We have replaced the high–power images as suggested. Notably, in the ventral tenia tecta (VTT) of the olfactory bulb the pattern of TSHR expression differed from other brain sites. The expression appeared densely packed within the neurons of the VTT as compared to the dispersed dotty expression in other regions of the brain.