Interplay between medial nuclear stalling and lateral cellular flow underlies cochlear duct spiral morphogenesis

  1. Mamoru Ishii
  2. Tomoko Tateya
  3. Michiyuki Matsuda
  4. Tsuyoshi Hirashima

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Abstract

A notable example of spiral architecture in organs is the mammalian cochlear duct, where the duct morphology is critical for hearing function. Molecular genetics has revealed the necessary signaling molecules for the formation of spirals in organs, but it remains unclear how cellular dynamics generate bending and coiling of the cochlear duct during development. Here we show two modes of multicellular dynamics underlying the morphogenetic process by combining deep tissue live-cell imaging, Förster resonance energy transfer (FRET)-based quantitation, and mathematical modeling. First, surgical separation of the cochlear duct revealed that bending forces reside primarily in the medial side of the duct. In the medial pseudostratified epithelium, we found that nuclei stall at the luminal side during interkinetic nuclear migration, which would cause the extension of the luminal side, thereby bending the duct. Second, long-term organ-scale FRET imaging of extracellular signal-regulated kinase (ERK) activity showed that helical ERK activation waves propagate from the duct tip concomitant with the reverse multicellular flow in the lateral side of the duct, resulting in advection-based duct elongation. We propose an interplay of distinct multicellular behaviors underpinning spiral morphogenesis in the developing cochlear duct.

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  1. eLife

    ###Reviewer #3:

    The manuscript by Ishii et al focuses on understanding how cellular dynamics drive the spiral shape of the cochlear duct in mammals. The authors use live imaging of inner ear explants to follow dynamics of interkinetic nuclear migration (IKNM) and ERK activity (using ERK FRET sensor) to track some of the processes that give rise to tissue bending during spiral duct formation. On the imaging side, the manuscript presents a technical tour de force, showing remarkable two photon imaging capabilities that provide insights into the dynamics underlying cochlear extension. These experiments reveal several new observations: (1) Medial epithelial layer (MEL) tends to bend more than the lateral epithelial layer (LEL) despite being more proliferative. (2) That nuclei of cells in the curved region of the cochlea tend to stay in the luminal side, following cell division, rather than migrate back to the basal side. (3) The cells migrate towards the apical lateral roof. (4) That there are orchestrated ERK waves that correlate with cell migration. Based on these observations and on mathematical modeling, the manuscript has two main claims: (1) that nuclear stalling on the luminal side following cell division leads to increased curvature which gives rise cochlear duct bending, and (2) that multicellular flow mediated by ERK signaling waves pushes cells towards the growing apex, supplying the cells required for luminal expansion. While the observations in the manuscript are certainly interesting, I worry however, that some of the claims are not sufficiently substantiated, and also the connection between the two observations is rather weak. Here are the detailed concerns:

    Major concerns:

    1. The authors argue that cell cycle arrest results in a decrease in the curvature of the cochlear duct, which supports the hypothesis that luminal nuclear stalling promotes MEL bending. This is fine, but luminal nuclear stalling can be a result and not a cause. Since in a bent region, the basal side is more packed, this density gradient can be the cause of nuclei stalling at the luminal side. The fact that the curvature decreased but not diminished after cell cycle arrest could suggest that nuclear stalling is not required for bending, but rather reinforces it.

    2. Since the authors discuss both cell proliferation and nuclear stalling, and cell migration, as forces that can drive bending and coiling, it hard to interpret the results of the mitomycin C experiment. Could it be that the tissue is less curved because there are less cells to supply the elongation tissue rather than less nuclear stalling? The authors should consider inhibiting either cell migration or the cytoskeletal machinery required for IKNM to dissect these effects.

    3. The authors present a mathematical model to demonstrate that nuclear stalling in the luminal side results in bending. To model nuclear motion they use a parameter, gamma, which controls the degree of basalward movement after IKNM. Modeling in such way means that other than gamma=1, the nucleus never fully returns to the basal side, but if I understand correctly this is not the case, as even if the nuclei that stall at the luminal side, eventually return to the basal side.

