The application of data-driven approaches to relevant problems in developmental biology depends on high quality input datasets of the tissue of interest. A data-driven analysis of cellular architecture thus requires high-resolution imaging followed by automated segmentation of individual cells. As developing embryos are large 3D specimens, it is beneficial to use a microscopy method that allows optical sectioning of defined axial z-planes. Here, we employed AiryScan FAST mode confocal microscopy (Huff, 2016) to achieve high signal-to-noise ratios and high axial resolution – both of which greatly facilitate 3D segmentation – whilst maintaining a sufficiently high acquisition speed for live imaging of a migrating tissue (~20 s/channel). In this way, we acquired a large set of single and dual channel volumes of wild-type primordia during migration, staged such that the pLLP is located above the posterior half of the embryo's yolk extension (32-36hpf, N = 173 samples in total). All of our samples expressed the bright cell membrane label cldnb:lyn-EGFP (Haas and Gilmour, 2006; Figure 2A, Figure 2—video 1) and many additionally expressed a red or far-red label for an internal cellular structure (see table in Supplementary file 1 for a complete overview).

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To segment cells, we combined commonplace image processing algorithms into a specialized automated pipeline that uses labeled membranes as its sole input (see materials and methods for details). We found that our pipeline reliably produces high-quality segmentations (Figure 2B, Figure 2—video 2) and that erroneously split or fused cells are rare. To further ensure consistent segmentation quality, each segmented stack was manually double-checked and rare cases of stacks where more than approx. 10% of cells had been either missed, split or fused were excluded (8 of 173; 4.62%). An expanded visualization of a representative segmentation (Figure 2C, Figure 2—video 3) allows the diversity of cell shapes in the lateral line and their relationship with overall tissue architecture to be appreciated in a qualitative fashion.

Overall, we collected n = 15,347 segmented cells from N = 165 wild-type primordia as the basis for our quantitative analysis. Unless otherwise specified, the results presented in this study are based on this dataset.

Most data science techniques require input data in a specific form, namely a feature space, which consists of a vector of numerical features for each sample and hence takes the shape of a 2D samples-by-features array (see Figure 1C). Even when segmented, confocal volumes of cells do not conform to this standard. Thus, numerical features must be extracted from volumetric image data. This can be achieved by simply measuring specific aspects of each cell such as volume or eccentricity (feature engineering) but for exploratory analysis it is preferable to derive an unbiased encoding of 3D image information into a 1D feature vector (feature embedding). This non-trivial task has previously been tackled in a number of ways (Pincus and Theriot, 2007; Peng and Murphy, 2011; Rajaram et al., 2012; Tweedy et al., 2013; Kalinin et al., 2018), however no readily applicable solution for feature embedding of both cell morphology and subcellular protein distributions from segmented 3D images has been described to our knowledge.

We therefore developed a novel method that allows feature embedding of arbitrary fluorescence intensity distributions. As a starting point, we took a standard workflow from the field of geometric morphometrics, which consists of four steps (Adams et al., 2013; Figure 3A):

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(1)conversion of image data – which is dense and regularly spaced in voxels – into a sparse point cloud of landmarks to facilitate computational transformations, (2) alignment of point clouds by registration to remove translational and rotational variance and hence retrieve pure shape information, (3) re-representation of landmark coordinates based on their deviation from a consensus reference common to all samples in order to arrive at features that are comparable across samples, and (4) dimensionality reduction by Principal Component Analysis (PCA) to find the most relevant features within the data. To adapt this classical workflow to make it applicable to 3D images of cells (Figure 3B, Figure 3—figure supplement 1), it was necessary for us to solve three key problems.

First, classical workflows extract point clouds based on specific landmarks that are consistent from sample to sample, such as the nose and eyes in facial shape analysis. As a heterogeneous population of cells does not display such common landmarks, we instead implemented a sampling strategy termed Intensity-Biased Stochastic Landmark Assignment (ISLA) inspired by Chan et al., 2018. ISLA automatically generates a point cloud that is a sparse encoding of the original image, with the local density of points representing local fluorescence intensity (Figure 3C). ISLA achieves this by treating normalized voxel intensity distributions as multinomial probability distributions and sampling them to obtain a predefined number of landmark coordinates. By adjusting the number of landmarks being sampled, the trade-off between precision and computational performance can be controlled.

Second, a solution was needed to prevent differences in cell location, size and rotation from obscuring shape information. We first removed translational variance by shifting all cells such that their centroids are placed at the origin coordinates (0,0) and then removed size variation by rescaling point clouds such that cell volumes are normalized. The removal of rotational variance, however, is usually achieved by spatial registration of cells (Pincus and Theriot, 2007), which is an ill-defined problem for a highly heterogeneous cell population. Therefore, we instead chose to re-represent the 3D point cloud of each cell in terms of the points' pairwise distances from each other, a representation that is intrinsically invariant to translation, rotation and handedness but retains all other information on the cloud's internal structure. We use the term Cell Frame Of Reference (CFOR) to refer to feature spaces where size and rotation have been normalized in this fashion, so only pure shape information is retained.

While it is beneficial to remove size and rotation when analyzing cell shape, these properties are themselves important for the organization of multicellular systems. For example, dedicated planar cell polarity pathways have evolved to control the rotational symmetry of cells within many tissues, including the mechanosensory hair cell organs deposited by the lateral line primordium (Mirkovic et al., 2012; Jacobo et al., 2019). Thus, in order to include this level of organization in our analysis, we also pursued a second approach wherein we register entire tissues (rather than individual cells) prior to feature embedding. In this case, rotational variance of individual cells is not removed but instead becomes biologically meaningful as it now reflects the actual orientation of cells within the tissue. Feature spaces resulting from this approach are referred to as Tissue Frame Of Reference (TFOR) and retain both cell size and relevant rotational information (Figure 3B).

Third and finally, given that point clouds extracted by ISLA are not matched across multiple cells, the classical method for defining a consensus reference across samples, which is to use the average position of matched landmarks (e.g. the average position of all nose landmarks in facial analysis), is not applicable here. To address this, we developed Cluster-Based Embedding (CBE) [inspired by Qiu et al., 2011, which works by first determining which locations in space contain relevant information across all samples and then measuring each individual sample's structure at these locations. More specifically, CBE proceeds by first performing k-means clustering on an overlay of a representative subset of all cells' point clouds. Using the resulting cluster centers as consensus reference points, CBE then re-represents each individual cell's point cloud in terms of a simple proximity measure relative to said cluster centers (Figure 3D). We validated CBE using a synthetically generated point cloud dataset, finding that it outperforms an alternative embedding strategy based on point cloud moments and that normalization of size and rotation (i.e. the cell frame of reference, CFOR) is beneficial in the detection of other shape features (Figure 3—figure supplement 2). For further details on feature embedding with ISLA and CBE as well as our evaluation using synthetic point cloud data see the materials and methods section.

In summary, Cluster-Based Embedding (CBE) of point clouds obtained from 3D images by Intensity-biased Stochastic Landmark Assignment (ISLA) is an expressive and versatile embedding strategy for transforming arbitrary 3D fluorescence distributions into 1D feature vectors. It thus provides a solution to one of the key challenges facing data-driven analysis of tissue development by unpacking the rich information encoded in image stacks into a format that is accessible for further quantitative analysis.

