Developmental Cell

Short Article The Notochord Breaks Bilateral Symmetry by Controlling Cell Shapes in the Zebrafish Laterality Organ Julien Compagnon,1 Vanessa Barone,1 Srivarsha Rajshekar,1,2 Rita Kottmeier,1,3 Kornelija Pranjic-Ferscha,1 Martin Behrndt,1 and Carl-Philipp Heisenberg1,* 1Institute

of Science and Technology Austria, 3400 Klosterneuburg, Austria address: Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA 3Present address: University of Mu ¨ nster, Institute of Neurobiology, 48149 Mu¨nster, Germany *Correspondence: [email protected] http://dx.doi.org/10.1016/j.devcel.2014.11.003 2Present

SUMMARY

Kupffer’s vesicle (KV) is the zebrafish organ of laterality, patterning the embryo along its left-right (LR) axis. Regional differences in cell shape within the lumen-lining KV epithelium are essential for its LR patterning function. However, the processes by which KV cells acquire their characteristic shapes are largely unknown. Here, we show that the notochord induces regional differences in cell shape within KV by triggering extracellular matrix (ECM) accumulation adjacent to anterior-dorsal (AD) regions of KV. This localized ECM deposition restricts apical expansion of lumen-lining epithelial cells in AD regions of KV during lumen growth. Our study provides mechanistic insight into the processes by which KV translates global embryonic patterning into regional cell shape differences required for its LR symmetry-breaking function.

INTRODUCTION Asymmetric positioning of organs along the left-right (LR) axis of the body in several vertebrate species relies on the formation of a transient symmetry-breaking organ at the end of gastrulation (Blum et al., 2009). The overall organization and function of laterality organs are highly conserved among vertebrates. In general, the vertebrate laterality organ consists of ciliated epithelial cells lining a fluid-filled cavity. The cilia are motile, oriented toward the posterior, and generate a leftward fluid flow within the lumen of the organ, which is essential for its LR patterning activity (Blum et al., 2009; Lee and Anderson, 2008). In zebrafish, the organ of laterality is named Kupffer’s vesicle (KV). It forms during early somitogenesis stages at the posterior end of the embryo from a cluster of around 60 progenitor cells, the so-called dorsal forerunner cells (DFCs). These cells undergo a mesenchymalto-epithelial transition to form the epithelial cyst-like structure of KV. In KV, ciliated cells surround the entire cavity of the cyst (Essner et al., 2005). To generate an efficient leftward flow and, hence, break LR symmetry, a higher density of cilia in anterior-

dorsal (AD) regions of KV is essential. As each lumen-lining epithelial cell only forms one cilium, higher cilia density in AD regions of KV is the result of a higher density of lumen-lining epithelial cells in this region (Okabe et al., 2008; Sampaio et al., 2014; Wang et al., 2011). However, the molecular and cellular mechanisms determining differential cell density within KV have only begun to be studied. Here, we show that regional differences in KV cell shape underlying cilia distribution in the organ are the result of localized extracellular matrix (ECM) deposition at the surface of the adjacent notochord, restricting apical expansion of lumen-lining cells within AD regions of KV in response to lumen growth. RESULTS AND DISCUSSION Lumen Growth Controls Regional Cell Shape Changes within KV To understand the origin of KV morphological asymmetry, we sought to quantitatively analyze the cell behaviors underlying its formation. KV asymmetry has previously been attributed to cells in AD regions being more densely packed and displaying a more columnar geometry than cells in the remainder of KV (Okabe et al., 2008; Figure 1A). This regional difference in cell packing and shape could, in principle, be due to differential migration of cells, differential regulation of cell division or cell death, and/or differential cell shape changes within the organ. Previous work has provided evidence that myosin-II-dependent cell shape changes and/or cellular rearrangements, but not differential cell division or cell death, are involved in the establishment of regional cell shape differences in KV (Wang et al., 2011, 2012). Consistent with a role of myosin II in locally affecting cell packing and shape in AD regions of KV, we found both myosin II light chain (MLC) fused to enhanced green fluorescent protein (EGFP) and phosphorylated MLC enriched at the apical surface of anterior KV cells (Figure S1A available online). To determine to what extent cell shape changes and/or cellular rearrangements are involved in KV acquiring its morphological asymmetry, we monitored the cellular behaviors underlying KV formation using high-resolution two-photon time-lapse imaging. As KV translocates on the surface of the spherical yolk sac during its formation, the anterior-posterior (AP) and dorsal-ventral (DV) axes, along which the organ is patterned, are rotating (Figure 1B; Movie S1). By combining KV cell nuclei

