Abstract
To enable robust patterning, morphogen systems should be resistant to variations in gene expression and tissue size. Here we explore how a Shh morphogen gradient in the ventral neural tube enables proportional patterning in embryos of varying sizes. Using a surgical technique to reduce the size of zebrafish embryos and quantitative confocal microscopy, we find that patterning of neural progenitors remains proportional after size reduction. Intriguingly, a protein necessary for Shh release, Scube2, is expressed far from the source of sonic hedgehog production. scube2 expression levels control Shh signaling extent during ventral neural patterning and conversely Shh signaling represses the expression of scube2, thereby restricting its own signaling. scube2 is disproportionately downregulated in size-reduced embryos, providing a potential mechanism for size-dependent regulation of Shh. This regulatory feedback is necessary for pattern scaling, as demonstrated by a loss of scaling in scube2 overexpressing embryos. In a manner akin to the expander-repressor model of morphogen scaling, we conclude that feedback between Shh signaling and scube2 expression enables proportional patterning in the ventral neural tube by encoding a tissue size dependent morphogen signaling gradient.
Summary Statement The Shh morphogen gradient can scale to different size tissues by feedback between Scube2 mediated release of Shh and Shh based inhibition of Scube2 expression
Author Contributions Z.M.C. conducted experiments and data analysis. Z.M.C and S.G.M. conceived the study, designed the experiments, and wrote the paper. K.I and Z.M.C. developed the size reduction technique. T.Y.C.T helped develop the image analysis technique and generated the tg(shha:memCherry) reporter line. S.G.M. supervised the overall study.
Introduction
When Lewis Wolpert first posed the “French Flag Problem”, he was seeking the answer to this fundamental question: What systems enable proportional patterning in embryos independent of embryo size? By the time Wolpert formalized this problem, developmental biologists had long known that embryos scale their patterning programs in response to changes in embryo size (Wolpert, 1969). For example, sea urchin larva pattern normally from a single blastomere up to the four-cell stage and amphibian embryos can survive bisection and pattern proportionally at a reduced size (Driesch, 1892; Morgan, 1895; Spemann, 1938; Cooke, 1981). Significant scaling of pattern formation to tissue availability seems to be a near universal property of developing organisms. Yet, as we pass the 50-year anniversary of Wolpert’s work, how morphogen gradients scale to pattern domains of varied sizes remains unclear in many systems.
Recent theoretical studies have proposed mechanisms that could account for scaling of morphogen-mediated patterning (Ben-Zvi and Barkai, 2010; Umulis and Othmer, 2013). Amongst the most prominent of these is a model termed expander-repressor integral feedback control (Ben-Zvi and Barkai, 2010). In this model, a morphogen represses the expression of another gene, known as the expander, that affects the range of the morphogen itself cell-non-autonomously. In such models, morphogen signaling will expand until it has reached an encoded equilibrium. This equilibrium is controlled by the morphogen’s repression of the expander, thus enabling “measurement” of the size of the domain in need of patterning. The first biological example of this mechanism was proposed in Xenopus axial patterning. In this original model, ADMP expands BMP signaling by binding Chordin and inhibiting shuttling of BMP towards the ventral side (Francois et al., 2009). However, more recent experimental work implicated another factor, Sizzled, which may play a central role in scaling in a mathematically equivalent manner (Ben-Zvi et al., 2014; Inomata et al., 2013). Expander-like relationships have also been proposed to regulate scaling of Dpp gradients during wing disc growth and even scaling of synthetic patterns in bacterial colonies (Ben-Zvi et al., 2011; Cao et al., 2016; Hamaratoglu et al., 2011).
Though scaling of early axis patterning following size reduction has been extensively studied, the molecular mechanisms through which tissues and organs subsequently scale their patterning has received less attention (Ben-Zvi et al., 2008; Inomata et al., 2013). Previously, scaling of patterning during organ growth has been considered in the fly wing disc, which grows remarkably in size while maintaining proportion (Averbukh et al., 2014; Ben-Zvi et al., 2011; Hamaratoglu et al., 2011). In vertebrates, the developing neural tube has been a powerful model to study morphogen-mediated patterning (Briscoe and Small, 2015). While neural tube patterning does not expand isometrically over time with growth, embryos of different species maintain consistent embryonic proportions in the face of significant variation in organ size during initial patterning (Kicheva et al., 2014; Uygur et al., 2016).
The vertebrate ventral neural tube is patterned by the morphogen Sonic Hedgehog (Shh; Marti et al., 1995; Roelink et al., 1995). Shh is produced by the notochord and floorplate and induces ventral cell fates over a long range in a dose-dependent manner (Briscoe et al., 2001; Zeng et al., 2001). Shh ligands themselves are dually lipid-modified and are highly lipophilic (Pepinsky et al., 1998; Porter et al., 1996a; Porter et al., 1996b). While mechanisms of Shh transport have long been disputed, biochemical evidence supports soluble Shh as a primary component of long-range signaling, and release of Shh ligands from cell membranes is critical for gradient formation (Burke et al., 1999; Chen et al., 2004; Zeng et al., 2001). Shh release was largely thought to be achieved by the protein Dispatched, but recent work has identified Scube2 as a more potent factor in promoting Shh release (Burke et al., 1999; Creanga et al., 2012; Kawakami et al., 2002; Tukachinsky et al., 2012).