Cell shape links cellular mechanics with tissue architecture and is thus key to understanding morphogenesis in model tissues like the developing lateral line primordium. To gain a data-driven view of cell shape in this system, we applied the ISLA-CBE workflow to the cell boundaries of our segmented dataset, deriving an embedded feature space representing cell morphology across the tissue, which we termed the pLLP's cellular shape space. We found that the resulting Principal Components (PCs) describe meaningful shape variation (Figure 4A–B) and that a small number of PCs is sufficient to capture most of the shape heterogeneity across the tissue (Figure 4C). Interestingly, the cells of the pLLP do not cluster into discrete morphological groups (Figure 4A–B,D), implying a continuous shape spectrum between biologically distinct cells such as those at the tissue's leading edge and those at the center of assembled rosettes.

Figure 4

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Embedded shape features are an unbiased encoding of shape without a pre-imposed biological meaning. It is thus interesting to ask what biological properties the most important shape features represent. To annotate the dimensions of the shape space with such biological properties, we correlated them against a curated set of simple engineered features (see materials and methods, and Supplementary file 2). Bigraph visualizations of these correlations reveal that the highest contributions to pLLP shape heterogeneity in the tissue frame of reference (TFOR) result from rotational orientation along different axes (PCs 1 and 2) as well as from absolute cell length in all three spatial dimensions (PCs 3, 5 and 6) (Figure 4E). Some of the PCs do not strongly correlate with any engineered features, implying that this information would have been lost without the use of unbiased feature embedding. Further manual inspection showed PC four to relate to additional rotational information for flat cells, PC seven to the curvature of cells along the tissue's dorso-ventral axis, and PC eight to cell sphericity. Overall, the dominance of orientation and size in the TFOR shape space is consistent with our previous observations on synthetic data (Figure 3—figure supplement 2A).

In the cell frame of reference (CFOR) (Figure 4F), which is invariant to size and rotation, cell shape heterogeneity is instead dominated by cell sphericity (PC 1), which accounts for nearly 50% of cell shape variation, with lesser contributions from cell surface smoothness (PC 2) and various minor factors that could not be annotated with a clear corresponding engineered feature. Cell sphericity and smoothness are thus important components of the pLLP's cellular architecture, a result that would have gone unnoticed without size- and rotation-invariant shape analysis. Intriguingly, both sphericity and smoothness are closely linked to cell surface mechanics, which have been little studied in the pLLP so far. The minor factors that remain unidentified in the CFOR shape space are likely mostly noise but PCs 3–5, which still explain a few percentage points of variance each (Figure 4C), may encode some meaningful shape information that is not obvious to the human eye and could only be retrieved using a computational approach.

The three-dimensional shape of cells in tissues is determined collectively via local cell interactions. Thus, one key advantage of image-based data is that extracted features are localized in the original spatial context of the tissue, allowing access to tissue-scale information that is lost in current single-cell omics approaches that require tissues to be dissociated. To analyze such tissue-scale patterns, we generated consensus maps of feature variation across primordia based on registered cell centroid positions (Figure 4G). As expected from our correlation analysis, features such as TFOR-PC1 (dorso-ventral orientation) are patterned along the corresponding axis of the tissue (top left panel). For TFOR-PC3 (cell height), the consensus map reveals an increase in cell height immediately behind the tissue's 'leading edge' (top right panel), which is consistent with the established notion that leader cells are flatter and follower cells transition into a more packed and columnar state (Lecaudey et al., 2008). This leader-follower progression is also reflected in CFOR-PC2 (cell surface smoothness), which displays a very similar pattern to TFOR-PC3 (Figure 4G, bottom right panel), possibly due to greater protrusive activity in leaders causing distortions in the cell's shape and thus reducing smoothness. These results provide unbiased quantitative support for previous observations on cell shape changes along the leader-follower axis but also indicate that these changes are continuous rather than discrete. This illustrates how our data-driven approach can provide a complete picture of the patterns of key features across a tissue, which would be challenging to achieve by other means.

As established above, we found that CFOR-PC1 (sphericity) is a key component of the pLLP's cellular architecture. The corresponding consensus pattern is more intricate than the others (Figure 4G, bottom left panel), implying that different processes affect cell sphericity in the primordium. Cells at the front of the tissue register as more spherical due to reduced cell height, again as a consequence of the aforementioned leader-follower transition. The more intriguing pattern is that amongst followers there is a tendency for cells at the center of the tissue to be more spherical than those at the periphery, which manifests as a horizontal stripe in the consensus map. This pattern has not been described previously and may point toward an unknown mechanical aspect of pLLP self-organization.

Taken together, these results illustrate how data-driven exploratory analysis of the cellular shape space can reveal interesting patterns of shape heterogeneity that are indicative of specific mechanisms of organization.

A key strength of the ISLA-CBE pipeline is that it is not limited to embedding objects with continuous surfaces such as then cell plasma membrane. Instead, it is capable of embedding arbitrary intensity distributions, including those of the cytoskeleton, of organelles, or of any other labeled protein. Thus, ISLA-CBE can be used to analyze, and integrate, many aspects of cellular organization beyond morphology. This is of particular benefit when studying behaviors that are driven by the interaction of many cellular mechanisms, as is the case for tissue and organ shaping.

Our dataset encompasses many dual-color stacks containing both labeled membranes and one of several other cellular structures, including nuclei (NLS-tdTomato, Figure 5A), F-actin (tagRFPt-UtrophinCH, Figure 5C), and the Golgi apparatus (mKate2-GM130, Figure 5E) (see table in Supplementary file 1 for a complete overview). For each of these structures we computed specific ISLA-CBE feature spaces (Figure 5B,D,F).

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We sought to combine this intracellular information into an integrated quantitative atlas of cellular architecture. Such atlas integration of image data is usually performed in image space by spatial registration of samples (Peng et al., 2011; Vergara et al., 2017; McDole et al., 2018; Cai et al., 2018). However, registration requires stereotypical shapes that can be meaningfully overlaid via spatial transformations, so it is not readily applicable to developing tissues with non-stereotypical cellular positions. Machine learning can be employed as an alternative strategy for atlas mapping that side-steps this problem. Using a training set of dual-color images that contain both reference features (i.e. cell shape derived from the membrane label) and target features (i.e. the intracellular distribution of a specific protein), a machine learning model is trained to predict the target from the reference. The trained model can then be run on samples where only the reference (i.e. membranes) was imaged to generate predictions for the target (i.e. an intracellular protein). This strategy can be applied directly to microscopy images by means of deep convolutional neural networks (Johnson et al., 2017; Christiansen et al., 2018). However, such deep learning typically requires very large datasets that are usually unobtainable in developmental biology studies. Here, we instead chose to work in the embedded feature space, predicting ISLA-CBE embeddings of protein distributions based on ISLA-CBE embeddings of cell shape. This simplifies the problem such that smaller-scale machine learning algorithms become applicable.

We trained different classical (non-deep) machine learning models to predict the embedded feature spaces of subcellular structures based on the shape space. We optimized and validated this approach using grid search and cross validation (see materials and methods). Focusing on feature spaces in the tissue frame of reference (TFOR), we found that multi-output Support Vector Regression (SVR) yields good predictions across different conditions (Figure 5—figure supplement 1). We therefore used SVR prediction to generate a complete atlas spanning the embedded TFOR feature spaces of all available markers.

