Dustin Revell and Zhang Li“Notch signaling regulates Akap12 expression and primary cilia length during renal tubule morphogenesis” Preprint posted to BioArchiv on September 9, 2019; doi: https://doi.org/10.1101/760181This preprint was reviewed as part of the Developmental Biology Journal Club at the University of Alabama Birmingham and the review is a summary of the group discussion.Mukherjee et al. used a combination of transgenic inducible mouse models as well as cell culture and spheroid models to demonstrate how Notch signaling regulates Akap12 expression to influence primary cilia length during renal tubule morphogenesis. The authors show that inhibition of Notch signaling through expression of dnMaml or the conditional deletion of RBPJ leads to kidney cysts and elongated cilia, mimicking the human disease Alagile syndrome. While these data are very interesting and lean towards the classification of Alagile syndrome as being one of the class of diseases termed ciliopathies , we found several concerns throughout the paper which are outlined below.Major Concerns:1) There is some confusion in the use of the different transgenic mouse models used in the paper. For Figures 1 and 2, the authors utilize the Pax8-rtTA; TRE-dnMamL model which will express dnMamL in the kidney of mice when treated with doxycycline. However, in Figure 5, the authors switch to a Rarb2-Cre; RBPJ(flox/flox) mouse without any introduction or explanation as to why. As Rarb2 is expressed in multiple tissues, not just the kidney, this may be influencing their results (https://www.ncbi.nlm.nih.gov/gene/5915).2) We feel that there should be some further validation to show that Notch signaling is truly reduced and in which cell types upon use of the Pax8-rtTA; TRE-dnMamL - This could be done via qRT-PCR looking at the common downstream genes HES and HEY. It is previously published that complete inhibition of Notch2 results in a lack of proximal tubules in murine kidney resulting in death at P0 (Kamath, Spinner, & Rosenblum, 2013; McCright et al., 2001). Figure 1D shows a large reduction in LTA positive proximal tubules suggesting that dnMamL might be causing an incomplete inhibition of Notch resulting in the renal phenotype.3) We are concerned as to the variability in the length of the primary cilia between cell culture experiments. In Figure 4C, the WT MDCK primary cilia were an average length of approximately 1.5microns, while in Figure7H, the WT cilia were less than 500nm in length. Primary cilia are generally between 3 to 5 microns in length (Keeling, Tsiokas, & Maskey, 2016), so this discrepancy leads to skepticism over the health of these MDCK cells and the conclusions made from these experiments.4) The authors conclude that inhibition of Notch signaling regulates Akap12 expression to increase cilia length during tubule morphogenesis. While the authors do show a clear demonstration that Akap12 is upregulated in the dnMamL MDCK cells and in E16.5-18.5 embryos from the Pax8-rtTA; TRE-dnMamL line, and that ectopic expression of Akap12 in MDCK cells is sufficient to increase cilia length, they do not provide conclusive evidence of the link between Notch and Akap12, the link between elongated cilia and cyst formation, or provide conclusive evidence that Akap12 overexpression induced cilia elongation is causal to cyst formation in vivo . In the discussion, the authors bring up a possible role of Akap12 to bind AuroraA and Plk1 to regulate spindle orientation, but fail to mention that AurA and Plk1 arethe major deciliogenesis pathways (Pugacheva, Jablonski, Hartman, Henske, & Golemis, 2007; Sanchez & Dynlacht, 2016) , and Akap12 overexpression could result in increased cilia length simply because AurA and Plk1 are no longer able to activate HDAC6 to reduce cilia length. In addition, Akap12 is also known to bind kinases such as PKA, which also is known to play a key role in cilia length maintenance through IFT protein phosphorylation (Abdul-Majeed, Moloney, & Nauli, 2012).Minor Concerns:1) In Figure 2, we wonder why A-D are H&E staining, while E-F are immunofluorescence. In addition, we wonder why the authors now stain for Megalin instead of LTL to detect proximal tubule segments.2) We think that the data presented in Figure 1 and Figure 2 would be strengthened by the addition of quantification of kidney size or cystic index, especially comparing the severity of the different induction timepoints of Figure 2.3) In Figure 3C, we are unsure what the X-axis labels (D2, B2, F3, A2, F2) are. Please clarify.4) In Figure 6, we are unsure what conclusion the authors are trying to draw. For instance, in the text they refer to a more “motile-like cilia phenotype”, yet Figure 6D, G, and H show 8 microtubule doublets with a misplaced doublet in the center, which happens in normal primary cilia as you image more distally from the cell body. They also lack the electron dense NDRC components and dynein arms which are present in motile cilia. To say whether or not the ciliary ultrastructure is disrupted, the authors would need to do 3D reconstruction using a technique such as scanning block face EM to ensure you are in the same region when comparing cilia.Abdul-Majeed, S., Moloney, B. C., & Nauli, S. M. (2012). Mechanisms regulating cilia growth and cilia function in endothelial cells.Cell Mol Life Sci, 69 (1), 165-173. doi:10.1007/s00018-011-0744-0Kamath, B. M., Spinner, N. B., & Rosenblum, N. D. (2013). Renal involvement and the role of Notch signalling in Alagille syndrome.Nat Rev Nephrol, 9 (7), 409-418. doi:10.1038/nrneph.2013.102Keeling, J., Tsiokas, L., & Maskey, D. (2016). Cellular Mechanisms of Ciliary Length Control. Cells, 5 (1). doi:10.3390/cells5010006McCright, B., Gao, X., Shen, L., Lozier, J., Lan, Y., Maguire, M., . . . Gridley, T. (2001). Defects in development of the kidney, heart and eye vasculature in mice homozygous for a hypomorphic Notch2 mutation.Development, 128 (4), 491-502.Pugacheva, E. N., Jablonski, S. A., Hartman, T. R., Henske, E. P., & Golemis, E. A. (2007). HEF1-dependent Aurora A activation induces disassembly of the primary cilium. Cell, 129 (7), 1351-1363. doi:10.1016/j.cell.2007.04.035Sanchez, I., & Dynlacht, B. D. (2016). Cilium assembly and disassembly.Nat Cell Biol, 18 (7), 711-717. doi:10.1038/ncb3370