Scube2 is a Signal sequence containing protein with a CUB domain and EGF-like repeats. The role of Scube2 in Shh signaling was first identified from work using the zebrafish you mutant which corresponds to scube2 (Hollway et al., 2006; Kawakami et al., 2005; van Eeden et al., 1996; Woods and Talbot, 2005; Yang et al., 2002). Interestingly, while scube2 mutants have defects in ventral patterning, scube2 is predominantly expressed in the dorsal and intermediate neural tube in both mice and zebrafish (Grimmond et al., 2001; Kawakami et al., 2005; Woods and Talbot, 2005). Additionally, epistasis experiments indicated that Scube2 acts upstream of Patched to stimulate Shh signaling (Woods and Talbot, 2005). This effect was also found to be cell-non-autonomous, as mosaic injection of scube2 mRNA was capable of rescuing Shh-signaling defects over a long range (Hollway et al., 2006; Woods and Talbot, 2005). Studies in cell culture then demonstrated that Scube2 releases Shh from secreting cells cell-non-autonomously (Creanga et al., 2012; Tukachinsky et al., 2012). Recent work concluded that Scube2 may be responsible for catalyzing the shedding of lipids from the Shh ligands, but this model is disputed by previous findings that released Shh remains dually lipid-modified (Creanga et al., 2012; Jakobs et al., 2014; Jakobs et al., 2016; Tukachinsky et al., 2012). Scube2’s cell non-autonomous role in Shh release and unexpected expression pattern led us to wonder whether Scube2 may regulate pattern scaling by acting as an expander, as has recently been hypothesized elsewhere (Shilo and Barkai, 2017). In this work, we use quantitative imaging of cell fate specification in zebrafish to investigate the scaling of ventral neural patterning and the regulatory role of Scube2.
Results
Ventral neural patterning scales with embryo size
Studying mechanisms of scaling during growth or between species of different sizes is difficult because many properties of the patterning system depend on stage or species-specific variables. To study scaling of pattern formation in embryos with comparable genetic backgrounds at matched time points, we developed a technique to reduce the size of zebrafish embryos inspired by classical work in amphibians, as we recently described (Ishimatsu et al., 2018; Morgan, 1895; Spemann, 1938). Two lateral cuts are made across the blastula stage embryo: one to remove cells near the animal pole, to avoid damaging signaling centers crucial to early D-V patterning, and a second to remove yolk near the vegetal pole. (Fig. 1A). With this technique, a significant fraction of embryos pattern normally and develop at a reduced size (Fig. 1B-C).
We measured scaling of neural patterning in size-reduced embryos using quantitative imaging (Megason SG, 2009; Xiong et al., 2013). High-resolution image-stacks of 18-24 hours postfertilization (hpf) stage matched zebrafish embryos were collected under identical settings, during the same imaging session, from matched Anterior-Posterior positions in control and experimentally perturbed embryos (Fig. 1D-E). Imaging volumes were analyzed with custom built software to manually demarcate the dorsoventral axis and width of the neural tube along the length of the dataset (Fig. 1F, Fig. S1). Image intensity values were extracted in a set number of bins along the D-V axis for the left and right halves of the neural tube to normalize for variability in neural tube height. This system allowed for the quantitative and unbiased comparison of 3-4 somite lengths of neural imaging data from multiple embryos.
Using this imaging platform, we compared the expression of patched2—a direct transcriptional target of Shh—with the tg(ptch2:kaede) reporter in wild type and size-reduced embryos (Fig. 1G-J) (Huang et al., 2012) . When quantified relative to their respective neural tube dorsal-ventral heights, tg(ptch2:kaede) response gradients maintained nearly identical intensity distributions despite neural tube height being 15.0% (+/- 2.8%) smaller in size-reduced embryos in this dataset (N=5), indicating that Shh responses scale following size reduction (Fig. 1I-J). When viewed on an absolute scale, control and size-reduced embryos show clear shifts in the response gradient as measured by the position at which 50% of mean control maximum intensity is reached (p=0.0076) (Fig. 1J). To quantify this effect at the level of cell fate specification, we utilized a triple transgenic imaging strategy based on reporter lines marking nkx2.2a (p3 progenitors), olig2 (pMN and some p3 progenitors), and dbx1b (p0, d6 progenitors) (Fig. 1K-L) (Gribble et al., 2007; Jessen et al., 1998; Kinkhabwala et al., 2011; Kucenas et al., 2008). Anterior-posterior averaged intensity profiles were then segmented to form cell fate profiles (see methods and Fig. S2). Using this method, we generated cell fate profiles which can be compared between embryos (Fig. S2). After normalizing for their altered D-V height (which was reduced in this population by 12.2% +/- 2.4% compared to controls), the average of these cell fate profiles of size-reduced embryos were virtually indistinguishable from those of full sized embryos (Fig. 1M). Furthermore, differences between progenitor domain boundary positions were visible when size normalization was removed (Fig. 1N). Statistically significant shifts in the positions of the p2 and pMN upper boundaries were observed only when compared in their absolute coordinates (Fig. 1N). This further demonstrates that ventral neural patterning adjusts to changes in total D-V height.