This atlas can be explored using the same visualizations shown previously for the shape space. As an example, we correlated the embedded features representing the Golgi apparatus (mKate2-GM130) with engineered features describing cell shape (Figure 5G). This shows that Golgi PCs 1 and 2 in the tissue frame of reference again relate to cellular orientation, whereas Golgi PC three correlates with cell height and a more columnar cell shape. Interestingly, the point cloud visualization reveals that Golgi PC three represents apical enrichment (Figure 5H,I) and the corresponding consensus tissue map shows that PC three is high in follower but not in leader cells (Figure 5J), indicating increased apical localization of the Golgi apparatus in followers. As apical Golgi localization is a hallmark of many epithelia (Bacallao et al., 1989), this finding provides unbiased quantitative support for a model where follower cells display increased epithelial character.

Besides subcellular protein distributions, changes in gene expression are key drivers of tissue organization. Indeed, one of the major goals of the field is to integrate gene expression data with cell and tissue morphology and behavior (Battich et al., 2013; Lee et al., 2014; Satija et al., 2015; Karaiskos et al., 2017; Stuart et al., 2019). We therefore investigated whether the machine learning approach to atlas construction we introduced here can be applied to any quantitative measurement that allows cell shape to be acquired simultaneously, including in-situ measurements of gene expression.

To this end, we performed single-molecule Fluorescence In-Situ Hybridization (smFISH) of pea3 RNA (Figure 6A), a marker of FGFR signaling activity associated with the progression of follower cell development (Aman and Piotrowski, 2008; Durdu et al., 2014). We acquired 2-color stacks of smFISH probes and cell membranes from fixed samples (N = 31, n = 3149), employed automated spot detection to identify and count RNA molecules (Figure 6—figure supplement 1A–E) and embedded cell shapes with ISLA-CBE. We then used SVR to predict smFISH spot counts based on cell shape and location, finding that by combining all available information (the tissue frame of reference shape space, the cell frame of reference shape space and cell centroid coordinates) the trained model is able to account for 38.2 ± 1.9% of pea3 expression variance (Figure 6B). The residual variance that could not be accounted for is due to high cell-to-cell heterogeneity in pea3 expression among follower cells that does not seem to follow a clear spatial or cell shape-related pattern (Figure 6C). Nevertheless, we were able to recover the graded overall leader-follower expression pattern of pea3 when running predictions across the entire atlas (Figure 6D). Our approach could therefore be used to superimpose at least the key patterns of gene expression in a tissue based on a set of in-situ labeling experiments, which in turn could potentially serve as a reference set to incorporate scRNA-seq data (Stuart et al., 2019).

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Data integration based on common reference measurements such as cell shape counters one of the major shortcomings of current-day fluorescence microscopy, which is the limited number of channels and thus of molecular components that can be imaged simultaneously. The approach demonstrated here shows how embedded feature spaces and machine learning can be utilized to perform such data integration even in non-stereotypically shaped samples.

Feature embedding and atlas mapping enable the conversion of images into rich multi-dimensional numerical datasets. To take full advantage of this, biologically relevant patterns such as the relationships between different cell types, cell shapes and tissue context need to be distilled from these data and presented in a human-interpretable form. Accomplishing this in an automated fashion rather than by laborious manual data exploration remains a challenging problem in data science.

Classically, data interpretation involves relating data to established knowledge. This prompted us to look for ways to encode contextual knowledge quantitatively and use it to probe our atlas in a context-guided fashion. When asking biological questions about pLLP development, it is useful to distinguish different cell populations: the leader cells that are focused on migration, the cells at the center of nascent rosettes that go on to form sensory hair cells later in development, the cells in the periphery of rosettes that will differentiate into so-called support and mantle cells and the cells in between rosettes (inter-organ cells) that will be deposited as a chain of cells between the maturing lateral line organs (Grant et al., 2005; Hernández et al., 2007; Nogare et al., 2017; Figure 7A). These four populations constitute simple conceptual archetypes that facilitate reasoning about the primordium's organization.

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To map archetypes into the tissue's cellular shape space, we manually annotated cells that constitute unambiguous examples for each archetype in a subset of samples (N = 26, n = 624) (Figure 7A). We then again used machine learning – specifically a Support Vector Classifier (SVC) – with either tissue or cell frame of reference shape features as input to predict the class of all unlabeled cells. For both sets of input features, we found that the classifier was readily able to distinguish leader cells, central cells and peripheral cells, confirming that these manually chosen archetypes are indeed morphologically distinct (Figure 7—figure supplement 1). The inter-organ cells, however, were frequently misclassified as peripheral or central cells, indicating that at this developmental stage they are not substantially different in terms of shape from other follower cells (Figure 7—figure supplement 1). This illustrates that mapping of manually annotated information into the atlas intrinsically provides an unbiased quality control of biological pre-conceptions, as classification fails for archetypes that are not distinguishable from others.

Importantly, the SVC not only predicts categorical labels but also the probabilities with which each cell belongs to each class, a measure of how much a given cell resembles each archetype. This provides an entry point for human-interpretable visualization of atlas data. Here, we performed PCA on the prediction probabilities to arrive at an intuitive visualization wherein cells are distributed according to their similarity to each archetype (Figure 7B,C). In contrast to unsupervised dimensionality reduction of the shape space (see Figure 4A,B,D), this new visualization is readily interpretable for anyone with basic prior knowledge of pLLP organization.

One directly noticeable pattern is in the number of cells found in intermediate states between the leader, central and peripheral archetypes (Figure 7C). Intermediates between leaders and peripheral cells are common, as would be expected given the leader-follower axis along the length of the primordium. Similarly, intermediates between peripheral and central rosette cells are common, reflecting the continuous nature of rosette structure along the inside-outside axis. However, cells in an intermediate state between the leader and central rosette archetype are rare, possibly indicating that a direct transition between the two states does not frequently occur.

Any other data available at the single-cell level can be overlaid as a colormap onto the archetype visualization to take full advantage of its intuitive interpretability (Figure 7D). When doing so, previously observed patterns such as the increased cell height in followers – particularly in central cells – are clearly visible. Notably, the same pattern can be seen for PC 3 of the embedded protein distribution of F-actin, which is part of the atlas. This reflects apical enrichment of F-actin in follower cells, an important feature of rosette morphogenesis (Lecaudey et al., 2008). By contrast, plotting CFOR-PC1 (cell sphericity) shows that it is elevated in central rosette cells, especially in a population furthest from the peripheral archetype, a finding that was not predicted by previous studies. This is consistent with the spatial distribution seen in Figure 4G and reinforces the novel finding that cell sphericity exhibits a non-trivial pattern related to rosette organization. Interestingly, the pattern of CFOR-PC2 (cell surface smoothness) may represent the additive combination of the leader-follower and central-peripheral patterns, supporting a model where distinct mechanisms act to determine cell shape along these two axes of the tissue.

While these observations were made through an exploratory data-driven approach, the archetype labels also allow them to be checked with classical statistical hypothesis testing. Doing so substantiates both the previously known leader-follower difference in cell height (Figure 7E) and the novel finding that inner rosette cells are significantly more spherical than outer rosette cells (Figure 7F).

Despite its relative simplicity, our archetype-based approach showcases how the mapping of contextual knowledge onto a large and complicated dataset can facilitate its intuitive visualization and biological interpretation, leading to novel hypotheses that can be further tested experimentally.

