Adaptive cell invasion maintains organ homeostasis

Julia Peloggia, Daniela Münch, Paloma Meneses-Giles, Andrés Romero-Carvajal, Melainia McClain, Y. Albert Pan, Tatjana Piotrowski

Posted on: 19 December 2020

Preprint posted on 5 December 2020

Hearing and navigation: role of adaptive cell invasion in maintaining organ homeostasis.

Selected by Mariana De Niz

Categories: cell biology, developmental biology, pathology

Background

The vestibular and auditory part of the vertebrate inner ear, and the lateral line sensory organ of aquatic vertebrates, contain hair cells that are immersed in fluid microenvironments essential for sensory transduction. Together, they allow different types of animals to detect sound and changes in body position, and/or are key for orientation, hunting, and avoiding predators. Changes in the ionic and electrical microenvironment in the ear or the lateral line can have important deleterious consequences for the vertebrate. Thus, several mechanisms that regulate ionic and osmotic homeostasis have evolved in vertebrates- this includes the presence of ion channels, and ionocytes. For homeostasis, the concentrations of multiple ions need to be regulated. This includes various channels and transporters in the inner ear of mammals, and the skin and gills of zebrafish. Neither in the vertebrate ear nor the lateral line, are the cellular and molecular basis of ion concentration control fully understood. The zebrafish sensory lateral line has been shown to be an excellent model system to study sensory organ development and regeneration in vivo. In their work, Peloggia, Münch et al (1) report the discovery of an invasive cell type in the zebrafish lateral line, possessing ionocyte characteristics (called Nm ionocytes), which migrate and persistently invade the post-embryonic and adult lateral line organs. Further, they show that the lateral line invasion process is adaptive, triggered and modulated by changing environmental stimuli. Altogether this model allows the study of physiological adaptation of vertebrate organs to changing environmental conditions.

Figure 1. Neuromast-associated ionocytes share morphological characteristics with skin ionocytes and are exposed to the external environment through an opening to the neuromast cupula. (From Ref 1.)

Key findings and developments

The authors began by describing previously uncharacterized cells in neuromasts (the sensory organs of the lateral line system), that express ionocyte markers. A combination of single-cell RNA-Seq and confocal microscopy led to the finding that some cells in the neuromasts are not labeled by lateral line markers. These appeared as fluorescence gaps. Notch pathway reporters were found to label 80% of the observed gaps in fluorescence. Using a combination of Notch-specific fluorescent reporters, FACS and scRNA-Seq analysis, allowed the authors to conclude that the Notch- cell in the pair of unlabeled cells in neuromasts is an ionocyte that expresses both HR (H+ secretion/Na+ uptake/NH4+ excretion) and NaR (Ca2+ uptake) ionocyte markers, while the Notch+ cell expresses a few ionocyte-associated genes, but lacks some morphological ionocyte characteristics. They labeled the pair of cells Neuromast-associated ionocytes (Nm ionocytes).

The authors then showed that Nm ionocytes are derived from skin cells surrounding the neuromast. Nm ionocytes share genes with skin ionocytes, but do not express neuromast markers, thus the authors wondered whether this points towards them having a different embryonic origin that the neuromast cells. To determine the embryonic origin of these cells, the authors used transgenic zebrafish that allow tracing cells throughout development, whereby clonally related cells have the same colour. Cre-induced recombination within the first few hours post-fertilization led to mosaically labeled lateral line primordia, which deposited mosaic neuromasts. Following up neuromasts over time led to two observations: a) within 2-3 days, some neuromasts contained pairs of cells in the poles with different colours than the other cells in the neuromast; b) the vast majority of neuromast cells become clonal in adult fish. Further reporter combinations allowed the authors to conclude that Nm ionocytes have a different embryonic origin than all other neuromast cells, and that the Nm ionocytes share their colour hue with skin cells immediately surrounding the neuromasts. This suggests that the skin cells might be the source of Nm ionocytes or at least share the embryonic origin. Further investigation regarding the progenitors led the authors to conclude that Nm ionocytes are likely derived from basal keratinocytes surrounding the neuromasts and differentiate once inside the sensory organ.

As all observations show that Nm ionocytes are not present at the time of neuromast deposition, the authors investigated the origin and dynamics of Nm ionocytes in vivo. For this study, the authors performed time lapse imaging to capture the moment when Nm ionocytes first appear in the neuromast. Time lapse imaging showed that Nm ionocytes migrate into neuromasts as pairs of cells. Tracking of individual pairs showed that after invasion some pairs of cells migrate to the other side of the organ while others remain closer to their site of entry. Spatial analyses of Nm ionocytes at 5 dpf show that these cells are eventually positioned stereotypically in the dorsal-ventral and anterior-posterior poles of primI- and primII-derived neuromasts, respectively. Moreover, the authors showed that migration and differentiation of Nm ionocytes was associated with a progressive increase in Notch signaling in one cell of the pair. Pharmacological inhibition of Notch signaling during neuromast development generated an excess of hair cells, while there was a decrease in support cells and a lack of ionocytes. Inhibition of Notch signaling after hair cell differentiation also showed a significant decrease in Nm ionocytes. This led to the conclusion that Notch signaling plays an essential role in the migration and differentiation of ionocyte progenitors invading mature neuromasts as pairs, and that it is also essential for their survival.