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tracking with lumen segmentation, we corrected these global movements and defined an organ-specific coordinate system. In this coordinate system, the origin was set to the center of mass of the lumen, and the axes were matched with the AP and DV axes of the embryo (Experimental Procedures; Figures S1B and S1C). To describe KV cell shape differences, we compared KV nuclei density variations between different regions of KV. As KV consists of a monolayered epithelium formed by cells with similar volume that are all in contact with the lumen, nuclei density is expected to inversely correlate with the lumen-facing apical area of KV cells. The apical area, in turn, should scale with cilia density, given that KV cells are monociliated (Essner et al., 2005); consequently, nuclei density can be used as a readout for cilia density, which is the ultimate morphological feature underlying the LR patterning activity of KV (Figures 1A and S1D). For analyzing KV nuclei density, we undertook a 4D quantitative analysis of their positions in a coordinate system that allows the registration of multiple independent samples. Our initial analysis of KV nuclei/cell density showed that the highest regional variation of nuclei density within KV was between the AD and the posterior-ventral (PV) quadrants (Figure 1C; Movie S2). We thus used the ratio of cell densities between these quadrants as a descriptor of KV morphological asymmetry, reflecting patterning along both the AP and DV axes. We found that, until the 4-somite stage (ss), there was no regional difference in cell density along the AP and DV axes of KV (Figures 1C, 1D, and S1E–S1G; Movie S2). After this stage, KV cells begun to accumulate in the anterior and dorsal sides of the organ, and this accumulation proceeded until the 7–8 ss (Figures 1C and 1D). Analysis of individual KV nuclei movements during this process showed that the relative positions of cells within the organ remained largely unchanged (Figures 1E, 1F, and S1B), suggesting that cell shape changes rather than large cellular rearrangements are responsible for KV acquiring its morphological asymmetry. As the appearance of KV asymmetry correlated with the expansion of KV lumen (Figures 1G and S1H), we sought to determine the potential influence of lumen expansion on KV regional cell shape changes. KV lumen growth relies on fluid secretion generated by cystic fibrosis transmembrane conductance regulator, which can be either inhibited by treatment with the Na+/K+-ATPase inhibitor ouabain or stimulated by treatment with IBMX and forskolin (Navis et al., 2013; Figure 1H). We found that inhibition of lumen growth or expansion beyond the maximal volume normally observed in unperturbed embryos reduced the ratio of cell density between the AD and PV regions of KV (Figure 1I). Notably, treated embryos did not show any recognizable changes in the architecture and/or molecular composition of the tissue(s) surrounding KV (Figure S4D), indicating that we were specifically probing the role of lumen growth in KV cell shape changes. Together, these observations suggest that regional cell shape changes within KV rely on the interplay between the intraluminal pressure and regional variation in contractility of the surrounding epithelium. To determine functional consequences of KV lumen growth perturbations and, thus, reduction in the ratio of cell density between the AD and PV regions of KV, on the bilateral symmetrybreaking function of KV, we analyzed LR patterning in embryos treated with drugs either increasing or decreasing KV lumen

volume (discussed earlier). We found that, in treated embryos, the expression of southpaw (spaw), the first indicator of LR asymmetric gene expression in zebrafish (Rebagliati et al., 1998), was no longer restricted to the left lateral plate mesoderm (Figures 1J and 1K). As ciliogenesis in treated embryos appeared unaffected (Navis et al., 2013), this suggests that lumen volume-dependent regional cell shape differences within KV are required for the bilateral symmetry-breaking function of KV. Interaction with the Notochord Induces Regional Cell Shape Differences within KV KV-forming cells must integrate AP and DV patterning information from their environment in order to break the bilateral symmetry of the embryo. Such patterning signals could be received by KV-forming cells at any time between the onset of gastrulation, when the DFCs, the progenitor cells of KV, are specified (Oteı´za et al., 2008), and the 4 ss, when regional differences in KV cell shape become apparent. To identify the origin of putative signal(s) patterning KV, we induced ectopic KVs by mosaic expression of the constitutively active Nodal receptor Acvr1b* (Activin receptor 1b; Figure 2A; Oteı´za et al., 2008). At bud stage, Acvr1b* mosaic embryos showed ectopic clusters of DFCs. These clusters formed apical foci (Figures S2A and S2B) and eventually transformed into structures, which, similarly to the endogenously located KV, were composed of sox17:GFP-expressing cells that formed a monolayered epithelium surrounding a lumen (Figures 2B and 2C). To test whether these ectopically induced KV-like structures have the same morphogenetic competence as their endogenous counterparts, we replaced the endogenous KV with an acvr1b*induced vesicle (see Experimental Procedures) and determined regional cell shape changes within the induced vesicle during lumen expansion. We found regional cell shape changes to be indistinguishable between the induced and endogenous KVs (Figure S2C), suggesting that the induced vesicle has the same morphogenetic competence as the endogenous KV. We then analyzed how KV cells change shape when induced in ectopic locations of the embryo. Surprisingly, we found that ectopically induced KVs, regardless of their position within the embryo, displayed regional cell shape differences similar to KV in its endogenous location, with cells in one region of KV being more columnar than in the remainder of the vesicle (Figure 2C). However, while in the endogenous KV, the region with the most columnar cells was consistently oriented toward the animal pole of the embryo, there was no such stereotypical orientation detectable in ectopic KVs (compare Figures 1A and 2C). This suggests that global embryo patterning cues along the AP and DV axes do not orient the asymmetry of KV within the embryo. To quantify the asymmetry of ectopic KV in the absence of obvious surrounding landmarks that allowed us to reliably place KV relative to the embryonic AP and DV axes, we determined the maximal cell density ratio obtainable between two halves of the organ, regardless of the actual orientation of the ectopic KV within the embryo (see Experimental Procedures). Strikingly, the maximal cell density ratio was similar in the ectopically induced organs compared to the average ratio observed in endogenously located KVs with similar lumen size (Figure 2D). Together, these observations suggest that there are local rather