Zebrafish (Danio rerio, RRID:ZFIN_ZDB-GENO-060919-1) were grown, maintained and bred according to standard procedures described previously (Westerfield, 2000). All experiments were performed on embryos younger than 3dpf, as is stipulated by the EMBL internal policy 65 (IP65) and European Union Directive 2010/63/EU. Live embryos were kept in E3 buffer at 27–30°C. For experiments, pigmentation of embryos was prevented by treating them with 0.002% N-phenylthiourea (PTU) (Sigma-Aldrich, St. Louis, US-MO) starting at 25hpf. For mounting and during live imaging, embryos were anaesthetized using 0.01% Tricaine (Sigma-Aldrich, St. Louis, US-MO).

All embryos imaged carried the membrane marker cldnb:lyn-EGFP (Haas and Gilmour, 2006), which was used for single cell segmentation. In addition, different subsets of the main dataset carried one of the following red secondary markers: cxcr4b:NLS-tdTomato (nuclei) (Donà et al., 2013), Actb2:mKate2-Rab11a (recycling endosomes), LexOP:CDMPR-tagRFPt (trans-Golgi network and late endosomes), LexOP:B4GalT1(1-55Q)-tagRFPt (trans-Golgi), 6xUAS:tagRFPt-UtrCH (F-actin) or atoh1a:dtomato (transcriptional marker for hair cell specification) (Wada et al., 2010). Furthermore, in two subsets of the data a red marker was injected as mRNA, namely mKate2-GM130(rat) (cis-Golgi) (Pouthas et al., 2008) and mKate2-Rab5a (early endosomes). Finally, one subset of samples was treated with 1 μM LysoTracker Deep Red (Thermo Fisher Scientific, Waltham, US-MA) in E3 medium with 1% DMSO for 90 min prior to imaging. The exact composition of the main dataset is tabulated in Supplementary file 1.

To drive expression of the UAS construct, those fish additionally carried the Gal4 enhancer trap ETL GA346 (Distel et al., 2009). To drive expression of LexOP constructs (Emelyanov and Parinov, 2008), those fish carried cxcr4b:LexPR (Durdu et al., 2014) and were treated with 10 μM of the progesterone analogue RU486 (Sigma-Aldrich, St. Louis, US-MO) from 25hpf.

Capped mRNA was produced by IVT using the mMESSAGEmMACHINE SP6 Transcription Kit (Thermo Fisher Scientific, Waltham, US-MA) according to the manufacturers’ instructions and was injected at 250 ng/μl into embryos at the 1 cell stage.

The following plasmids were generated by MultiSite Gateway Pro Cloning (Invitrogen, Waltham, US-MA) based on the Tol2kit (Kwan et al., 2007): LexOP:CDMPR-tagRFPt (with cry:mKate2 transgenic marker), LexOP:B4GalT1(1-55Q)-tagRFPt (with cry:mKate2 transgenic marker), Actb2:mKate2-Rab11a (with clmc2:GFP transgenic marker), 6xUAS:tagFRPt-UtrCH (with cry:mKate2 transgenic marker), sp6:mKate2-GM130(rat) and sp6:mKate2-Rab5a. tagRFPt (Shaner et al., 2008) and UtrCH (Burkel et al., 2007) were kindly provided by Jan Ellenberg and Péter Lénárt, respectively. Zebrafish CDMPR, B4GalT1, Rab5a and Rab11a were cloned by extraction of total RNA from dechorionated 48hpf zebrafish embryos using the RNeasy mini kit (Qiagen, Hilden, Germany) and QIAshredder (Quiagen, Hilden, Germany) according to manufacturer's instructions, followed by reverse transcription with SuperScript III Reverse Transcriptase (Thermo Fisher Scientific, Waltham, US-MA) using both random hexamers and oligo-dT simultaneously, according to the manufacturer's instructions, and finally amplification of genes of interest from cDNA with the following oligonucleotides (Sigma-Aldrich, St. Louis, US-MO) (template-specific region underlined):

• CDMPR FOR: GGGGACAAGTTTGTACAAAAAAGCAGGCTGGATGTTGCTGTCTGTGAGAATAATCACT

• CDMPR REV: GGGGACCACTTTGTACAAGAAAGCTGGGTCCATGGGAAGTAAATGGTCATCTCTTTCCTC

• B4GalT1 FOR: GGGGACAAGTTTGTACAAAAAAGCAGGCTGGATGTCGGAGTCGGTGGGATTCTTC

• B4GalT1 REV: GGGGACCACTTTGTACAAGAAAGCTGGGTCTTGTGAATTAACCATATCAGAGATAAATGAAATGTGTCG

• Rab5a FOR: GGGGACAGCTTTCTTGTACAAAGTGGCTATGGCCAATAGGGGAGGAGCAACAC

• Rab5a REV: GGGGACAACTTTGTATAATAAAGTTGCTTAGTTGCTGCAGCAGGGGGCT

• Rab11a FOR: GGGGACAGCTTTCTTGTACAAAGTGGCTATGGGGACACGAGACGACGAATACG

• Rab11a REV: GGGGACAACTTTGTATAATAAAGTTGCCTAGATGCTCTGGCAGCACTGC

Embryos were manually dechorionated with forceps at 30-34hpf and anaesthetized with 0.01% Tricaine (Sigma-Aldrich, St. Louis, US-MO), then transferred into 1% peqGOLD Low Melt Agarose (Peqlab, Erlangen, Germany) in E3 containing 0.01% Tricaine and immediately deposited onto a MatTek Glass Bottom Microwell Dish (35 mm Petri dish, 10 mm microwell, 0.16–0.19 mm coverglass) (MatTek Corporation, Ashland, US-MA). No more than 10 embryos were mounted in a single dish. A weighted needle tool was used to gently arrange the embryos such that they rest flatly with their lateral side directly on the glass slide. After solidification of the agarose, E3 containing 0.01% Tricaine was added to the dish. Embryos were imaged at 32-36hpf, when the pLLP was located above the posterior half of the embryo's yolk extension.

The microscope used for imaging was the Zeiss LSM880 with AiryScan technology (Carl Zeiss AG, Oberkochen, Germany), henceforth LSM880. High-resolution 3D stacks (voxel size: 0.099 μm in xy, 0.225 μm in z) were acquired with a 40 × 1.2 NA water objective with Immersol W immersion fluid (Carl Zeiss, Oberkochen, Germany). Imaging in AiryScan FAST mode (Huff, 2016) with a piezo stage for z-motion and bi-directional scanning allowed acquisition times for an entire volume to be lowered to approximately 20 s (40 s for dual-color stacks using line switching). Deconvolution was performed using the LSM880's built-in 3D AiryScan deconvolution with 'auto' settings.

Note that optimal image quality could only be achieved by adjustment of the stage to ensure that the cover glass is exactly normal to the excitation beam. For each dish we imaged, we used 633 nm reflected light and line scanning to get a live view of the cover glass interface, which allowed us to manually adjust the pitch of the stage to be completely horizontal. This process was repeated for both zx and zy line scans.

Single molecule Fluorescence In-Situ Hybridization (smFISH) was performed according to standard protocols (Durdu et al., 2014; Raj et al., 2008) using previously published Quasar 670-conjugated Stellaris smFISH probes (LGC, Biosearch Technologies, Hoddesdon, UK) designed to target pea3 mRNA, listed below.

