Two-Photon Microscopy Reveals Stationary Podocytes in Living Zebrafish Larvae

Aug 28, 2026

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Podocytes form the core of the glomerular filtration barrier and display complex, actin-based cytoskeletal architecture in vivo, while their true motility status in living organisms remains debated. Extended intravital two-photon microscopy was used to observe translucent casper zebrafish larvae expressing eGFP specifically in podocytes. Time-lapse imaging from 5–7 days post fertilization shows podocyte cell bodies and major process branching patterns remain unchanged for up to 23 hours, indicating stationary behavior. The study supports podocytes as stationary cells in the intact glomerulus.

LanguageEnglish

CategoryResearch & Report

FormatPDF

Pages6

Reading Time15 min

AccessFree to download

Background

Podocyte motility in vivo vs in vitro

Methods

Intravital two-photon microscopy in transgenic zebrafish

Imaging time points and resolution

Results

Podocyte morphology and eGFP labeling

Time-lapse dynamics of cell bodies and processes

Conclusion

Podocytes are stationary in intact zebrafish glomeruli

BRIEF COMMUNICATION [www.jasn.org](www.jasn.org) Two-Photon Microscopy Reveals Stationary Podocytesin Living Zebraﬁsh Larvae Nicole Endlich,* Ole Simon,* Achim Göpferich,† Henny Wegner,* Marcus J. Moeller,‡ Elisabeth Rumpel,* Ahmed M. Kotb,* and Karlhans Endlich* *Department of Anatomy and Cell Biology, Universitätsmedizin Greifswald, Greifswald, Germany; †Department of Pharmaceutical Technology, Universität Regensburg, Regensburg, Germany; and ‡Division of Nephrology and Immunology, Rheinisch-Westfälische Technische Hochschule, Aachen University Hospital, Aachen, Germany ABSTRACT Podocytes are an essential component of the glomerular ﬁltration barrier and cover the outer aspect of glomerular capillaries. They form a complex actin-based cytoskeleton in vivo and show prominent motility in vitro, but whether podocytes are stationary or mobile in vivo is debated. To address this question, the pronephros of translucent zebraﬁsh larvae (casper) expressing enhanced green ﬂuorescent protein (eGFP) speciﬁcally in podocytes (wt1a:eGFP larvae) was observed by intravital two-photon microscopy over extended periods of time. Podocyte cell bodies and the interdigitating branching pattern of major processes could be resolved with a resolution of approximately 1 mm in the xy-plane. Time-lapse imaging of zebraﬁsh larvae at 5–7 days after fertilization demonstrated that podocytes neither migrated nor changed the branching pattern of their major processes over a time period of up to 23 hours. In summary, we show by extended intravital two-photon microscopy that podocytes are stationary cells in the intact glomerulus of a translucent zebraﬁsh with ﬂuorescently-labeled podocytes. J Am Soc Nephrol 25: 681–686, 2014 . doi: 10.1681/ASN.2013020178 Podocytes cover the outer aspect of the glomerular capillaries forming actinbased interdigitating foot processes (FPs) with neighboring podocytes in a zipperlike manner. 1 This unique three-dimensional organization of FPs is required for proper ﬁltration of the blood and an intact ﬁltration barrier. Ultrastructural changes of this sophisticated morphology of FPs, such as effacement or retraction of FPs, lead to severe glomerular diseases, often resulting in ESRD. Previous studies showed that FPs express actin and actin-associated proteins, such as cortactin, formin, coﬁlin, and RhoGTPases that are involved in the nucleation, polymerization, and depolymerization of actin ﬁlaments.2–6 It is still not completely understood why podocytes possess such a prominent actin cytoskeleton. For many years, researchers have debated, mainly stimulated by in vitro ﬁndings,7–11 whether podocytes in vivo are actively moving cells or rather stationary. Recent observations in mice suggest that podocytes may migrate several micrometers within minutes. 12 This would imply that there is a coordinated migration of neighboring podocytesto guarantee a continuously formed ﬁltration slit. Resolving this question is important to understand the role and the inﬂuence of the actin-cytoskeleton for the function of podocytes in vivo. For this purpose, podocytes need to be visualized in living animals with high resolution. In the past, two groups observed kidney tissue in living mice by the use of multiphoton microscopy.12–14 However, because podocytes were only negatively stained (by dye exclusion), primary and secondary processes of podocytes have not been visualized with sufﬁcient resolution. More recently, Khouri et al. have used transgenic mice expressing green ﬂuorescent protein (GFP) in a podocyte-speciﬁc fashion to resolve podocytes by multiphoton microscopy in more detail.15 By using transgenic mice that expressed GFP only in a fraction of podocytes, Grgic etal. have even succeeded in visualizing the structural details down to the FPs by standard laser-scanning confocal microscopy.16 Likewise, Höhne et al. could visualize FPs by two-color labeling of podocytes in transgenic mice.17 However, in the latter studies

FAQs

Why is podocyte motility in vivo important?

Podocytes maintain the filtration barrier through coordinated structure of their actin-based foot processes. Determining whether they migrate or remain stationary clarifies how the actin cytoskeleton supports filtration slit formation.

How were podocytes visualized in this study?

Translucent casper zebrafish larvae expressing eGFP specifically in podocytes (wt1a:eGFP) were imaged with intravital two-photon microscopy over extended periods.

What did the time-lapse imaging show about podocyte behavior?

From 5–7 days post fertilization, podocyte cell bodies and the branching pattern of major processes did not migrate or change over up to 23 hours.

Aug 28, 2026

Uploaded by Đào