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Figure 1. Lumen Growth Drives Cell Shape Change within KV (A) Coronal sections of KV in a Tg(sox17:LMA-tdTomato) embryo, which expresses plasma membrane-targeted tdTomato in KV cells and was injected with h2afva-tagBFP mRNA to label cell nuclei; upper panel, 1 ss (10.3 hpf); lower panel, 6 ss (12 hpf). (B) Sagittal sections of sequential time points during KV formation in a Tg(sox17:GFP; actb2:LMA-tdTomato) embryo starting at 1 ss (10.3 hpf). (C) KV nuclei density distribution along the AP and DV axes during KV formation in a Tg(sox17:GFP) embryo injected with h2afva-mCherry and LMA-tagBFP mRNA at 2 ss (10.6 hpf; upper plot) and 6 ss (12 hpf; lower plot). (D) KV nuclei density ratio between the AD and PV quadrants of KV between 1 ss and 10 ss (10.3–14 hpf); mean ± SEM; n = 11 embryos; data were polled from Tg(sox17:GFP) and Tg(sox17:LMA-tdTomato) embryos. (E and F) In (E), a lateral nuclei density plot is shown of KV in a Tg(sox17:GFP) embryo injected with h2afva-mCherry and LMA-tagBFP mRNA at the 2 ss (10.6 hpf) showing the color code used in (F) to plot changes in cell position along a normalized AP axis between 2 ss and 6 ss (10.3–12 hpf). (G) KV nuclei density ratio between the AD and PV quadrants as a function of average KV cell apical surface in 1 ss to 10 ss embryos (10.3–14 hpf; mean ± SEM); n = 11 embryos; data were polled from Tg(sox17:GFP) and Tg(sox17:LMA-tdTomato) embryos. (H) Average KV cell apical surface in control (n = 31 time points), ouabain-treated (n = 14 time points), and IBMX + forskolin-treated (n = 7 time points) Tg(sox17:GFP) embryos; data were polled from 5 ss to 7 ss embryos (11.6 - 12.5 hpf). (I) KV nuclei density ratio between the AD and PV quadrants in control (n = 31 time points), ouabain-treated (n = 14 time points), and IBMX + forskolin-treated (n = 7 time points) Tg(sox17:GFP) embryos; data were polled from 5 ss to 7 ss embryos (11.6–12.5 hpf). (legend continued on next page)