Briefly, embryos were fixed overnight in 4% PFA in PBS-T (PBS with 0.1% Neonate-20) at 4°C, then rinsed three times in PBS-T and subsequently permeabilized with 100% methanol overnight at −20°C. Embryos were rehydrated with a methanol series (75%, 50%, 25% Methanol in PBS-T, 5 min per step) and rinsed three times with PBS-T. The yolk was manually removed using forceps. Next, samples were pre-incubated with hybridization buffer (0.1 g/ml deyxtrane sulfate, 0.02 g/ml RNase-free BSA, 1 mg/ml E. coli tRNA, 10% formamide, 5x SSC, 0.1% Neonate-20 in ddH2O) at 30°C for 30 min and subsequently hybridized with pea3 probe solution (0.1 μM in hybridization buffer) at 30°C overnight in the dark. After probe removal, embryos were stained with DAPI (1:1000) in washing buffer (10% formamide, 5x SSC, 0.1% Neonate-20 in ddH2O) for 15 min at 30°C and finally kept in washing buffer for 45 min at 30°C.

Stained embryos were mounted on glass slides using VECTASHIELD HardSet Antifade Mounting Medium (Vector Laboratories, Burlingame, US-CA) and imaged immediately to prevent loss of signal due to photobleaching. Imaging was performed with a 63 × 1.4 NA oil immersion objective on the LSM880 in FAST mode with 488 nm and 639 nm excitation lasers. Stacks were acquired using 8x averaging with 0.187 μm z-spacing and a pixel size of 0.085 μm, then deconvolved with the built-in 3D AiryScan deconvolution on 'auto' settings.

The following smFISH probes were used:

• 1: aaggaagacggacagaggca, 2: ctgtgttttaatgagctcca, 3: cttaaccgtttgtggtcatt,

• 4: ccatccatcttataatccat, 5: agtataaggcacttgctggt, 6: atttccttgcgacctattag,

• 7: tcaacagtctatttaggggc, 8: atgtatttcctttttgtcgc, 9: aagaggtcttcagattcctg,

• 10: cctgaagttggcttaaatcc, 11: ggaacttgagcttcggtgag, 12: aacaaactgctcatcgctgt,

• 13: cactgagttctctgagtgaa, 14: ttcttaatcttcacaggcgg, 15: tagctgaagctttgcttgtg,

• 16: tcataggcactggcgtaaag, 17: ctggacatgagctcttagat, 18: ttgggggaataatgctgcat,

• 19: tgagggtggattcatatacc, 20: cggaagggaacctggaactg, 21: agagtgttgccgatggaaac,

• 22: tgctgaggaggataaggcaa, 23: ccatgtactcctgcttaaag, 24: tcctgtttgaccatcatatg,

• 25: caggttcgtaagtgtagtcg, 26: tgtgatggtacatggatggg, 27: aaacatgtagccttcactgt,

• 28: tggcacaacacgggaatcat, 29: tcacctcaccttcaaatttc, 30:accttcacgaaacacactgc,

• 31: tagttgaagtgagccacgac, 32: gaagggcaaccaagaactgc, 33: atgcgatgaagtgggcattg,

• 34: atgagtttgaattccatgcc, 35: ttgtcatagttcatggctgg, 36: gtaacgcaaagagcgactca,

• 37: ttttgcataattcccttctc, 38: aggttatcaaagcttctggc, 39: cgctgattgtcgggaaaagc,

• 40: gttgacgtagcgctcaaatt, 41: aagaaactccctcatcgagg, 42: tacatgtagcctttggagta,

• 43: aaaggagaatgtcggtggca, 44: gtggtaaactgggatgggaa, 45: atacaagaggatggggtggg,

• 46: gaatgcagagtccctaatga, 47: agataggcctcagaagtgag, 48: gcaatctcttgaaccacagt.

The software for this study was developed using the Anaconda distribution (Anaconda, Inc, Austin, US-TX) of python 2.7.13 (64-bit) (Python Software Foundation, Beaverton, US-OR) (Van Rossum, 1995).

The following scientific libraries and modules were used: numpy 1.11.3 (Travis and Oliphant, 2006) and pandas 0.19.2 (McKinney, 2010) for numerical computation, scikit-image 0.13.0 (van der Walt et al., 2014) and scipy.ndimage 2.0 (Jones and Oliphant, 2001) for image processing, scikit-learn 0.19.1 (Pedregosa and Varoquaux, 2011) for machine learning, matplotlib 1.5.1 (Hunter, 2007) and seaborn 0.7.1 (Waskom et al., 2016) for plotting, networkx 1.11 (Hagberg et al., 2008) for graph-based work, tifffile 0.11.1 (Gohlke, 2016) for loading of TIFF images, and various scipy 1.0.0 (Jones and Oliphant, 2001) modules for different purposes. Parallelization was implemented using dask 0.15.4 (Team, 2016).

Jupyter Notebooks (jupyter 1.0.0, notebook 5.3.1) (Kluyver et al., 2016) were utilized extensively for prototyping, workflow management and exploratory data analysis, whereas refactoring and other software engineering was performed in the Spyder IDE (spyder 3.2.4) (Raybaut et al., 2018). Version control was managed with Git 2.12.2.windows.2 (Torvalds and the Git contributors, 2018) and an internally hosted GitLab instance (GitLab, San Francisco, US-CA).

Following AiryScan 3D deconvolution with 'auto' settings on the LSM880, images were converted to 8bit TIFF files using a custom macro for the Fiji distribution (Schindelin et al., 2012) of ImageJ 1.52 g (Schneider et al., 2012). The minimum and maximum values determining the intensity range prior to 8bit conversion were selected manually such that intensity clipping is avoided. Care was taken to apply the same values to all samples of a given marker to ensure consistency.

Samples with the cxcr4b:NLS-tdTomato nuclear label exhibited a degree of bleed-through into the lyn-EGFP membrane label channel. To prevent this from interfering with single-cell segmentation, we employed a linear unmixing scheme in which the contribution of NLS-tdTomato ($C$, the contaminant image) is removed from the green channel ($M$, mixed image), resulting in the cleaned membrane channel ($U$, unmixed image). Our approach assumes that the signal in $M$ is composed according to Equation 1, implying that $U$ can be retrieved by subtraction of an appropriate contamination term (Equation 2).

To compute the optimal bleed-through factor $a$ we minimized a custom loss function (Equation 3), which is essentially simply the Pearson Correlation Coefficient ($PCC$) of the contaminant image $C$ and the cleaned image $U$ given a particular candidate factor $a_i$. To ensure that unreasonably high values of $a$ are punished, we centered the values of the cleaned image onto their mean and converted the result to absolute values, causing overly unmixed regions to start correlating with $C$ again.

We found that this approach robustly removes NLS-tdTomato bleed-through, producing unmixed images that could be segmented successfully.

3D single-cell segmentation was performed on membrane-labeled stacks acquired, deconvolved and preprocessed as detailed in the sections above.

The pipeline for segmentation consists of the following steps, applied sequentially:

1. 3D median smoothing with a cuboid 3 × 3×3 vxl structural element to reduce shot noise.

2. 3D Gaussian smoothing with σ = 3 pxl to further reduce noise and smoothen structures.