Next, the authors performed 3D modeling of Nm ionocytes to study their morphological characteristics. They used a combination of correlative light, and serial block face scanning electron microscopy (SBEM) to find and 3D reconstruct the pair of cells. 3D modeling of the cell membranes of the Notch+ and Notch- cells revealed the tight associations between these cells and their morphology. Key observations include that the cells are in close contact with each other along their whole apico-basal axis, that their nuclei are located basally, and that the Notch+ cell is smaller than the Notch- cell. Both cells have extensions that reach the apical surface of the neuromast. The pair forms a crypt at the top of the apical extensions with microvilli projecting only from the Notch- cell into the lumen. This crypt was found to be exposed to the gelatinous cupula microenvironment that covers the cuticular plate and hair cilia of live animals. Altogether, the morphology of Nm ionocytes resembles that of gill and skin ionocytes.

Following the observation that Nm ionocytes are in direct contact with the overlying cupula, the authors went on to explore their role in the interaction with the ionic microenvironment. Their first finding was that the percentage of ionocyte containing neuromasts, and the number of Nm ionocytes per neuromast significantly increases throughout larval and fish development. Further investigation led the authors to suggest that Nm ionocytes continue to invade neuromasts as animals age. Moreover, the authors noted that increased Nm ionocyte frequency appears to be directly correlated with moving the animals from an ion-rich medium, to an ion-poor one. This was confirmed by detailed investigation of salinity changes and the frequency of Nm ionocyte invasion. These findings support the hypothesis that Nm ionocytes respond to salinity changes and are involved in regulating ion homeostasis.

The authors found that incubation in MilliQ water led to increased Nm ionocytes, but also decreased hair cell numbers. To test whether cell death modulates Nm ionocyte numbers, the authors killed hair cells using antibiotics, and saw no differences in Nm ionocyte frequency. Moreover, they went on to investigate whether Nm ionocytes could be involved in maintaining the pH in the hair cell microenvironment, and found that Nm ionocyte number and percentage of neuromasts with ionocytes increased with decreasing pH. This points towards an additional involvement of Nm ionocytes in acid/base secretion.

Finally, the authors then went on to functionally abrogate Nm ionocytes, by identifying candidates that when knocked out caused Nm ionocyte loss. One such candidate was foxi3a, a transcriptional master regulator that specifies skin ionocytes from epidermal precursors. Moreover, the authors showed that the absence of Nm ionocytes in foxi3a mutants affects hair cell function. Altogether, they show that Nm ionocytes are key players for regulating the ionic microenvironment surrounding lateral line hair cells.

What I like about this preprint

I find the question investigated (namely, understanding organ homeostasis), and the plethora of methods used, fascinating. I enjoyed very much reading the preprint, and I think the findings will be exciting to various scientific communities.

doi: https://doi.org/10.1242/prelights.26558

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Author's response

Julia Peloggia, Daniela Münch shared

Open questions

Your work was very interesting and I have lots of questions- some specific to your work, and others regarding the parallelism to mammals.

Thank you very much for your interest in our paper! We are happy to share some of our excitement with the community and answer all questions you may have.

1.You mention along your introduction the parallelisms between the fish lateral line, and the mammalian inner ear. You describe later on the presence of ionocytes in various organs such as the mammalian kidneys and lungs. Is it known whether the process of adaptive cell invasion is similar among mammals and fish, and across organs? is there anything known regarding this phenomenon in mammals, and their role in maintenance of organ homeostasis?

That is a very interesting question that we are also curious about. While it is known that ionocytes in other organs, like the airway epithelium, are also derived from basal cells, the timing of their differentiation is still poorly understood (2, 3). In the lung there is no cell invasion, the cells are specified and differentiate within the epithelium. The accessibility and tractability of the lateral line has provided us with an almost unique opportunity to observe in vivo the invasive nature of these cells, particularly post embryogenesis. While in the lung the source of ionocytes is known, more investigation is needed to understand if and how environmental stimuli can modulate their differentiation. Similarly, in other organs, like the fish inner ear, the precise source of ionocytes, as well as their timing of differentiation and the associated cellular behaviours still remain to be determined.

Basal keratinocytes are quite plastic cells that are important for homeostasis and regeneration of other organs in mammals. Two examples are hair follicle and taste bud regeneration (4, 5). In both cases, after injury basal cells can migrate and differentiate into the lost cell types of these organs to promote regeneration. Interestingly, in our case, basal keratinocytes do not differentiate into placodally-derived neuromast cells, but rather into a different cell type that changes the cellular composition of the organ. It would be interesting to investigate ionocyte differentiation dynamics in the lung and inner ear and understand how similar they would be compared to what we described in the lateral line.

2.From your observation in foxi3a mutants, are there known diseases/mutations (investigated either in mammalian or fish models) whereby ionocyte migration is impeded?