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than global cues patterning KV in both ectopic and endogenous locations. To identify potential structures that locally pattern KV, we analyzed in detail the morphology of cells surrounding the ectopic KVs. Interestingly, we found cellular structures directly adjacent to the most columnar KV cells that were highly reminiscent of a notochord. To test whether these structures indeed were formed by notochordal cells, we analyzed the expression of notail-a (ntl-a), a gene expressed in notochord precursor cells, using ntla:CFP transgenic embryos. We found in all analyzed cases that the most columnar cells within the ectopically formed KV were adjacent to ntla:CFP-expressing notochordal cells, reminiscent to the situation in endogenous KVs (Figure 2E). This points at the notochord as a structure potentially capable of locally inducing KV morphological asymmetry. To investigate whether the notochord is, indeed, conferring KV asymmetry, we attempted to induce ectopic KV in the absence of adjacent notochordal cells. Analysis of the notochordal structures adjacent to the ectopically induced KV revealed that the notochordal cells were typically not expressing Acvr1b* (Figure 2E). This suggests that they are secondarily induced by ectopic Nodal signals emanating form the Acvr1b*-expressing KV cells, given that Nodal signaling can induce the expression of its own ligands (Meno et al., 1999). To block secondary notochord induction and, thus, uncouple KV from notochord induction, we thus decided to induce ectopic KV in maternal-zygotic one eye pinhead (MZoep) mutant embryos defective in Nodal signal reception. Consistent with our assumption that notochordal cells next to ectopic Acvr1b*-expressing KV cells in wild-type embryos are secondarily induced by Nodal signals emanating from those cells, we found that mosaic expression of Acvr1b* in MZoep mutant embryos expressing the notochord fate marker ntla:CFP typically led to the induction of ectopic KVs without adjacent notochordal cells (Figure S2D). Strikingly, in such instances, KV did not display any cell shape differences as those seen in the endogenous KV where cells in one region of KV remained more columnar than in the remainder of the vesicle (Figure 2F). Accordingly, in all cases where an ectopic KV without an adjacent notochord was induced, the maximal cell density ratio was significantly lower than in cases where an adjacent notochord was formed, suggesting that the notochord is critical for KV morphological asymmetry (Figure 2H). In very rare cases, however, we also found notochordal cells next to the ectopic KV in MZoep mutant embryos (Figure S2D). Unlike the situation in wild-type embryos, these notochordal cells were expressing Acvr1b* themselves, suggesting that Acvr1b* expression triggered KV and notochord cell fate specification in adjacent cells. Interestingly, KV cells adjacent to the notochord in MZoep mutant embryos remained columnar (Figure 2G), and the maximal cell density ratio was higher than in cases without notochord (Figure 2H). This suggests that MZoep mutant cells expressing Acvr1b* are, in principle, competent to display regional cell shape differences in the presence of adjacent notochordal cells. To further exclude that the lack of endogenous

Nodal signaling in Acvr1b*-expressing MZoep mutant cells interferes with their morphogenetic competence to form KV, we treated wild-type embryos with the Nodal-receptor inhibitor SB505124. Consistent with previous findings that Nodal signaling is required for DFC specification (Essner et al., 2005), we found that blocking Nodal signaling at pregastrula stages (4 to 10 hr postfertilization [hpf]) efficiently interfered with DFC specification (Figure S2E). In contrast, when treating embryos with Nodal-signaling inhibitors from bud stage (10 hpf) onward, we did not detect any changes in KV morphogenesis (Figure S2F), indicating that endogenous Nodal signaling has no major function in KV morphogenesis. Collectively, these findings suggest that the notochord is the structure locally inducing regional cell shape differences in KV. To further test whether the notochord has an instructive function in patterning KV, we sought to determine the effect of putting a notochord next to posterior KV cells, which normally display a squamous morphology. To this end, we induced a second notochord in a wild-type embryo by injection of Acvr1b* in one marginal blastomere at the 16-cell stage and then selected embryos in which the duplicated axis was not accompanied by the induction of ectopic DFCs (Figure 2I). In several such cases, where the induced secondary notochord was in contact with posterior KV cells at the end of epiboly, the apical surface size of those posterior cells—and, consequently, cilia density—became similar to that of cells facing the primary notochord (Figures 2J, 2K, S2G, and S2H; Movie S3). Together, these findings suggest that the notochord provides instructive signals inducing regional cell shape differences and cilia distribution within the forming KV. Notably, the relative position of the notochord and laterality organ is a feature conserved among different vertebrates (Blum et al., 2009; Lee and Anderson, 2008), pointing to the intriguing possibility that the function of the notochord in regulating KV morphology constitutes a common mechanism in vertebrate LR patterning. The Notochord Induces Regional KV Cell Shape Differences by Locally Accumulating ECM ECM has recently been shown to regulate epithelial cell shape and lumen formation by influencing the extracellular physical environment. As KV cells are expressing ECM receptors (Ablooglu et al., 2010) and several ECM components accumulate at the notochord surface, we hypothesized that the notochord might induce cell shape differences within KV by triggering ECM deposition close to the AD region of KV. Analyzing laminin-a1b1g1 and fibronectin (FN)-1 deposition around KV showed that these components strongly accumulated at the axial-paraxial boundary next to the more densely packed KV cells in AD regions of the vesicle (Figures 3A–3D). Moreover ECM deposition around ectopic KV was associated with the presence of a coinduced notochord (Figures 3E and S3B). This correlation suggests a functional link between the position of the notochord, ECM accumulation, and cell shape changes within KV.

(J) Phenotypic classes defined for scoring the lateralization of spaw expression by in situ hybridization between 18 ss and 22 ss (18–20 hpf). (K) Quantification of the lateralization of spaw expression in control (n = 77 embryos), ouabain-treated (n = 24 embryos), and IBMX + forskolin-treated (n = 33 embryos) Tg(sox17:GFP) embryos. A, anterior; P, posterior; L, left; R, right; V, ventral; D, dorsal; An, Animal pole; Veg, vegetal pole; ctrl, control. Scale bars in (A) and (B), 20 mm. See also Figure S1.