3. Thresholding to retrieve a binary mask of foreground objects (i.e. the membranes). To automatically determine the appropriate threshold, we use a custom function inspired by a semi-manual approach for spot detection (Raj et al., 2008). Starting from the most frequent value in the image histogram as a base threshold, we iteratively scan a limited range of positive offsets (usually 0 to 10 in steps of 1, for slightly lower-quality images 0 to 40 in steps of 2) and count the number of connected components in the inverse of the binary mask resultant from applying each threshold. This roughly represents the number of cell bodies in the stack that are fully enclosed in cell membranes at a given threshold. We consider the threshold producing the largest number the best option and use it to generate the final membrane mask.

4. Removal of disconnected components by morphological hole filling.

5. Labeling of connected components on the inverted membrane mask. This ideally yields one connected component per cell, i.e. the cytoplasm.

6. Removal of connected components smaller than 1'000 voxels (artifacts) and re-labeling of connected components larger than 1'000'000 voxels as background objects.

7. Watershed expansion using the labeled connected components as seeds and the smoothed input image as topography (with additional 3D Gaussian smoothing on top of steps 1 and 2, with σ = 3 pxl). The background objects surrounding the primordium are also expanded.

8. Assignment of the zero label to background objects and removal of any objects disconnected from the primordium by retaining only the single largest foreground object.

We manually optimized the parameters of this pipeline for our data by inspecting the output during an extensive set of test runs.

Finally, we also manually double-checked all segmentations and discarded rare cases where more than approx. 10% of cells in a stack had been missed or exhibited under- or oversegmentation.

Intensity-biased Stochastic Landmark Assignment (ISLA) (Figure 3A–C, Figure 3—figure supplement 1A) was applied to cropped-out bounding boxes of single segmented cells. To capture cell shape, the 6-connected inner hull of the binary segmentation mask was used as input image for ISLA. To capture intensity distributions, voxels outside the segmentation mask were set to zero and a simple background subtraction was performed to prevent landmarks from being assigned spuriously due to background signal. The background level was determined as the mean intensity within the masked cell and was subtracted from each voxel's intensity value, with resulting negative values set to zero.

With the inputs so prepared, voxel intensities were normalized such that their sum equals one by dividing each by the sum of all. Then, landmarks were assigned by considering the normalized voxel intensities as the probabilities of a multinomial distribution from which 2000 points were sampled (with replacement). This number was determined through test runs in which we found that, for volumes of our size, diminishing returns set in around 500 points and using more than 2000 points no longer improved the performance of various downstream analyses.

Following ISLA sampling, landmark coordinates were scaled from pixels to microns to account for anisotropic image resolution.

Note we recently published another study that utilizes a simplified version of ISLA for some of the data analysis, albeit in a very different way from how it is used here (Wong et al., 2020).

To place cells in a matched Tissue Frame of Reference (TFOR) prior to feature embedding (Figure 3—figure supplement 1B, left route), primordia were aligned using a simple PCA-based approach that does not require full image registration. To this end, 3000 landmarks were sampled from a given primordium's binary overall segmentation mask using ISLA and a modified PCA was applied to the resulting point cloud. Given that the pLLP's longest axis is always its front-rear axis and the shortest axis is always the apicobasal axis, PCA transformation snaps primordia that had been acquired at a slight angle into a consistent frame of reference. Our modification ensured that 180° flipping could not occur in this procedure. To complete the alignment, the primordial point clouds were translated such that the frontal-most point becomes the coordinate system's origin. Finally, the ISLA point clouds extracted from individual cells as described in the previous paragraph were transformed using the same PCA, thus matching their orientation to the common tissue frame of reference.

To create a Cell Frame of Reference (CFOR) that is invariant to size, rotation and handedness (Figure 3—figure supplement 1B, right route), point cloud volumes were first normalized such that the sum of the magnitudes of all centroid-to-landmark vectors is 1. Second, cellular point clouds were cast into a pairwise distance (PD) representation. In the PD space, each point of the cloud is no longer characterized by three spatial coordinates but instead by the distances to every other point of the cloud. This representation is rotationally invariant but also extremely high-dimensional (an LxL array, where L is the number of landmarks). To reduce dimensionality, only the 10th, 50th and 90th percentiles of all pairwise distances for each point were chosen to represent that point (resulting in an Lx3 array), which we reasoned would encode both local and global relative spatial location.

To determine reference cluster centers for Cluster-Based Embedding (CBE) (Figure 3D, Figure 3—figure supplement 1B), point clouds from multiple samples were centered on their respective centroids and overlaid. K-means clustering was performed on this overlaid cloud (using scikit-learn's MiniBatchKMeans implementation) with k = 20. The resulting cluster centers were used as common reference points for the next step.

Several measures were taken to improve the robustness and performance of this cluster detection. First, individual cellular point clouds were downsampled from 2000 points to 500 points prior to being overlaid, using k-means clustering with k = 500 clusters, the centers of which were used as the new landmarks. Second, not all available cells were used in the overlay. Instead, a representative random subset of primordia (at least 10, at most 25) was selected and only their cells were used in the overlay. The resulting cluster centers were used as reference points across all available samples. Third, the entire overlaid point cloud was downsampled using a density-dependent downsampling approach inspired by Qiu et al., 2011 (see below), yielding a final overlaid cloud of at most 200'000 points, which allowed reference cluster centers to be computed reasonably efficiently.

Density-dependent downsampling was performed using a simplified version of the algorithm described by Qiu et al., 2011. First, the local density ($LD$) at each point is found, which is defined as the number of points in the local neighborhood, i.e. a sphere whose radius is the median pairwise distance between all points multiplied by an empirically determined factor (here 5). Next, a target density ($TD$) is determined, which in accordance with Qiu et al. was set to be the third percentile of all local densities. Now, points are downsampled such that the probability of keeping each point is given by Equation 1. If necessary, the resulting downsampled distribution is further reduced by random sampling in order to reach the maximum of 200'000 points.

Density-dependent downsampling was chosen to avoid cases where high-density agglomerations of landmarks in a particular region accumulate multiple clusters and thus deplete lower-density regions of local reference points; density-dependent downsampling preserves the overall shape of the overlaid point cloud whilst reducing local density peaks.

Following the determination of common reference points, CBE proceeds by extracting features describing the local landmark distribution around said reference points for each separate cellular point cloud. Several such features were implemented, including the number of landmarks in the local neighborhood of each reference point, the number of landmarks assigned to each reference point by the k-means clustering itself, the local density of landmarks at each reference point determined by a Gaussian Kernel Density Estimate (KDE), and either the magnitude or the components of the vector connecting each reference point to the centroid of its 25 nearest neighbors. The results were similar across all of these approaches but we ultimately chose to proceed with the last entry in the above list (the vector components; see Figure 3D for a simple 2D example) based on its inclusion of some additional directional information.

The feature extraction described above yields an n-by-3k latent feature space, where n is the number of cells and k the number of shared reference clusters (here k = 20). As a final step, this space was transformed by Principal Component Analysis (PCA) to bring relevant variation into focus and reduce dimensionality.

In addition to CBE, an alternative embedding based on the moments of the TFOR or CFOR point clouds was also generated as a comparably simple baseline. We computed the 1 st raw moments (Equation 5), the 2nd centralized moments (Equation 6) and the 3rd to 5th normalized moments (Equation 7) (55 features in total) and once again used PCA to arrive at a compact and expressive feature space.