Scube2 levels control Shh signaling
Based on its role in the cell-non-autonomous regulation of Shh release and its dorsal expression pattern, we hypothesized a potential role for Scube2 in enabling scaling of Shh gradients. This hypothesis depends on scube2 expression levels having a dose dependent effect on Shh signaling. However, previous work concluded that Scube2 is only required for Shh signaling as a permissive factor (Kawakami et al., 2005; Woods and Talbot, 2005). To examine the role of Scube2 in ventral neural patterning, we performed a morpholino knockdown of scube2 in tg(ptch2:kaede) reporter embryos using a previously validated translation inhibiting morpholino (Fig. 2A-C) (Woods and Talbot, 2005). We observed a decrease in Shh signaling following morpholino injection, as demonstrated by a statistically significant suppression of maximum ptch2:kaede intensity (Fig. 2C)(Woods and Talbot, 2005)(Woods and Talbot, 2005)(Woods and Talbot, 2005)(Woods and Talbot, 2005)(Woods and Talbot, 2005). Additionally, quantification of nkx2.2a, olig2, and dbx1b domain sizes in embryos injected with scube2 morpholino showed a contraction of ventral progenitor domains (Fig. 2D-F). Ventral shifts in the upper boundaries of the pMN and p3 domains were statistically significant, due in part to near complete elimination of the nkx2.2a+ p3 domain (Fig. 2F). Unexpectedly, expansion of the p1-2 domain dorsally was also observed, potentially implying long range Shh signaling is required for complete p1-2 induction.
Previous work concluded that Scube2 was a permissive factor based on a lack of ectopic expression of downstream Shh signaling markers as observed by whole mount in situ hybridization experiments (Woods and Talbot, 2005). However, our high-resolution quantitative imaging reveals that injection of scube2 mRNA leads to the expansion of Shh signaling, as shown by broader distributions of tg(ptch2:kaede) fluorescence (Fig. 2G-I). Embryos injected with scube2 mRNA showed significant dorsal shifts in the position of half control maximum tg(ptch2:kaede) intensities (p=0.0047), a measure of absolute Shh signaling range (Fig. 2H). Maximum tg(ptch2:kaede) fluorescence in scube2 overexpressing embryos was somewhat higher on average, but not statistically significant (p=0.0512), which may suggest the stronger effect of scube2 overexpression is to extend the range of Shh signaling. In addition, scube2 overexpression affected cell type patterning in the ventral neural tube, as measured in triple transgenic nkx2.2a, olig2, and dbx1b reporter embryos (Fig. 2J-L). Quantification of these cell fate profiles revealed large increases in p3 and pMN domain sizes, a decrease in the size of the p2-p1 domains, and unchanged patterning of the p0-d6 domains and more dorsal cell types. Ventralization was measured by comparing dorsal boundaries of the p3 and pMN, which were statistically significantly shifted (Fig. 2L). These data indicate that not only is Scube2 required for long range Shh signaling, but that scube2 overexpression amplifies endogenous Shh signaling. Additionally, this suggests that Scube2-stimulated Shh release is a limiting factor in normal patterning.
Shh negatively regulates Scube2 expression over a long-range
To study Scube2’s expression, we developed the tg(scube2:moxNG) reporter line containing 7.6KB of the endogenous regulatory sequences driving the extremely bright moxNeonGreen fluorescent protein (Fig. 3A) (Costantini et al., 2015). The expression of tg(scube2:moxNG) we observed is consistent with previously reported in situ hybridizations (Grimmond et al., 2001; Kawakami et al., 2005; Woods and Talbot, 2005). Tg(scube2:moxNG) embryos showed very low expression close to the sources of Shh in the floor plate and notochord—as visualized with a transgenic shh:memCherry reporter line—and high levels of expression in the dorsal-intermediate neural tube (Fig. 3A-C). Time lapse imaging of tg(scube2:moxNG) embryos revealed weak mesodermal expression in the early embryo, which faded during the onset of neurulation and was replaced by high levels of expression in the dorsal and intermediate neural tube (Fig. 3D-G, Movie S1-2).