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Figure 2. The Notochord Affects KV Cell Shapes (A) Single blastomere injection of constitutively active acvr1b (acvr1b*) mRNA at the 32/64-cell stage (1.75/2 hpf) leads to the formation of ectopic KV at the end of gastrulation; mRNA of h2afva fused to a fluorescent protein (FP) is coinjected to label nuclei in the progeny of the injected blastomere. (B) Control (left) and acvr1b*-mRNA-injected Tg(sox17:GFP) embryo at 6 ss (12 hpf); white arrow, endogenous KV; purple arrow, ectopic KV. (legend continued on next page)

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To test whether ECM deposition directly affects KV cell shape, we knocked down laminin-g1, a prominent ECM component accumulating at the axial-paraxial notochord boundary, using previously characterized morpholino antisense oligonucleotides (laminin-g1 MO). In contrast to the laminin-receptor integrin, whose knockdown has recently been shown to interfere with KV epithelial integrity and lumen formation (Ablooglu et al., 2010), interfering with laminin-g1 function had no obvious effects of KV epithelialization and lumen formation (Figures S3A and S3D), allowing us to analyze the function of laminin-g1 in establishing regional cell shape difference within KV. We found that, in laminin-g1 morphant embryos, laminin-a1b1g1 failed to accumulate around KV (Figure S3A), and Myl12.1-EGFP enrichment in the anterior part of KV was reduced (Figure S3C). Analyzing KV asymmetry in morphant embryos further showed that the ratio of cell density between the AD and PV regions of KV was reduced (Figures 3F and S3E). We also found that ciliogenesis was defective in KV cells of laminin-g1 morphant embryos, consistent with previous observations in laminin-b1 mutants (Hochgreb-Ha¨gele et al., 2013). To determine whether the effect on KV cell shape in laminin-g1 morphant embryos was due to a cell-autonomous or nonautonomous function of laminin y1, we injected laminin y1 MOs specifically into the yolk syncytial layer (YSL), previously shown to affect gene expression in KV cells but not other deep cells of the embryo (Amack and Yost, 2004). KV-specific knockdown of laminin-g1 affected ciliogenesis (Figure 3G), consistent with a cell-autonomous function of laminin in ciliogenesis, but had no recognizable effect on regional cell shape differences within KV (Figure 3F). This suggests that laminin-g1 is cell-nonautonomously required for KV cells to adapt regional differences in shape. To test whether interfering with FN deposition has consequences for KV morphogenesis similar to knocking down laminin-g1, we injected mRNA encoding truncated versions of FN-1a and -1b, previously shown to have a dominant-negative function (see Experimental Procedures). Ubiquitous expression

of truncated FN-1a and -1b reduced the ratio of cell density between the AD and PV regions of KV, suggesting that FN, like laminin-g1, is needed for KV cells to adapt regional differences in shape (Figure 3F). However, unlike laminin-g1, interfering with FN in KV cells did not affect ciliogenesis, allowing us to evaluate the role of ECM-dependent regional differences in KV cell shape for the LR symmetry-breaking function of KV (Figure 3H). We found that ubiquitous expression of truncated FN-1a and -1b interfered with the lateralization of spaw expression within the lateral plate (Figure 3I). This, together with our findings on the requirement of regional cell shape differences for the function of KV in breaking LR symmetry, suggests that ECM-dependent cell shape changes are critical for KV function. To further test how ECM affects KV cell shape, we analyzed the response of individual purified KV progenitors when placed on ECM-coated substrates in vitro. We found that these cells changed their shape and reorganized their actomyosin cytoskeleton on ECM-coated substrates in an integrin-dependent manner (Figures S4A–S4C), suggesting that ECM-integrin signaling affects KV cell shape by modulating the actomyosin cytoskeleton. The local deposition of the ECM at the axial-paraxial notochord boundary next to AD KV cells could have either a permissive function, allowing KV cells to acquire regional differences in cell shape, or, alternatively, could directly instruct AD cells to adapt a specific shape. To distinguish between these possibilities, we sought to create an artificial gradient of ECM around KV and test how such gradient would affect the regional distribution of KV cells. To this end, we injected mRNAs encoding the truncated versions of FN-1a and -1b into two-cell-stage embryos in order to perturb FN-1 function in only the left or right half of the embryo (see Experimental Procedures). To validate our approach, we monitored how truncated FN expression locally affects somite formation, a process previously shown to depend on FN function (Koshida et al., 2005). We found that expression of truncated FN-1a and -1b in the left or right