In Equations 5, 6, 7, $C_d$ is the array of all point cloud coordinates along the spatial dimension $d$, $M_m$ is the set of raw (${rM}_m$), centralized (${cM}_m$) or normalized (${nM}_m$) moments of the $m$-th order, and $[i,j,k]$ includes all combinations of length 3 drawn from the integer range $\left[0,\dots ,m\right]$ that satisfy $i+j+k=m$. All operations are element-wise except $mean(\dots )$ and $std(\dots )$, which compute the mean and standard deviation across a given array.

In order to benchmark the capability of CBE for latent feature extraction, we required a gold standard dataset to test whether CBE recovers known latent parameters underlying a population of shape objects. We thus wrote a generator to automatically create a large synthetic dataset of cell-like point clouds. This generator functions in four stages:

1. Generate a spine (five parameters)The spine is defined by its height along the z dimension (i.e. the apicobasal axis), by an offset, which is the distance and angle by which the end point of the spine is shifted in xy compared to the starting point, and by a connecting line between endpoints, which is a 2^nd degree polynomial.

2. Generate the cell surface (nine parameters)The surface of the cell is determined based on its distance d to the spine as a function of z. We used the logit-normal as this distance function on the grounds that it has only two parameters, can be asymmetric, monomodal or bimodal, crosses (0, 0) and (1, 0), and has a range d > 0 within the domain 0 > z > 1. For each cell, three different logit-normals were used to describe the surface at three uniformly spaced angles around the spine. Additionally, each of those functions was independently scaled by multiplication with another parameter.

3. Sampling of point clouds (0 parameters)The actual surface point cloud to be used in feature embedding was generated by sampling 2000 points with the following scheme: first randomly sample a position along z and an angle, then determine the corresponding radius by linear interpolation between the logit-normal values of the two adjacent angles at which they are defined. Finally, angle and radius are converted to the Cartesian coordinate system.

4. Centering, scaling and rotation (three parameters)Finally, point clouds are centered on their centroid and cell size is adjusted by multiplication with a scaling parameter, followed by rotation using a homogeneous transformation matrix with two angle parameters.

All in all, this generator requires 17 parameters of which one modifies only size (not shape) and two modify only rotation. For each synthesized cell, the specific parameter values for the generator were sampled from normal or uniform distributions with hyperparameters set based on empirical experimentation such that a varied population of cell shapes is generated and unreasonably deformed shapes are avoided.

Using the generator described above, we synthesized a set of n = 20'000 cellular point clouds and embedded them using both CBE and the moment-based alternative embedding strategy. We then tested how well the values of the 17 known generative parameters could be retrieved from the embedded feature spaces using different scikit-learn implementations of multivariate-multivariable regression, specifically k-Nearest Neighbor (kNN) regression, multi-output linear Support Vector Regression (l-SVR) and multi-output radial basis function kernel Support Vector Regression (r-SVR). Optimal hyperparameters for the two SVRs were determined using cross-validated grid search with GridSearchCV, scanning 5 orders of magnitude surrounding the defaults of C (penalty) and epsilon as well as gamma (RBF kernel coefficient) for r-SVR.

This synthetic experiment showed that cell size and orientation largely obscure other shape features if CFOR-normalization is not performed (Figure 3—figure supplement 2A). Furthermore, features generated by CBE enable similar or better predictive performance than moments-based features regardless of the method used for prediction (Figure 3—figure supplement 2B). Interestingly, kNN performs markedly better on CBE-embedded spaces, indicating that CBE more meaningfully encodes point cloud similarity as local neighborhood in the embedded space.

We extracted various explicitly engineered features from entire tissues, segmented cell volumes, and segmented cell point clouds. The features used in this study are listed and briefly described in Supplementary file 2.

To construct the multi-channel atlas, we used the secondary markers present in many of our samples (see Supplementary file 1), embedded them with ISLA and CBE (see the corresponding sections above) and then trained multivariate-multivariable regressors to predict those embeddings based on the corresponding cell shape embeddings as input features. All embeddings were standardized and PCA-transformed prior to machine learning and only the first 20 PCs were considered. Predictions were performed from shape TFOR to secondary marker TFOR and from shape CFOR to secondary marker CFOR.

Because expression of the secondary markers was sometimes heterogeneous across the primordium, only cells with a secondary marker intensity above the 33rd percentile were used as training data. Furthermore, to prevent rare extreme outliers (resulting from segmentation artifacts) in either the shape or secondary marker feature space from affecting the prediction, such anomalies were detected and removed from the training data using the isolation forest algorithm (sklearn.ensemble. isolation_forest) with contamination = 0.05.

To select the best machine learning model, the following regressors were tested using 3-fold cross-validation based on scikit-learn's ShuffleSplit function: k-nearest neighbors regression (sklearn. neighborsKNeighborsRegressor), random forest regression (sklearn.ensemble.RandomForest Regressor), elastic net regression (sklearn.linear_model.ElasticNet), Lasso regression (sklearn. linear_model.Lasso), a multi-layer perceptron (sklearn.neural_network.MLPRegressor), and a support vector regressor with an RBF-kernel (sklearn.svm.SVR). Relevant hyperparameters were optimized on the NLS-tdTomato nuclear marker using a 5-fold cross-validated grid search (sklearn.model_selection. GridSearchCV) of 5 orders of magnitude surrounding the scikit-learn defaults. Performance was evaluated across different secondary channels and latent feature embeddings (Figure 5—figure supplement 1A), with the primary aim being high explained variance but also giving some consideration to computational efficiency (training and prediction time). After finding that TFOR predictions perform substantially better than CFOR predictions, possibly because the TFOR shape space incorporates more relevant information for intracellular marker prediction and/or because CFOR spaces contain more noise and more specific information that is challenging to predict, we focused further analysis on TFOR for the time being. We selected the RBF SVR as the regressor of choice because of its consistently high performance.

For atlas prediction across the entire dataset, the SVR model was trained for each secondary channel (using the corresponding best hyperparameter set) on all available data for that channel and then applied to predict that channel's embedded space for all other cells.

Single-cell segmentation and cell shape feature embedding was performed on the pea3 smFISH dataset in the same fashion as for the live imaging dataset, resulting in 3'149 cells from 31 samples.

The blob_log spot detector from scikit-image was used (skimage.blob.blob_log) to identify smFISH spots (Figure 6—figure supplement 1A–D). Parameters were optimized by manual testing and by scanning different values for the threshold and min_sigma parameters, arriving ultimately at min_sigma = 1, max_sigma = 4, num_sigma = 10, threshold = 0.22, overlap = 0.5, and log_scale = False, which produced average counts per cell that are reasonably consistent across different primordia (Figure 6—figure supplement 1E) and closely approximate previously reported pea3 smFISH spot counts in the pLLP (Durdu et al., 2014). However, we also found that optimal settings can vary between different smFISH experiments and thus recommend parameter optimization and careful evaluation of the results for every experiment. Furthermore, in this dataset the spot detector failed to detect a reasonable number of spots in some exceptional samples, so we excluded primordia with a mean count per cell of less than two spots from further analysis, leaving 2906 cells from 29 primordia in the dataset.

Because smFISH must be performed on fixed samples, we checked for fixation effects on cell shape, which would make cross-predictions with the live sample atlas more challenging. In bulk comparisons of key shape features (Figure 6—figure supplement 1F–H) we found no significant differences between live and fixed samples. However, such bulk comparisons may miss subtle fixation effects, making the development of methods to quantify, pinpoint and correct such effects an important future goal.