To test whether Scube2 is downregulated by Shh signaling, we injected mRNA encoding a potent activator of Shh signaling, dnPKA, at the single cell stage and observed the resulting embryos (Hammerschmidt et al., 1996). Embryos injected with dnpka mRNA showed near complete ablation of neural tg(scube2:moxNG) expression (Fig. 4 A-C). To test whether Shha ligands themselves were capable of suppressing Scube2 expression at a distance, we mosaically overexpressed shha in tg(scube2:moxNG) embryos by injecting a single blastomere at the 16-cell stage with either memmTagBFP2 alone or with shha mRNA (Fig. 4 D-F). We expected local inhibition of Scube2 reporter activity near secreting cells within a few cell diameters. Surprisingly, tg(scube2:moxNG) expression was nearly completely eliminated in these embryos indicating potent cell-non-autonomous repression of Scube2 by Shh. When quantified, these embryos demonstrate a highly significant reduction of peak tg(scube2:moxNG) intensities (Fig. 4F). To test whether Shh’s inhibition of Scube2 is required for its endogenous low ventral expression, we treated embryos with sonidegib, a potent Smoothened antagonist starting at the dome stage. Resulting embryos showed expanded scube2 expression towards the floor plate and notochord (Fig. 4G-I). Shifts in ventral boundaries were quantified by measuring the D-V position at which 50% of the maximum intensity of the control population was reached. These measurements were statistically significantly shifted in sonidegib-treated embryos relative to controls, indicating that endogenous Shh signaling is responsible for a lack of ventral scube2 expression (Fig. 4I). A ventral expansion tg(scube2:moxNG) was also observed following Cyclopamine treatment, which is consistent with the previous findings of a genomewide screen for genes regulated by Shh signaling (Fig. S3) (Xu et al., 2006). To further probe the transcriptional regulation of Scube2’s expression we performed a small scale CRISPR mutagenesis screen and found that Pax6a/b are necessary for driving Scube2 expression. Coinjection of pax6a and pax6b sgRNAs with Cas9 caused significant downregulation of tg(scube2:moxNG) relative to control embryos injected with a sgRNA targeting tyrosinase, an unrelated pigment gene (Fig. S4).
Scube2 diffuses over long distances during patterning
While Scube2 is known to act cell non-autonomously from transplantation experiments and Scube2-conditioned media has a potent Shh release stimulating effect in vitro, the localization of Scube2 protein during development is unknown (Woods and Talbot, 2005; Creanga et al., 2012). In vitro, Scube2 is thought to associate with Heparin Sulfate Proteoglycans, and Scube2 had been hypothesized to diffuse from secreting cells in the dorsal neural tube, the need for which was later disputed (Jakobs et al., 2016; Kawakami et al., 2005; Hollway et al., 2006). To examine Scube2’s localization, we developed Scube2 fluorescent fusion proteins by tagging the C-terminus as previously validated in cell culture with other tags (Fig. 5A) (Creanga et al., 2012). The resulting Scube2-mCitrine fusion proteins were functional and rescued Scube2 CRISPR mutants at comparable rates to wildtype Scube2 (Fig. S5). Mosaic injection of scube2-mCitrine mRNA at the 32-64 cell stage revealed that Scube2-mCitrine diffuses distantly from producing cells (Fig. 5B). Following single cell mRNA injection, Scube2-mCitrine fusions were secreted and did not remain associated with cell membranes, as demonstrated by their presence in the extracellular space between cells marked with mem-mCherry (Fig. 4C).
To assay Scube2’s rate of diffusion we performed Fluorescence Recovery After Photobleaching (FRAP) at the dome stage, during which cell movement is minimal. FRAP was performed in a 100μm x 100μm region and recovery was observed at 10 second intervals over 5 minutes (Fig. 5 D-E, Movie S3-4). Image data from the bleached region was then normalized to its initial intensity and fitted to recovery curves using standard methods (Munjal et al., 2015). We find that the addition of Scube2 to mCitrine causes only minor changes to its diffusion (Fig. 5F-G). No significant differences were observed between the calculated mobile fractions of Scube2-mCitrine (0.283 +/- 0.018) and Sec-mCitrine (0.338+/- 0.019; unpaired t-test p=0.0698). When the time to 50% recovery of the mobile fraction is calculated, Scube2-mCitrine has modestly slower diffusion time than Sec-mCitrine alone (Fig. 5 F-G; p=0.0405). These data further support Scube2’s diffusion in the extracellular space, which likely mediates its long range of effect.
To observe distributions of Scube2 during development, we generated a transgenic line expressing the full length Scube2 protein fused to moxNeonGreen under control of Scube2 regulatory sequences (Fig. 5H-K). We validated the functionality of this Tg(scube2:scube2-moxNG) line using a morpholino which bound only endogenous Scube2 at the splice junction of exon6, and not Tg(scube2:scube2-moxNG) derived RNA which lacks this splice junction (Figure S6). Tg(scube2:scube2-moxNG) embryos were markedly resistant to treatment with this morpholino, validating the in vivo functionality of this construct (Figure S6). Tg(scube2:scube2-moxNG) embryos showed broad distributions of Scube2 during patterning (Fig. 5H-K). Throughout early patterning Scube2-moxNeonGreen is visible near ventral cells marked by tg(shha:mem-mCherry), although Scube2 is expressed largely in the dorsal neurectoderm at this timepoint (Fig. 5H-I, Movie S5-6). By 24 hpf tg(scube2:scube2-moxNG) fluorescence is found distributed throughout the embryo, although expression from tg(scube2:moxNG) is localized to the dorsal-intermediate neurectoderm. These data further suggest that Scube2’s long range of effect can be explained by diffusion from secreting cells in the intermediate and dorsal neural tube to the source of Shh in the floor plate and notochord.
Feedback regulation of Scube2 levels is necessary for pattern scaling
To examine the regulation of Scube2 in size-reduced embryos, we performed our size reduction technique on tg(scube2:moxNG; shha:mem-mCherry) embryos and imaged them at 20 hours post fertilization. Unlike other observed patterning genes, scube2 expression levels did not scale in size-reduced embryos but were instead severely reduced (Fig. 6A-C). This finding is consistent with an expander-repressor-like model of Scube2-Shh. In this regime, inhibition of scube2 expression in size-reduced embryos would contract Shh signaling, enabling adjustment of Shh signaling for a decreased tissue size (Fig. 6D-E).