(C) Ectopic KV induced in a Tg(sox17:GFP; actb2:LMA-tdTomato) 6 ss embryo (12 hpf); left panel, 3D rendering; right panel, single plane; arrow points toward the half of the ectopic organ with the highest nuclei density. (D) Maximal nuclei density ratio between organ halves in endogenous (n = 3 embryos) and ectopically (n = 5 embryos) located KV with comparable average cell apical surfaces (between 100 and 120 mm2) in Tg(sox17:GFP) embryo at 6 ss (12 hpf); mean ± SEM. (E) Endogenous (left panel) and ectopic (right panel) KVs in Tg(Ola.Actb:Has.HRAS-EGFP; -1ntla:CFP) embryos at 6 ss (12 hpf); arrow points toward the half of the organ with the highest nuclei density. (F) Ectopic KVs in MZoep; Tg(Ola.Actb:Has.HRAS-EGFP) embryos at 6 ss (12 hpf) located between the yolk and the epiblast (left panel) and between the enveloping layer and the epiblast (right panel). (G) Ectopic KV in a MZoep; Tg(Ola.Actb:Has.HRAS-EGFP) embryo at 6 ss (12 hpf) adjacent to a coinduced notochord (labeled by H2afva-mcherry, yellow dashed line); arrow points toward the half of the ectopic organ with the highest nuclei density. (H) Maximal nuclei density ratio between organ halves in ectopic KV at 6 ss (12 hpf) in control Tg(sox17:GFP) (n = 5 embryos), MZoep; Tg(Ola.Actb:Has.HRASEGFP) (n = 4 embryos), and MZoep; Tg(Ola.Actb:Has.HRAS-EGFP) with coinduced notochord (n = 2 embryos) embryos with comparable average cell apical surfaces (between 100 and 120 mm2); mean ± SEM; *p < 0.05 (Mann-Whitney test). (I) Single marginal blastomere injection of acvr1b* mRNA at the 16-cell stage (2 hpf) leads to the formation a secondary axis, whose notochord contacts the posterior side of endogenous KV at the end of gastrulation; upper panel schematizes a gastrulating embryo with both endogenous and induced axes; lower left panel shows a Tg(sox17:GFP) embryo at 80% epiboly (8.5 hpf) injected with Acvr1b* mRNA at the 16-cell stage (2 hpf) with both primary (purple arrow) and secondary (yellow arrow) axes. (J) KV adjacent to the notochord from both the primary (purple dashed line) and secondary (yellow dashed line) axes in a Tg(sox17:GFP; actb2:LMA-tdTomato) embryo at 6 ss (12 hpf). (K) a-Acetylated tubulin staining of KV in Tg(sox17:GFP) control embryos (upper panels) at 6 ss (12 hpf) and in similar staged embryos with a secondary axis where KV is adjacent to the notochord from both the primary (white dashed line) and secondary (yellow dashed line) axes; panels at the right show the corresponding cilia distribution based on z-projections. An, animal pole; Veg, Vegetal pole; noto, notochord; Ac. Tubulin, acetylated tubulin; ctrl, control. Scale bars for (C), (E), (F), (G), (J), and (K), 20 mm. See also Figure S2B.

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Figure 3. The Notochord Affects KV Cell Shape by Polarizing Laminin Deposition around KV (A) a-Laminin-a1b1g1 antibody staining of KV in Tg(sox17:GFP) embryos at 1 ss (10.3 hpf; coronal section, A.1) and 6 ss (12 hpf; sagittal section, A.2 and A.3); A.4 and A.5 show insets of the region boxed in upper panels. (legend continued on next page)

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half of the embryo severely disrupted somitogenesis in the expressing side (Figure 4A). Notably, we also found that KV cells were differently arranged along the LR axis of the organ, with cell density at the side of KV that was adjacent to surrounding cells expressing truncated FN-1a and -1b being lower than the density of cells on the opposite side (Figures 4B and 4C; Movie S4). These observations support our hypothesis that KV cell shape changes are induced by asymmetric ECM deposition around the organ. Our findings so far suggest that the notochord affects KV cell shape by modulating ECM deposition. To further validate this assumption, we analyzed laminin-a1b1g1 deposition around KV in embryos where both an endogenous notochord and a secondarily induced notochord were in contact with KV (Figures 2I–2K). In such cases, we found ECM accumulating at the border between the notochord and paraxial mesoderm close to both contact sites (Figure 4H), supporting our assumption that the notochord modulates ECM deposition around KV. To determine whether ECM needs to specifically accumulate at the border between notochord and paraxial mesoderm in order to induce regional differences in cell shape within KV, or whether other ECM-rich borders might be equally instructive, we sought to expose the forming KV to an ECM-rich border forming independently of the notochord. To this end, we turned to floating head (flh) mutant embryos, in which the notochord fails to form. In flh mutants, the mesodermal axial-paraxial border is absent, and KV cells are, instead, directly in contact with the ventral side of the posterior neural keel, a border where ECM components, such as laminin-a1b1g1 and FN-1, were also accumulating (Figures 4D and 4E). Our initial analysis of regional differences in KV cell shape in flh mutant embryos turned out to be inconclusive, since the size of KV lumen in mutant embryos was reduced below the level where a morphological asymmetry becomes detectable in wild-type embryos (Figure 4D). Therefore, we attempted to rescue KV lumen growth in flh mutants by treating embryos with IMBX and forskolin to stimulate fluid secretion into KV lumen (Navis et al., 2013). In treated flh mutant embryos, lumen growth was restored (Figure 4F). Further analysis of KV cell shape revealed ectopic regional KV cell shape differences, with the most columnar cells being positioned adjacent to the border between KV and the ventral neural keel (Figure 4F). Accordingly, the ratio of cell density between the side adjacent to the neural keel and the opposing side of KV increased with