Loss of functional ion-regulating cells is associated with many pathologies such as cystic fibrosis (2, 3), distal renal tubular acidosis and hearing deficiencies (6-8). However, not much is known about ionocyte migration per se. We hope that our paper and our results will motivate researchers to pay attention to this aspect of ionocyte development.

3. A more “big picture” question directly related to your model organism: you showed in your work how the external environment such as salinity and pH, influence Nm ionocyte numbers, frequency of migration, etc. With the continuous pollution of water bodies world-wide, is it known if and how different pollutants have affected fish populations by altering processes such as adaptive cell invasion?

That is such an interesting point. We jokingly discussed planning a field trip to study Nm ionocytes in wild zebrafish populations and see how these cells behave. The fish in the lab are kept under quite comfortable and constant conditions – which might be why these cells eluded discovery for so long. Regarding the pollutants, ionocytes appear to be quite resilient and the adaptive process seems to be robust. With anthropogenic sources increasingly affecting ion homeostasis of several water bodies across the world, it is tempting to speculate that adaptive cell invasion of Nm ionocytes could help maintain lateral line hair cell function in fish across different ranges of salinity.

4.You present here the Notch-mediated signaling pathway as a trigger for adaptive cell invasion. You also measured in your work the speed and total displacement of Notch+ and Notch- cells. Are there other signals/factors mediating cell migration and invasion – for instance the usual chemokine/cytokine-based signals? Are these signals/factors the same throughout the entire development of the fish? (you mentioned that this process continues throughout larval development and fish development). Can these be affected by exogenous situations such as infections or other forms of disease?

Thank you very much for asking this question. We are also really interested in these aspects and we are already working on a follow up of our story looking into the induction and recruitment signals for these cells. We still do not have an answer for the readers, but we are checking the usual culprits such as chemokine/cytokines, and we hope people will stay tuned. In mammals, skin ionocyte numbers and mammalian fluid homeostasis are controlled by hormones. We are interested in further dissecting how these global signals can be selectively interpreted by cells and trigger adaptive cell invasion.

5.You mention in your discussion that findings on Nm ionocytes could be helpful to better understand pathologies affecting the inner ear. Can you expand further on this?

The importance of maintaining a specialized ionic microenvironment surrounding hair cells is shared between the inner ear and the lateral line. Having an open and accessible system like the lateral line neuromast allows us to directly manipulate factors that play pivotal roles in inner ear pathologies, such as altered ionic composition and pH of the surrounding fluid. We can then investigate their effect on hair cell function in the presence or absence of functional ionocytes. The modulatory aspect of Adaptive Cell Invasion adds a new element to hair cell ion homeostasis that may assist us in further dissecting phenotypes and understanding hearing deficits – now we know of more things to look for!

References

Peloggia J, Münch D, et al, 2020. Adaptive cell invasion maintains organ homeostasis, bioRxiv.
Montoro, D.T., Haber, A.L., Biton, M., Vinarsky, V., Lin, B., Birket, S.E., Yuan, F., Chen, S., Leung, H.M., Villoria, J. and Rogel, N., 2018. A revised airway epithelial hierarchy includes CFTR-expressing ionocytes. Nature, 560(7718), pp.319-324.
Plasschaert, L.W., Žilionis, R., Choo-Wing, R., Savova, V., Knehr, J., Roma, G., Klein, A.M. and Jaffe, A.B., 2018. A single-cell atlas of the airway epithelium reveals the CFTR-rich pulmonary ionocyte. Nature, 560(7718), pp.377-381.
Donati, G. and Watt, F.M., 2015. Stem cell heterogeneity and plasticity in epithelia. Cell stem cell, 16(5), pp.465-476.
Barlow, L.A. and Klein, O.D., 2015. Developing and regenerating a sense of taste. In Current topics in developmental biology(Vol. 111, pp. 401-419). Academic Press.
Karet, F.E., Finberg, K.E., Nelson, R.D., Nayir, A., Mocan, H., Sanjad, S.A., Rodriguez-Soriano, J., Santos, F., Cremers, C.W., Di Pietro, A. and Hoffbrand, B.I., 1999. Mutations in the gene encoding B1 subunit of H+-ATPase cause renal tubular acidosis with sensorineural deafness. Nature genetics, 21(1), pp.84-90.
Lorente-Cánovas, B., Ingham, N., Norgett, E.E., Golder, Z.J., Frankl, F.E.K. and Steel, K.P., 2013. Mice deficient in H+-ATPase a4 subunit have severe hearing impairment associated with enlarged endolymphatic compartments within the inner ear. Disease models & mechanisms, 6(2), pp.434-442.
Norgett, E.E., Golder, Z.J., Lorente-Cánovas, B., Ingham, N., Steel, K.P. and Frankl, F.E.K., 2012. Atp6v0a4 knockout mouse is a model of distal renal tubular acidosis with hearing loss, with additional extrarenal phenotype. Proceedings of the National Academy of Sciences, 109(34), pp.13775-13780.

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Adaptive cell invasion maintains organ homeostasis

Background

Key findings and developments

What I like about this preprint

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