Next, we examined whether feedback control of scube2 expression levels by Shh signaling is required for pattern scaling by saturating scube2 levels by over-expression. If Scube2 is responsible for adjusting Shh signaling during scaling, we would expect scube2-overexpressing size-reduced embryos to have the same absolute Shh response profiles as controls, which would fail to scale following size normalization (Fig. 6F-G). If scaling of ventral patterning is not dependent on Scube2, we would expect maintenance of pattern scaling with size-proportionate increases in ptch2:kaede distributions in both populations. We overexpressed scube2 by mRNA injection in ptch2:kaede reporter embryos. When normalized for differences in D-V heights, size-reduced scube2-overexpressing embryos showed a disproportionate expansion of the Ptch signaling gradient compared to normal-sized scube2-overexpressing embryos (Fig. 6H-K). Dorsal expansion of Shh signaling is quantified using the position of 50% of average maximum control intensity, which is statistically significantly shifted dorsally in size-reduced embryos (Fig. 6J). Importantly, when tg(ptch2:kaede) response profiles are plotted on an absolute rather than relative scale, they nearly exactly overlap (Fig. 6K). This overlap without size normalization suggests that scube2 overexpression encodes a response profile which is independent of embryo size and is not secondarily tuned by another scaling related factor. This strongly suggests that control of scube2 expression levels is required for scaling the Shh response gradient.
Discussion
Our work uncovers that the morphogen Sonic Hedgehog can self-regulate to enable scale-invariant patterning through linking morphogen signaling to inhibition of Scube2. We discovered that patterning of the neural tube adjusts to tissue availability following surgical size reduction in zebrafish embryos. Using overexpression experiments we demonstrate that Scube2’s activity during patterning is not just permissive—overexpression of scube2 quantiatively enhances Shh signaling (Woods and Talbot, 2005). Utilizing a transgenic reporter line which we developed, we characterized the expression of Scube2 during neural patterning and found that Shh signaling is responsible for its repression in the ventral neural tube. Using Scube2 fluorescent fusion proteins we found that Scube2 is broadly distributed from secreting cells, explaining its previously reported cell non-autonomous activity (Creanga et al., 2012; Woods and Talbot, 2005). Unlike other patterning genes, scube2 responds to changes in neural tube height by disproportionately decreasing its expression, and overexpression of Scube2 inhibits scaling of the Shh signaling gradient by circumventing its feedback control. The expression of scube2 thus can be seen as comparable to the “size-dependent factor” Sizzled, which is thought to enable scaling in early D-V patterning by tuning its expression levels to embryo size by chordin-dependent feedback inhibition (Inomata et al., 2013).
The relationship between Scube2 and Shh has important similarities to proposed “expander-repressor” models of morphogen scaling (Barkai and Ben-Zvi, 2009; Ben-Zvi and Barkai, 2010; Inomata et al., 2013). As with expanders in these models, scube2 enhances morphogen range, is repressed by morphogen signaling, and acts cell non-autonomously at a distance from its source. However, Scube2’s reported role in morphogen release may be distinct from the proposed mechanism of expanders. Expanders extend the range of morphogens by promoting their diffusion or inhibiting their degradation (Ben-Zvi and Barkai, 2010). While release of Shh ligands from secreting cells would support their transport, the potentially irreversible nature of this effect and local action at the morphogen source would make distinct predictions for Scube2’s effects on morphogen distributions. Nonetheless our study marks the first observation of an expander-repressor-like relationship outside of the BMP/Dpp signaling pathway in a developing organism. This finding raises the possibility that expander-repressor-like relationships may be common motifs in the regulation of morphogen gradients.
We began this work in part due to interest in the discrepancy between the area of Scube2’s activity in the ventral neural tube and its expression in the dorsal neural tube. Our work with Scube2 fluorescent protein fusions revealed that Scube2 is diffusive and is distributed broadly from producing cells. Scube2’s diffusion from producing cells could easily account for the distance between its expression domain and area of effect (Figure 5). Scube2’s broad distribution and considerable extracellular diffusivity bolsters the hypothesis that Scube2 may serve as a chaperone for Shh during its transport as hypothesized previously (Tukachinsky et al., 2012). Cell culture experiments have indicated that Scube2 cooperates with Dispatched in a cholesterol-dependent “hand off” by binding different domains on Shh’s cholesterol moiety (Tukachinsky et al., 2012). Continued binding of Scube2 to the hydrophobic sterol domain may facilitate the un-hindered diffusion of Shh through the extracellular millieu. This model is consistent with the dose dependency we observe in our Scube2 overexpression experiments (Figure 2G-L) and may help solve the puzzle of the long-range transport of dually lipid modified hedgehog in vertebrates. Further investigation of the strength and duration of Scube2 and Shh’s binding in vivo may shed light on this relationship. Unfortunately, direct imaging of this phenomenon is hampered by the lack of fully functional Shh fluorescent protein fusions (Chamberlain et al., 2008).