lumen size in treated flh mutant embryos and typically aligned with the direction of the maximal cell density ratio (Figure 4G). These findings suggest that ECM components locally accumulating at the border between the ventral neural keel and KV can induce ectopic regional KV cell shape differences in flh mutant embryos. Collectively, our experiments unveil an instructive role of surrounding ECM asymmetry in regulating KV cell shape. Considering this, together with our finding of the nonautonomous requirement of ECM in regulating KV cell shape (Figure 3), we conclude that the notochord induces KV morphological asymmetry by forming a border with the paraxial mesoderm that provides a local source of ECM components close to KV cells in AD regions of the vesicle. EXPERIMENTAL PROCEDURES KV Live Imaging and Morphometric Analysis Dechorionated embryos were mounted in 0.7% low-melting-point agarose and imaged on a Lavision Biotec TrimScope II two-photon microscope with optical parametric oscillator using a Zeiss 20 3 1.0 water-dipping lens (Olympus BX51 WI stand). Live embryos were maintained at 28.5 C during imaging. Image analysis was performed using Imaris (Bitplane) and MATLAB (Mathworks). KV nuclei were annotated and tracked manually, and the tracks were corrected for translational and rotational drift of the organ. The lumen was manually segmented. The result of such annotated data set in Imaris is shown in Figure S1B. KV nuclei coordinates, lumen volume, lumen surface, lumen center of mass coordinates, time intervals, and stages corresponding to the first time point were subsequently exported for analysis. The lumen center of mass was corrected for drift and then used as the origin of the coordinate system. For KV in endogenous locations, the rotation parameters were visually determined using the yolk and notochord as references, followed by data set rotations to align the xy and zy planes with the coronal and sagittal planes. With this, the KV nuclei coordinates were transformed into a KV-specific coordinate system, as illustrated in Figure S1C. For KV in an ectopic location, the rotation parameter was determined to maximize cell density ratio between the organ halves. For KV in flhn1/n1, the rotation parameter was visually determined using the interface of KV cells with the ventral side of the neural keel. For all analyzed cases, nuclei density ratios were calculated and exported along with density plots. Additional information is available in the Supplemental Experimental Procedures. SUPPLEMENTAL INFORMATION Supplemental Information includes Supplemental Experimental Procedures, four figures, and four movies and can be found with this article online at http://dx.doi.org/10.1016/j.devcel.2014.11.003.

(B) Thickness of the KV epithelium [n = 42 measurements on 3 Tg(sox17:GFP) embryos]; box and whisker plot. (C) Basal a-laminin-a1b1g1 antibody staining intensity along KV epithelium in the same sections used for (B); n = 6 lines measurements; see dashed line in (A) for location; box and whisker plot; red line, trend based on the local average method. (D) a-FN-1 antibody and DAPI staining on 6 ss (12 hpf) Tg(sox17:GFP) embryo (coronal section). (E) a-Laminin-a1b1g1 antibody and DAPI staining on ectopic KV in 6 ss (12 hpf) Tg(sox17:GFP) (left panel), MZoep; Tg(sox17:GFP) (middle panel), and MZoep; Tg(sox17:GFP) with a coinduced notochord (arrow in right panel) embryos. (F) Ratio of nuclei densities between the AD and PV quadrants of KV as a function of average KV cell apical surface; mean ± SEM. Control n = 11 embryos; laminin g1-MO1-cell n = 15 embryos; laminin g1-MODFC n = 7 embryos; embryos injected with 40 pg truncated FN 1a and 1b mRNA n = 4 embryos. Data were polled from Tg(sox17:GFP) and Tg(sox17:LMA-tdTomato) embryos between 1 ss and 10 ss (10.3–14 hpf). (G) Projected cilia length in control embryos and embryos injected with laminin g1 MO either into the YSL between the 512- and 1,000-cell stages or at the 1-cell stage. ****p < 0.0001 (t test); n > 140 cilia for each condition; n > 5 embryos for each condition. (H) Projected cilia length in control embryos and embryos injected with 40 pg truncated FN 1a and 1b mRNA; n > 89 cilia for each condition; n > 3 embryos for each condition; ns, p value (t test) > 0.05 (I) Quantification of the lateralization of spaw expression between 18 ss and 22 ss (20–22 hpf) by in situ hybridization in control (n = 18 embryos) and embryos injected with 40 pg truncated FN 1a and 1b mRNA (n = 36 embryos). A, anterior; P, posterior; L, left; R, right; V, ventral; D, dorsal; ns, not significant; YSL, yolk syncytial layer; ctrl, control; trunc. fibro., embryos injected with 40 pg truncated FN 1a and 1b mRNA. Scale bars in (A), (D), and (E), 20 mm. See also Figure S3.

