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\begin{document}

\title{Pre-review of~``Notch signaling regulates Akap12 expression and primary
cilia length during renal tubule morphogenesis''}



\author[1]{Zhang Li}%
\author[2]{dzrevell}%
\affil[1]{University of Alabama at Birmingham}%
\affil[2]{Developmental Biology Journal Club}%


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  \date{May 25, 2024}


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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; \textbf{doi:} https://doi.org/10.1101/760181

This 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 \emph{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 \emph{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 \emph{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 are\emph{the
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.\emph{Cell Mol Life Sci, 69} (1), 165-173.
doi:10.1007/s00018-011-0744-0

Kamath, B. M., Spinner, N. B., \& Rosenblum, N. D. (2013). Renal
involvement and the role of Notch signalling in Alagille
syndrome.\emph{Nat Rev Nephrol, 9} (7), 409-418.
doi:10.1038/nrneph.2013.102

Keeling, J., Tsiokas, L., \& Maskey, D. (2016). Cellular Mechanisms of
Ciliary Length Control. \emph{Cells, 5} (1). doi:10.3390/cells5010006

McCright, 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.\emph{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. \emph{Cell, 129} (7), 1351-1363.
doi:10.1016/j.cell.2007.04.035

Sanchez, I., \& Dynlacht, B. D. (2016). Cilium assembly and
disassembly.\emph{Nat Cell Biol, 18} (7), 711-717. doi:10.1038/ncb3370

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