Kupffer's vesicle size threshold for robust left-right patterning of the zebrafish embryo

Abstract

Background: Motile cilia in the "organ of asymmetry" create directional fluid flows that are vital for left-right (LR) asymmetric patterning of vertebrate embryos. Organ function often depends on tightly regulated organ size control, but the role of organ of asymmetry size in LR patterning has remained unknown. Observations of the organ of asymmetry in the zebrafish, called Kupffer's vesicle (KV), have suggested significant variations in KV size in wild-type embryos, raising questions about the impact of KV organ size on LR patterning.

Results: To understand the relationship between organ of asymmetry size and its function, we characterized variations in KV at several developmental stages and in several different zebrafish strains. We found that the number of KV cilia and the size of the KV lumen were highly variable, whereas the length of KV cilia showed less variation. These variabilities were similar among different genetic backgrounds. By specifically modulating KV size and analyzing individual embryos, we identified a size threshold that is necessary for KV function.

Conclusions: Together these results indicate the KV organ of asymmetry size is not tightly controlled during development, but rather must only exceed a threshold to direct robust LR patterning of the zebrafish embryo.

Keywords: cilia; congenital disease; developmental noise; left-right patterning; organ size control.

Figure 1. Analysis of variation in development of Kupffer’s vesicle in wild-type embryos

(A–C) Representative maximum projections of confocal images of KV cells labeled with aPKC antibodies (magenta) to mark the KV lumen and acetylated Tubulin antibodies (green) to visualize cilia at 8 ss. These examples show a wild-type embryo with small KV size and few cilia (A) and embryos with an intermediate (B) or larger (C) lumen size. (D–F) Quantification of KV cilia number (D), cilia length (E) and lumen area (F) at 4 ss (n=26 embryos), 6 ss (n=23), 8 ss (n=30) and 10 ss (n=30). The horizontal line in the box and whisker plots indicates the mean and the whiskers show the minimum and maximum values. (G) Variations in cilia number, cilia length, and lumen size at each developmental stage are represented as one standard deviation converted to a percentage of the mean.

Figure 2. The amount of variation in Kupffer’s vesicle development is similar among different wild-type and transgenic strains

(A–C) Images of KV at 8 ss from three different transgenic strains. KVs appeared similar in most strains, including Tg(myl7:GFP) (A) and Tg(sox17:GFP) (B), but were smaller inTg(h2afx:EGFP-rab11a) (C). See text for descriptions of each strain. (D–F) Analysis of KV cilia number (D), cilia length (E) and lumen area (F) at 8 ss for different wild-type (TL and AB) and several transgenic strains. Data from wild-type TAB strain at 8 ss presented in Figure 1 is included for comparison. The number of embryos (N) analyzed for each strain is indicated in (G). (G) Variations in cilia number, cilia length, and lumen size are represented as one standard deviation as a percentage of the mean. (H–I) Analysis of potential correlations between KV lumen area and cilia number (H) and KV lumen area and cilia length (I). Each point represents a single embryo (n=292 embryos). The R² (coefficient of determination) value is listed for all strains pooled.

Figure 3. Role of Kupffer’s vesicle size in LR patterning

(A–D) Modulating Cftr activity was used to alter KV lumen size at 8 ss. The Cftr activating drug CFTact-09 increased KV size (B), whereas a loss-of-function mutation (C) or morpholino (MO) interference (D) reduced KV size relative to controls (A). Dashed circles represent approximate KV lumen boundaries. (E–H) Possible outcomes of RNA in situ hybridization analysis of the LR patterning marker spaw in lateral plate mesoderm (arrowhead) at 18 ss are normal left-sided (E), bilateral (F), reversed right-sided (G) or absent (H) expression. Dashed lines indicate the embryonic midline. L=left; R=right. (I–K) Quantification of KV area (I), cilia number (J) and cilia length (K) in embryos with altered Cftr function and controls. (L) Analysis of spaw expression in Cftr modulated embryos. Error bars represent one standard deviation. n=number of embryos analyzed. * indicates p < 0.05

Figure 4. Identification of a Kupffer’s vesicle size threshold necessary for robust LR patterning

(A–D) Representative DIC images of different sized KVs in living wild-type embryos at 8 ss. The KV^max area is indicated for each representative embryo. (A′–D′) spaw expression (arrowhead) at 18 ss in the same embryos. Dashed lines indicate the embryonic midline. L=left; R=right. (E–H) Analysis of spaw asymmetry in embryos in which KV was measured at 8ss (E–F) or 3 ss (G–H). (E, G) Normal (left-sided) and altered (bilateral, reversed or absent) spaw outcomes were plotted based on KV^max area. Each point represents a single embryo. (F, H) Observed spaw expression in embryos within specific ranges of KV^max areas. n=number of embryos analyzed.