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Developmental Cell The Notochord Shapes Kupffer’s Vesicle

A

B

D

E

F

G

C

H

sox17:GFP

Figure 4. Polarized Distribution of ECM around KV Determines Regional Differences in KV Cell Shape (A) Sections on the dorsal side of a Tg(sox17:GFP) embryo at 6 ss (12 hpf) injected at the two-cell stage with h2afva-mCherry mRNA and 40 pg truncated FN 1a and 1b mRNA. Left panel, DAPI staining; right panel, a-FN-1 antibody staining. (B) Coronal KV section on Tg(sox17:GFP) embryos at 6 ss (12 hpf) injected at the two-cell stage with h2afva-mCherry and 40 pg truncated FN 1a and 1b mRNA. (C) Ratio of cell densities between the mRNA-injected side and the noninjected side; mean ± SEM; data were polled from Tg(sox17:GFP) between the 6 ss and 8 ss (12–13 hpf); p < 0.05, Mann-Whitney test. Control, n = 14 embryos; embryos injected with 20 pg truncated FN 1a and 1b mRNA, n = 6; embryos injected with 40 pg truncated FN mRNA, n = 5. (D) a-FN-1 antibody staining (left) and a-laminin-a1b1g1 antibody staining (right) in 6 ss (12 hpf) flh; Tg(sox17:GFP) embryos. (E) Basal a-laminin-a1b1g1 antibody staining intensity along KV epithelium; n = 6 lines measurements on 3 flh; Tg(sox17:GFP) embryos; box and whisker plot; red line, trend based on the local average method. (F) KV in a flh; Tg(sox17:GFP) embryo at 6 ss (12 hpf) treated with 40 mM IBMX and 10 mM forskolin from 1 ss (10.3 hpf) onward; neural keel side is up. (G) Ratio of cell densities between neural keel and the opposing side as a function of average apical surface (mean ± SEM); data were polled from 3 ss to 7 ss (11– 12.5 hpf) flh; Tg(sox17:GFP) embryos treated with 40 mM IBMX and 10 mM forskolin from 1 ss (10.3 hpf) onward; n = 6 embryos. (H) a-Laminin-a1b1g1 antibody staining of KV adjacent to the notochord (white dashed lines) of both the primary and secondary axes in Tg(sox17:GFP) embryos at 6 ss (12 hpf). A, anterior; P, posterior; L, left; R, right; V, ventral; D, dorsal; crtl; control; trunc. fibro., embryos injected with 40 pg truncated FN 1a and 1b mRNA. Scale bars for (A), (B), (D), (F), and (H), 20 mm.

782 Developmental Cell 31, 774–783, December 22, 2014 ª2014 Elsevier Inc.

Developmental Cell The Notochord Shapes Kupffer’s Vesicle

AUTHOR CONTRIBUTIONS J.C. and C.-P.H. developed the ideas and experimental approaches and wrote the manuscript. J.C. performed the experiments with help from V.B., S.R., R.K., and K.P.-F. V.B. and M.B. generated the Tg(sox17:LMA-Tomato) and Tg(actb2:mCherry-Hsa.UTRN) lines, respectively. ACKNOWLEDGMENTS We are grateful to members of the C.-P.H. lab, M. Concha, D. Siekhaus, and J. Vermot for comments on the manuscript and to M. Furutani-Seiki for sharing reagents. This work was supported by the Institute of Science and Technology Austria and an Alexander von Humboldt Foundation fellowship to J.C.

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Received: April 14, 2014 Revised: October 9, 2014 Accepted: November 4, 2014 Published: December 22, 2014

Okabe, N., Xu, B., and Burdine, R.D. (2008). Fluid dynamics in zebrafish Kupffer’s vesicle. Dev. Dyn. 237, 3602–3612.

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