We trained an RBF-kernel SVR to perform multivariate regression of pea3 smFISH counts based on as much other information available about each cell as possible (Figure 6B, Figure 6—figure supplement 1E), namely a combined feature space incorporating the first 10 shape TFOR PCs, the first 10 shape CFOR PCs, and the x, y and z coordinates of cell centroids in TFOR. Hyperparameters C (penalty), epsilon, and gamma (RBF kernel coefficient) were again optimized using GridSearchCV, whereas ShuffleSplit was used to randomly split data into training and test data for evaluation as shown in Figure 6B.

The four archetypes were manually annotated in 26 primordia (Figure 7A) using Fiji's multi-point selection tool, yielding 93 leader cells, 241 outer rosette cells, 182 inner rosette cells and 108 between-rosette cells (624 cells in total). Only the clearest examples of the respective archetypes were labeled.

Cell archetype prediction was performed using scikit-learn's Support Vector Classifier (SVC) with a Radial Basis Function (RBF) kernel, using either TFOR- or CFOR-embedded cell shapes as input features. We optimized the SVC hyperparameters for each embedding using scikit-learn's GridSearchCV with 5-fold cross-validation, testing whether or not to use feature standardization, whether or not to use PCA and keep the first 15, 30 or 50 PCs, as well as screening 5 orders of magnitude surrounding the scikit-learn default values for C (penalty) and gamma (RBF kernel coefficient). The resulting best estimator was used for all further training and prediction.

The confusion matrices in Figure 7—figure supplement 1 were produced by splitting the annotated cells into a training set (436 cells) and a test set (188 cells) using scikit-learn's StratifiedShuffleSplit to balance labels. Predictions for the entire atlas dataset were generated following training with all 624 manually annotated cells.

The archetype space was constructed by inferring the classification probabilities for each class (using sklearn.svm.SVC.predict_proba) and performing a PCA on them. The 3D and 2D visualizations in Figure 7 were then generated by plotting the first three or two principal components, respectively.

Fiji's Straighten tool was used to align angled samples with the main image axes for the purpose of illustration in Figures 1, 2, 5, 6 and 7 and in Figure 2—video 1, 2, 3 but never as part of an image analysis pipeline. Maximum projections were created with Fiji whereas 3D videos were rendered with Imaris 7.7.2 (Bitplane, Belfast, UK).

The expanded view of the segmented pLLP (Figures 1B and 2C) was generated by first determining the centroids of each segmented cell and then shifting them apart by scaling of their x and y coordinates by a single user-specified factor. The cells were then pasted into an appropriately scaled empty image stack at the new centroid locations, leaving them shifted apart uniformly but not individually rescaled or otherwise transformed. A python implementation of this approach called tissueRipper is available under the MIT open source license on GitHub at github.com/WhoIsJack/tissueRipper.

The corresponding bigraphs (Figures 4E,F and 5G) were generated using a custom plotting function based on the networkx module. The edges were colored according to the signed value of the Pearson correlation coefficient and sized according to its magnitude. Edges with an absolute correlation coefficient smaller than 0.3 were omitted. The nodes of the engineered features were sorted to reduce edge crossings and group similar nodes, which was achieved by minimizing the following custom loss function:

where $f_E$ and $f_L$ are the number of engineered and latent features, respectively. $C$ is the current sort order of the engineered features, that is a permutation of the integer interval $[0,f_E]$. Finally, $\left|pcc\left(E_i,L_j\right)\right|$ is the absolute Pearson correlation coefficient of the values of the i-th engineered feature and the $j$-th latent feature. In essence, this loss function is the sum of all Euclidean rank distances between engineered and latent features, weighted by their corresponding absolute Pearson correlation coefficients. Minimization was performed by random shuffling of the sort order and retaining only shuffles that reduced the loss until no change was observed for 2000 consecutive shuffles.

Note that since the sign of principal components is not inherently meaningful, we flipped it for shape TFOR-PCs 1, 3, 5, six and shape CFOR-PC one across all analyses presented in this study to ensure that PCs positively correlate with their most defining engineered feature(s), facilitating discussion of the results.

Consensus maps of feature variation (Figures 4G, 5J and 6C,D) were based on an overlay of TFOR centroid positions of cells across all relevant samples.

The cut-off for the consensus tissue outline was determined by computing the local density of these overlaid centroids using scipy's Gaussian kernel density estimation with default settings. Regions with densities below 10% of the range between the minimum and maximum density were considered outside of the primordium and are shown as white in the plot.

The local consensus feature values were computed by applying a point cloud-based Gaussian smooth across the individual cells' feature values, using the 0.5th percentile of all pairwise distances between centroids as σ. This smoothed distribution was then plotted as a tricontourf plot with matplotlib using up to 21 automatically determined contour levels.

Generally, we use N to refer to the number of embryos/primordia and n to the number of cells.

Statistical significance for comparisons between two conditions was estimated without parametric assumptions using a two-tailed Mann-Whitney U test (scipy.stats.mannwhitneyu with keyword argument alternative='two-sided') with Bonferroni multiple testing correction (Haynes, 2013) where appropriate. We considered p>0.01 as not statistically significant.

Significance tests with large sample sizes such as those encountered during single-cell analysis tend to indicate high significance regardless of whether the difference between populations is substantive or technical (Sullivan and Feinn, 2012), which is why we also report estimates of effect size for any comparison that is statistically significant. Effect sizes were estimated using Cohen, 1977. The resulting values can be described as no effect (d≈0.0), a small effect (d≈0.2), a medium effect (d≈0.5) or a large effect (d≈0.8) (Cohen, 1977).

Requests for experimental resources and reagents should be directed to and will be fulfilled by Darren Gilmour (darren.gilmour@imls.uzh.ch).

All raw and processed data are available freely and openly via the Image Data Resource repository (idr.openmicroscopy.org) under accession number idr0079. Code is available under the MIT open source license on GitHub at github.com/WhoIsJack/data-driven-analysis-lateralline. Note that we aim to update the core algorithms to python 3 and make them available as a readily reusable module in the near future. Inquiries regarding data and code should be directed to Jonas Hartmann (jonas.m.hartmann@protonmail.com).

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

We thank Sabine Görgens and Andreas Kunze for their support with fish and lab maintenance, respectively. We thank the EMBL Advanced Light Microscopy Facility (ALMF) and the UZH Center for Microscopy and Image Analysis (ZMB) for maintenance of and assistance with microscopes. We thank Alejandra Guzman Herrera for generating the Act2b:mKate2-Rab11a line. We thank Francesca Peri and Stefano De Renzis for kindly providing temporary lab space. We thank Christian Tischer and Marvin Albert for helpful discussion on image analysis and numerical computation. We thank Stefano De Renzis, Daniel Krueger, Marvin Albert and Andrew Kennard for critical reading of the manuscript. JH and EG were supported by the EMBL International PhD Programme (EIPP), M.W. was supported by an EMBO Long-Term Fellowship and the EMBL Interdisciplinary Postdoc (EIPOD) Program under Marie Curie COFUNDII Actions. The Gilmour lab was supported by the European Molecular Biology Laboratory (EMBL), the University of Zurich (UZH) and Swiss National Science Funds Grant 31003A_176235.

Animal experimentation: All experiments on zebrafish embryos were performed between 0-3 days post-fertilization according to the rules of the European Molecular Biology Laboratory, following the guidelines of the European Commission, Directive 2010/63/EU.

© 2020, Hartmann et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.