While some evidence suggests that Scube2 plays a role in lipid-shedding, these observations conflict with previous HPLC analysis and the findings of independent groups which demonstrated that Shh species released by Scube2 are dually lipid modified (Creanga et al., 2012; Tukachinsky et al., 2012). If correct, a model of Scube2 in which it acts only transiently at the cell surface of producing cells—either by enabling the formation of multimeric Shh complexes or lipid shedding—would have interesting implications for its role as an expander. Expanders are often formalized as having a dose dependent reversible effect on morphogen spread, while a transient role of Scube2 in Shh multimeric complex formation or shedding would be localized and irreversible. Mathematical modeling may reveal interesting implications of each proposed mechanism in Scube2-Shh feedback interactions during pattern scaling.
Scube2 is one of several recently identified elements of the Shh signaling pathway that exerts cell non-autonomous effects. Recent work has shown that Hhip—initially characterized as a membrane-tethered hedgehog antagonist—acts over a long range that cannot be explained by ligand sequestration (Kwong et al., 2014). Additionally, the Hedgehog receptor, Patched, may also have cell non-autonomous inhibitory effects on Smoothened through regulating inhibitory sterols or sterol availability (Bidet et al., 2011; Roberts et al., 2016). Together with known feedback relationships and the diffusivity of Scube2 that we demonstrated here, these mechanisms interlink Shh signaling between neighboring cells and may enable tissue level properties, such as the scaling of pattern formation we observed.
However, scaling of neural patterning is unlikely to be achieved by regulation of Shh signaling alone. BMP signaling in the dorsal neural tube is known to pattern dorsal progenitors. Scaling of BMP signaling in dorsal neural patterning may be achieved via a similar or carry-over mechanism to early DV axis patterning and should be explored in further studies. In the early D-V patterning system, both existing models propose expander-like relationships between elements of the BMP signaling pathway. The first model proposed ADMP as a scaling related factor, while more recent research has demonstrated that Sizzled has an indispensable role in scaling (Ben-Zvi et al., 2008; Ben-Zvi et al., 2014; Inomata et al., 2013). During neural patterning, the BMP antagonists Noggin, Follistatin, and Chordin are expressed in the notochord while BMP ligands are expressed in the roof plate. Intriguingly, while Sizzled does not seem to be expressed during neural patterning, ADMP is expressed in the notochord and thus may play a role in the scaling of BMP-mediated patterning of the dorsal neural tube (Willot et al., 2002).
BMP signaling is known to increase the thresholds for Shh-dependent cell fate specification, making signaling integration between these pathways a potential additional candidate regulator of scaling (Liem et al., 2000; McHale et al., 2006). Inhibition of either Shh or BMP signaling causes expansion of signaling by the alternative program. In normal patterning, cells do not measure ratios of BMP and Shh. In fact, Dbx1 positive progenitors in the medial neural tube require little to no Shh or BMP signaling present in order to be specified (Pierani et al., 1999). In addition, recent experiments with precise control of Shh and BMP concentrations in an explant system have shown that cells choose either ventral or dorsal fates in the presence of significant BMP and Shh signaling (Zagorski et al., 2017). Regulation of scube2 expression may be another way to enable crosstalk between signaling pathways, as scube2 is not expressed in the dorsal most cells of the spinal cord, suggesting repression by dorsal factors. Specification of the dorsal boundary of scube2 expression may encode yet more information about the size of the tissue which would then affect Shh spread.
Methods
Generation of Transgenic Lines
The construct used to make tg(scube2:moxNG) was generated by isothermal assembly of PCR-amplified scube2 regulatory elements obtained from the CHORI-211 BAC library. Regulatory elements were in part chosen based on annotations of H3K4me1 and H3K4me3 binding (Aday et al., 2011). Selected regulatory sequences spanned 1677bp of upstream intergenic sequence and 5962bp of the area spanning exons 1-5 scube2. Regulatory sequences were cloned into a pMT backbone by placing a zebrafish codon-optimized moxNeonGreen fluorescent protein and sv40 poly-A tail just downstream of the endogenous scube2 Kozak sequence (Costantini et al., 2015). The construct used to make tg(scube2:scube2-moxNG) was generated using the same regulatory sequences as tg(scube2:moxNG), with the addition of cDNA corresponding to exons 6-23 of the Scube2 coding sequence downstream of exon 5 and moxNeonGreen attached at the c-terminus with a 10 amino acid long GA rich linker. The construct used to make tg(shh:mem-mCherry) was derived from the previously reported tg(shh:GFP), by replacement of GFP with mem-mCherry (Megason, 2009; Shkumatava et al., 2004).
Transgenic lines were generated by injecting plasmid DNA for each construct along with Tol2 mRNA into wild type (AB) embryos at the single cell stage, as described previously (Kawakami, 2004). moxNeonGreen positive embryos were then selected for raising. Upon reaching sexual maturity, F0s were outcrossed and screened for founders. Founders were isolated and raised as single alleles. Monoallelic versions of each line are shown throughout the paper.
