Biological systems are increasingly viewed through a quantitative lens that demands accurate measures of gene expression and local protein concentrations. CRISPR/Cas9 gene tagging has enabled increased use of fluorescence to monitor proteins at or near endogenous levels under native regulatory control. However, due to typically lower expression levels, experiments using endogenously-tagged genes run into limits imposed by autofluorescence (AF). AF is often a particular challenge in wavelengths occupied by commonly used fluorescent proteins (GFP, mNeonGreen). Stimulated by our work in C. elegans, we describe and validate Spectral Autofluorescence Image correction By Regression (SAIBR), a simple, platform-independent protocol and FIJI plugin to correct for autofluorescence using standard filter sets and illumination conditions. Validated for use in C. elegans embryos, starfish oocytes and fission yeast, SAIBR is ideal for samples with a single dominant AF source, and achieves accurate quantitation of fluorophore signal and enables reliable detection and quantification of even weakly expressed proteins. Thus, SAIBR provides a highly accessible, low barrier way to incorporate AF correction as standard for researchers working on a broad variety of cell and developmental systems.

We investigate planar cell polarity (PCP) in the Drosophila larval epidermis. The intricate pattern of denticles depends on only one system of PCP, the Dachsous/Fat system. Dachsous molecules in one cell bind to Fat molecules in a neighbour cell to make intercellular bridges. The disposition and orientation of these Dachsous–Fat bridges allows each cell to compare two neighbours and point its denticles towards the neighbour with the most Dachsous. Measurements of the amount of Dachsous reveal a peak at the back of the anterior compartment of each segment. Localization of Dachs and orientation of ectopic denticles help reveal the polarity of every cell. We discuss whether these findings support our gradient model of Dachsous activity. Several groups have proposed that Dachsous and Fat fix the direction of PCP via oriented microtubules that transport PCP proteins to one side of the cell. We test this proposition in the larval cells and find that most microtubules grow perpendicularly to the axis of PCP. We find no meaningful bias in the polarity of microtubules aligned close to that axis. We also reexamine published data from the pupal abdomen and find no evidence supporting the hypothesis that microtubular orientation draws the arrow of PCP.

10We investigate the mechanisms of planar cell polarity (PCP) in the larval epidermis of 11 Drosophila. Measurements of the amount of Dachsous across the segment find a peak 12 located near the rear of the anterior compartment. Localisation of Dachs and 13 orientation of ectopic denticles reveal the polarity of every cell in the segment. We 14 discuss how well these findings evidence a zigzag gradient model of Dachsous activity. 15 Several groups have proposed that Dachsous and Fat fix the direction of PCP via 16 oriented microtubules that transport PCP proteins to one side of the cell. We test this 17 proposition in the larval cells and find that most microtubules grow perpendicularly 18 to the axis of PCP. We find no significant bias in the polarity of those microtubules 19 aligned close to that axis. We also reexamine published data from the pupal abdomen 20 and fail to find evidence supporting the hypothesis that microtubular orientation 21 draws the arrow of PCP. 22As cells construct embryos and organs they need access to vectorial information that 24 informs them, for example, which way to migrate, divide, extend axons or how to 25 orient protrusions. In Drosophila there are (at least) two conserved genetic systems 26 Page 2 of 32 that generate vectorial information, and here we are concerned with only one of those, 27 the Dachsous/Fat system. Dachsous (Ds) and Fat (Ft) are large atypical cadherin 28 molecules that form heterodimeric bridges from cell to cell that help build planar cell 29 polarity (PCP) in one cell and also convey polarity between cells (Ma et al., 2003; 30 Matakatsu and Blair, 2004; Casal et al., 2006; Lawrence and Casal, 2018). The 31 activity of Ft is increased and Ds is reduced when they are phosphorylated by a third 32 molecule, Four-jointed (Fj), a Golgi-based kinase (Ishikawa et al., 2008; Brittle et al., 33 2010; Simon et al., 2010). The distribution of Ds, Ft and Fj and the interaction 34 between these molecules together determine what we describe as "Ds activity", by 35 which we mean the propensity of Ds on one cell to bind to Ft in the neighbouring cell. 36 Experiments suggest that, using the disposition and orientation of Ds-Ft bridges, each 37 cell compares the Ds activity of its two neighbours and points its denticles towards the 38 neighbour with the higher Ds activity. In this way, the local slopes in a landscape of Ds 39 activity determine polarities of all the cells (Casal et al., 2002; see Lawrence and 40 Casal, 2018 for more explanation). 41 A model: the ventral epidermis of the Drosophila larva 42Each segment of the larva is divided by cell lineage into an anterior (A) and a 43 posterior (P) compartment. In the ventral epidermis, a limited region of the segment 44 makes rows of thorny denticles that are polarised in an almost invariant pattern, while 45 the larger part of each segment makes no denticles and therefore its polarity is not 46 known (Figure 1-figure supplement 1). The Ds/Ft system is alone effective in 47 48 the "core" or Stan/Fz system (Casal et ...