a Schematic overview of miR interaction with mRNA. b Summary of miRs known to respond to specific stimuli in chondrocytes, and the genes which are affected by changes to these miR expression levels. The mechanical stimulus found to alter miR expression is listed on the left, the miR and how its expression is altered is displayed in the central column and the downstream gene targets of each miR are shown in the right column. ⁽¹⁾Dunn et al. 2009, ⁽²⁾Guan et al. 2011, ⁽³⁾Cheleschi et al. 2017

Zebrafish craniofacial cartilage has key components found in human articular cartilage. a Schematic representation of the cartilage elements which make up the zebrafish craniofacial skeleton at 5 dpf. Orientation compass included in bottom right, with A anterior and P posterior. b, c Ventral view confocal image stacks of 5 dpf craniofacial skeleton immunostained for type II collagen (b) and type I collagen (c) in the ECM surrounding chondrocytes. Inset shows zoom of jaw joint. Scale bar = 100 μm. Images in b and c previously published in (Lawrence et al. 2018). d–f Ventral paraffin sections of 5 dpf craniofacial skeleton stained with Safranin O (d) to show proteoglycans, Masson’s trichrome (e) to show the presence of collagen, and alcian blue (f) to show the presence of glycosaminoglycans in the chondrocyte ECM which makes up the cartilage. MC Meckel’s cartilage, JJ jaw joint, PQ palatoquadrate, C ceratohyal, Ch example of a chondrocyte surrounded by ECM, EP ethmoid plate

Tools available in zebrafish to facilitate spatiotemporal study of miRNA targets at a cellular level. a–c Ventral view confocal images of the craniofacial cartilages of Tg(4.9Sox10:EGFP;col2a1aBAC:mcherry) (a), Tg(col2a1aBAC:mcherry;col10a1aBAC:citrine)hu7050 (b), Tg(- 4.9Sox10:EGFP; 4xGTIIC:eGFP) (c) imaged live. Scale bar = 100 μm. d’, d’’ Ventral view confocal images of the craniofacial cartilages of Tg(Sox10:GAL4- VP16; UAS:Kaede) with photoconverted chondrocytes shown in magenta and annotated with a white arrowhead in the same fish at 4 dpf (d’) and 5dpf (d’’). Scale bar = 100 μm. e’–e’’ Confocal images of the lower jaw joint in Tg(Sox10:GAL4- VP16; UAS:Kaede) with photoconverted chondrocytes shown in magenta and annotated with a white arrowhead in the same fish at 3 dpf (e’) and 5dpf (e’’). Scale bar = 50 μm, images in e’ and e’’ previously published in (Brunt et al. 2017). f, g Ventral view confocal images of the craniofacial cartilages of Tg(5xBMPRE-Xla.Id3:GFP; col2a1aBAC:mCherry) (f) and Tg(7xTCF.XlaSiam:nlsGFP;col2a1aBAC:mCherry) (g) imaged live. Scale bar = 100 μm. h Finite element model of maximum principal (E. max) strains in 5dpf wild-type zebrafish viewed from a dorsal orientation. E. max represents tensional strains with cooler colours (blue) on the scale corresponding to lower strain and red corresponding to higher strain values. MC Meckel’s cartilage, JJ jaw joint, PQ palatoquadrate, C ceratohyal