Zebrafish Strains
For wild type lines, AB fish were used. All fish were kept at 28°C on a 14-hour-light/10-hour-dark cycle. Embryos were collected from natural crosses. All fish-related procedures were carried out with the approval of Institutional Animal Care and Use Committee (IACUC) at Harvard University. tgBAC(ptch2:kaede) (Huang et al., 2012; renamed from ptch1 due to a change in zebrafish gene nomenclature), tg(nkx2.2a:mGFP) (Jessen et al., 1998), tg(olig2:GFP) (Shin et al., 2003), tg(olig2:dsRed) (Kucenas et al., 2008), and tgBAC(dbx1b:GFP) (Kinkhabwala et al., 2011) have been described previously.
Size Reduction Technique
Size reduction was performed as described in our previous report (Ishimatsu et al., 2018). Embryo sizes were reduced by sequentially removing ~1/3 of the cells from the animal cap, then wounding the yolk. These surgeries are performed in 1/3 ringers solution, and embryos are immobilized in a thin layer of 2% methyl cellulose. Surgeries can be performed either with glass needles – as previously described – or using a loop of thin stainless-steel wire that is inserted through a glass capillary tube and mounted on a halved chopstick as done here. Healthy uninjected embryos show a maximum success rate of ~60% while embryos which have undergone injection or were spawned by older females have significantly lower success rates. In each size reduction experiment, embryos are screened for health and the largest size reductions; those with insufficient size reduction or morphological defects are discarded.
Construct Generation and Injections of mRNAs and Morpholinos
Scube2-mCitrine was generated from cDNA obtained from the Talbot lab (Woods and Talbot, 2005). Fluorescent protein fusions were made by attaching mCitrine or moxNeonGreen with a 10 amino acid GA rich linker to the c-terminus of Scube2. Membrane-mTagBFP2 constructs were generated using membrane localization tags reported previously (Megason, 2009; Subach et al., 2011). These constructs were each sub-cloned into a pMTB backbone. mRNA for all experiments was synthesized from pCS or pMTB backbones via in vitro transcription using the mMESSAGE mMACHINE system (Ambion). Embryos were injected at the single cell stage using a Nanoject system set to 2.3nl of injection volume containing 70-90pg of RNA for each mRNA injected. Injected embryos were then screened for brightness, and damaged embryos were removed. Scube2 morpholino injections were performed with 7ng of Scube2 MO2 and 3.5ng of p53 MO to control for phenotypic variability, while control morpholino injections were performed using 10.5ng of p53 MO only (Gerety and Wilkinson, 2011; Woods and Talbot, 2005).
Sonidegib and Cyclopamine Treatment
Stock solution of 1 mM Sonidegib suspended in DMSO was used for treatment as generously given by the lab of Rosalind Segal. Embryos were placed in egg water containing a concentration of 50μM for the treatment condition, and equal parts DMSO were added to the sham control. Cyclopamine was dissolved in 100% ethanol to make 50mM stock solution and was diluted for treatment in egg water to 100 μM. Treatment began at 7 hpf and continued until imaging at 22 hpf.
Confocal Imaging
For quantitative imaging, embryos were staged and mounted in our previously described dorsal mount (Kimmel et al., 1995; Megason, 2009; Xiong et al., 2013) in egg water with 0.01% tricaine (Western Chemical, Inc.). Embryos were manipulated for proper positioning with hair loops, before gently lowering the coverslip. Embryos were not depressed by the coverslip or impinged by the mold, enabling imaging of their normal proportions. Imaging was performed on embryos staged at 18-24 hpf, unless otherwise noted in corresponding figure legends. Live imaging was performed using a Zeiss 710 confocal/2-photon microscope, Zen image acquisition software, and C-Apochromat 40X 1.2 NA objective. For fluorescent protein excitation, 405 nm (BFP), 488 nm (GFP/moxNeonGreen), 514 nm (mCitrine), 561 nm (mCherry/dsRed) and 594 nm (mCardinal) lasers were used. The imaging field was centered in each embryo on the somite 6/7 boundary for consistent positioning between images. For quantitative analysis, imaging datasets are only compared between sibling embryos imaged on the same day with the same settings. This approach aims to avoid clutch effects or variability in detector sensitivity and laser power that occur over time. Typical imaging settings with the 40x objective were as follows: image size of 1024x1024 pixels with .21μm per pixel and an interval of 1μm in the Z direction. For display purposes, images are rendered in cross sectional views (X-Z axis) which are then rotated for display, with image intensities for co-injection markers adjusted evenly within datasets for displayable brightness. FRAP, early stage embryo imaging and time-lapses were performed using a 1.0 NA 20x objective. Brightfield and widefield fluorescence images of whole embryos were obtained using an Olympus MVX10 and a Leica MZ12.5 dissecting microscope.
FRAP Experiments and Analysis
Imaging for FRAP was performed using a 1.0 NA 20x objective at dome stage. Bleaching was performed for two minutes in a 100μmx100μm area in the center of the frame with a 488nm Argon laser. Imaging was performed with a low laser power to reduce bleaching, and images were obtained at 10 second intervals over five minutes to quantify recovery. FRAP data in the bleached region was then normalized to the minimum and maximum intensity for each respective time trace. Normalized recovery intensities were then fitted to the following exponential to determine the mobile fraction (A) in MATLAB: y = A(1−e−τt) (Munjal et al., 2015). These fitted traces were also used to determine the point at which 50% maximum recovery was reached in Figure 5G.