    4. Furthermore, for luminal nuclear stalling, the authors tracked only the nuclei of dividing cells. This makes the data in Fig 2D' much clearer. However, in their model the authors show only these nuclei and not all nuclei. In addition, they show many crowded nuclei in the model, yet this is not observed in the images provided in the manuscript. Therefore, it seems the model does not represent the morphology of the tissue properly. The authors should model the process with non-dividing cells at the basal side.

    5. In lines 250-252 the authors claim that the higher volumetric growth measured at the MEL should cause an opposite curvature relative to the innate one. This is true if EdU intensity is proportional to volumetric growth, but cells in the MEL and LEL may not be the same size. For example, cells in the MEL could be smaller than cells in the LEL. The authors should therefore measure the nuclei number density and the volumetric cell density to clarify this. If the number density of the nuclei is indeed higher at the MEL, it may also explain the higher structural integrity of the MEL relative to the LEL demonstrated in figure 1C.

    6. The authors show the effect of ERK inhibition on tissue flow speed. This is a very important observation and raises several important questions. What is effect of ERK inhibition on curvature? On tissue length? On proliferation? These will provide a more complete understanding of the effect of RK inhibition.

    7. The authors should also test the effect of mitomycin C on cell flow and ERK activity. AS mentioned above, it is not clear whether the effect of mitomycin C is a result of less nuclear stalling or perhaps less cells that flow towards the apex.

    8. In Figure 3 the authors analyze the EdU distribution over the cochlear duct. This analysis is done using the maximum intensity projection of the stack. It seems that a more accurate way to quantify would be to use the summed intensity image rather than the maximum intensity image. This may reveal additional details that were missed by throwing away all other layers except the one at maximum intensity.

    9. In Figure 4 the colors used for the ERK activity analysis are very hard to see for color-blind people. It would be easier for this audience if the authors changed 1 of these color to green/red/yellow.

  2. eLife

    ###Reviewer #2:

    The paper by Hirashima and colleagues shows some interesting cellular mechanisms they conclude drive the spiraling and outgrowth of the mammalian cochlea. The two cellular mechanisms they propose are supported by experiments and modeling. The spiraling ERK wave and the contrasting movement of lateral cells was very intriguing. However, the ERK wave and lateral cell movements seem disconnected from the bending forces discussed. Are the authors saying that the ERK mediated lateral cell movements are important for cochlear growth while the MEL is important for the bending? The two mechanisms they discuss seem insufficient to explain all of cochlear spiraling. Other cellular mechanisms such as cell proliferation and convergent extension are mentioned but their roles are not incorporated into their discussion. Are they not required? How do they complement their results?

    1. While the authors talk about bending forces, the paper has no measurements of the forces generated by different tissues. I also feel there are other cellular mechanisms that are mentioned but never incorporated into their proposed explanation for duct coiling such as convergent extension and actomyosin based basal shrinkage. Proliferation is discussed quite a bit but seems to be dismissed as a force. In the introduction they mention how Shh mediated proliferation is required for duct elongation while Fgf10 null mutants have a shortened duct yet normal proliferation. So what is the role for proliferation? Maybe they can answer this in the context of their interesting observation that there is more proliferation in the roof than the floor which would be predicted to bend the cochlea along that axis. When combined with the medial lateral bending could these two forces result in the spiraling? It also seems like this differential proliferation between the floor and roof was in more than just the epithelium correct? Could the cartilaginous capsule around the duct guide the bending as well? In their culture experiments, if too much of the capsule was removed then normal duct development was disrupted.

    2. Their demonstration that the bending forces are in the medial half is interesting but the only tissue whose mechanism is studied is the MEL. Could convergent extension in other medial tissues such as the prosensory domain (which Wang et al. showed was occurring in this tissue) and surrounding mesenchyme be the main force generator for the bending of the medial half of the cochlear duct? Does the MEL cultured by itself bend? They say that cell intercalation can drive ductal elongation but not bending (line 83) but can't convergent extension occur asymmetrically in the tissue? Such as by occurring in the overlying medial mesenchyme but not in the medial epithelium. It should be noted that the bending by the epithelium does not have to provide high forces as long as the force provided by other tissues are similar across the medial lateral axis, the bending in the epithelium could bias the mass of tissue to bend.