Image Analysis
Images were analyzed using a custom MATLAB-based image analysis software that enables rapid segmentation of neural tube imaging data. Neural imaging data is segmented by the user sequentially from anterior to posterior. Over a set step size (usually 50 pixels), the user selects points at the base of the floor plate cell and top of the roof plate cell that divide the neural tube into its two halves (Fig. S1A). The user then selects the widest point of the neural tube in each image. Imaging data from mature neurons, found laterally, and within the lumen of the neural tube, found medially, are disregarded using a set percentage of neural width (Fig. S1B). Once these positions are recorded, imaging data is then recovered as average pixel intensity in 25 bins from ventral to dorsal across 3-4 somites of A-P length. This binning and averaging strategy enables comparison of data between embryos that accounts for variations in neural tube D-V height. During the segmentation process, the researcher is blinded to the title of the dataset which contains information about its treatment condition. For distribution plots, binned intensities are reported for each embryo as the average intensities for each bin along the entire AP axis of the imaging volume. Each embryo’s average intensity profile is then treated as an independent sample and averaged for displayed distribution profiles and standard deviations. To avoid artifacts caused by rounding in the calculations of half maximum control intensity positional values are extracted from the spline-interpolated intensity profiles for each individual dataset.
Progenitor domain segmentation is performed on average intensity profiles from each embryo in a dataset in the following manner: first, all intensity profiles in the data set undergo background subtraction and cross channel fluorescence caused by the extreme brightness of the dbx1b:GFP line is removed from the olig2:dsred channel. Intensity profiles are then fed to a peak finding algorithm to identify local maxima. Both dbx1b+ and nkx2.2a+ progenitor domains are found in the green channel, so a maximum of two peaks is allowed. In the red channel, only one peak is specified to identify olig2:dsred signal. Average peak intensity values for each domain are then calculated for the entire control dataset, and 50% of this value in the case of the nkx2.2a and dbx1b domains is used as the threshold for calculating domain width. Given its greater spread along the D-V axis, a threshold of 25% of peak height is used in calculating width of the olig2+ domain. Domain widths are then extracted from spline-interpolated intensity profiles to avoid errors introduced by rounding to the next bin. Segmented widths and positions of nkx2.2a, olig2, and dbx1b expression are then averaged for plotting purposes. Domain plots are generated by assigning all nkx2.2a+ progenitors to the p3 fate, olig2+ progenitors lacking nkx2.2a expression overlap to the pMN fate, and dbx1b+ progenitors to the p0-d6 fate. These domain sizes and positions are then used to reconstruct domains in-between or flanking them, which include the p2-p1 domain between pMN and p0-d6, the floorplate below p3, and the d5-roofplate above p0-d6. These heights and positions are then used to generate the stacked bar plots shown.
Statistical Analysis
Statistical comparisons of maximum average intensity and position of 50% maximum intensity are performed by an unpaired T-test. Although each dataset contains hundreds of measurements of each binned intensity value over the A-P axis of a z-stack, only the average of these measurements for each embryo is treated as a data-point for calculation of the standard deviation and statistical significance tests. This is done to avoid oversampling that would exaggerate statistical significance. In all measurements, statistical significance is markedly increased if analysis is performed by treating all underlying intensity measurements as samples. Thresholds for calculating the position of half maximum are determined from the average maximum of the corresponding control dataset for each experiment. Position is then determined from the fitted trend-line to avoid inaccuracies due to rounding. To calculate the significance of shifts in boundary positions, upper domain boundaries for each embryo were compared in an un-paired t-test between embryos from each population. When the progenitor domain segmentation algorithm finds there is no domain present, the boundary is set to 0.
CRISPR Screen for Scube2 Regulators
Cas9 protein was generated and purified in lab as described (Gagnon et al., 2014). Three guide RNA sequences targeting the first one-to-three exons of each gene were selected based on their quality using the web-tool CHOP-CHOP and synthesized using standard methods (Gagnon et al., 2014). Equivalent guide RNA and Cas9 protein concentrations were used in all samples for mosaic knockout. Phenotypes were assessed at 18-20 hpf by confocal microscopy.
Competing interest
The authors declare no competing or financial interests.
Funding
Z.M.C was supported in part by the program in Biological and Biomedical Sciences at Harvard University. S.G.M. and Z.M.C. were supported by R01-GM107733 and R01-DC015478. T.Y.-C.T. was supported by the Damon Runyon Cancer Foundation Fellowship.
Supplemental Movies
Movie S1- tg(scube2:moxNG; shh:mem-mCherry) timelapse transverse view
Movie S2- tg(scube2:moxNG; shh:mem-mCherry) timelapse maximum intensity projection dorsal view
Movie S3- Scube2-mCitrine FRAP imaging in a dome stage embryo
Movie S4- Sec-mCitrine FRAP imaging in a dome stage embryo
Movie S5- tg(scube2:scube2-moxNG; shh:mem-mCherry) timelapse transverse view
Movie S6- tg(scube2:scube2-moxNG; shh:mem-mCherry) timelapse maximum intensity projection dorsal view
Acknowledgments
We thank Lisa Goodrich, Rosalind Segal, and Wolfram Goessling for their comments and helpful discussion. The Scube2 construct was a gift from the Talbot lab.