    3. The mathematical modeling for the luminal bending is less convincing than the mathematical modeling for the ERK and Cell flow coupling. The simulated curves in Fig. 2K are quite different from the Experimental measure in Fig. 2M, especially for the Mitomycin C condition. I feel that the values plugged in for the Numerical simulation, the standard parameter set were not well justified. What happened to the simulations as these values changed? Was the parameter space for acceptable values broad? In contrast the parameters for the numerical simulation of the ERK activation waves and cell flows were well justified. The parameters chosen might explain the big differences between simulation and experimental in figure 2.

    4. For the cell tracking experiments in the lateral region the resolution was 4-5 cells. The resulting cell flow patterns were very interesting but why didn't the authors track single cells? Segmenting individual cells via cytoplasmic labeling is much trickier but the nuclei are identifiable and the Imaris software they used in the paper has a cell tracking feature for such labeling. I would think that individual cell movements might provide more insights. In line 303 they say they can see cell contractions which I assume is for individual cells? How were cell contractions identified? Movie 5 was excellent and very informative. Do the cell flows correlate at all with the proliferation seen with Edu staining?

  3. eLife

    ###Reviewer #1:

    This is a fascinating manuscript that explores for the first time the potential mechanisms underlying cochlear morphogenesis. The authors have used a combination of modeling, beautiful imaging and ERK-FRET reporter mice reporter mice to suggest at least two processes may be at play in cochlear shaping - differential interkinetic nuclear migration and a cellular flow that appears to correlate with ERK activation.

    I have no major concerns with this lovely piece of work. The imaging and quantification is meticulous, and the observations made by the authors are novel and will of great interest to cell biologists interested in morphogenesis, no just aficionados of the inner ear.

    The one suggestion I would make is for the authors to clarify the relationship between cell proliferation and ERK activation. When they reference the inner ear literature, they point out that FGF pathway mutants have deficient cochlear morphogenesis and proliferation, and they hypothesize that FGF-induced ERK activation may be responsible for their propagating waves. However, they also reference work suggesting that cellular extension during collective migration can also induce ERK activation and also suggest SHH-induced proliferation as another causative factor in promoting ERK activation through proliferation. I think the authors should try and clarify this - both in their explanation, but also by comparing the effects of the MEK inhibitor PD0325901 on ERK activity and tissue flow speed (Fig 4I and S3F) with the effects of the FGFR inhibitor SU5402, and also Shh inhibitors like cyclopamine. If the effects they see are directly due to FGF signaling, one would expect a change in ERK activation and cell flow with the same kinetics as with PD0325901. However, if Shh-induced proliferation is responsible, the change in ERK activation would take much longer to achieve. I think these experiments should be possible to do in a relatively short period of time.

  4. eLife

    ###This manuscript is in revision at eLife

    The decision letter after peer review, sent to the authors on September 9 2020, follows.

    Summary

    The mammalian cochlear duct is a spiral-shaped organ. This study investigated the mechanisms underlying the bending of the cochlear duct. Using two-photon live imaging and mathematical modeling, it was reported that the bending of the cochlear duct is caused by stalling of nuclei in the luminal side of the medial cochlear duct during interkinetic nuclear migration. Using FRET-based imaging, cochlear duct elongation is attributed to an oscillatory wave of ERK activity originating from the cochlear tip.

    All three reviewers were impressed by the imaging results. Although the reviewers and editors find the concept and approach interesting, blocking cell proliferation may be too crude a method to address the authors' hypothesis and many questions were raised by the results of blocking cell division. Second, the relationship between cell proliferation and ERK-driven migration is also unclear. Please see comments from Reviewer #2 and #3 for specifics. Third, what is the relationship between SHH-induced proliferation and ERK activation as suggested by the authors (see comments from Reviewer #1)? Additionally, it is problematic to illustrate a difference in the bending force between medial and lateral cochlear duct that is presumably occurring at E12.5 and E14.5 with a cochlear dissection at E17.5. The tissue architecture is completely different between E12.5 and E17.5. The surgery basically removed a specific region of the cochlear duct, the stria vascularis, rather than medial versus lateral halves of the cochlear duct.

  5. Version published to 10.1101/2020.07.24.219469 on bioRxiv