Journal: Development (Cambridge, England)

Article Title: Analysis of cranial tenocyte heterogeneity reveals a role for Wnt signaling in tendon attachments

doi: 10.1242/dev.205047

Figure Lengend Snippet: Subcluster and is HCR analyses of dermal bone and pectoral fin muscle attachments reveals spatial heterogeneity of MTJ and enthesis tenocytes. (A) Diagram of lateral view of cranial musculoskeletal tissues in a 72 hpf embryo showing tenocytes (magenta), ligamentocytes (red), muscle fibers (blue) and chondrocytes (green) with cl-SH and pectoral fin tenocytes highlighted. (B-E,I-L,N-Q,U-W) Lateral views of 72 hpf embryos showing is HCR at the cl-SH tendon for ifitm5 (C,E), klf2a (D,E,K,L), col22a1 (P,Q) and thbs4b (V,W) and immunofluorescence for anti-mCherry (B,E,I,L,N,Q,U,W) and anti-phosphomyosin (P-myosin) (J,L,O,Q). (F,M) Magnified views of the region outlined by dotted lines in E and L, respectively, showing 3D-voxel volumes of tenocyte nuclei with mCherry staining, expression of ifitm5 and klf2a . (G) Violin plots with overlayed box plots showing expression of scxa , ifitm5 , klf2a and col22a1 across the re-clustering of cluster 30. P -values calculated using Wilcoxon rank sum test. **** P <0.0001. The box region comprises 50% of cells representing the interquartile range (IQR), while the bottom and top regions represent 25th and 75th percentile. The whiskers represent the spread of data outside the IQR within the normal variability of gene expression. (H) UMAP plot showing tenocyte cluster 30 (dermal bone tenocytes) re-clustered to display separation of distinct enthesis and MTJ domains marked by expression differences in G and T. (R,S,X,Y) Magnified views of the regions marked by blue and orange lines in Q and W displaying 3D voxel volumes of tenocyte nuclei with mCherry signal, and is HCR expression of col22a1 and thbs4b . (T) Feature plot displaying relative expression distributions of scxa , klf2a (enthesis markers) and thbs4b (MTJ marker) across cluster 30. Scale bars: 30 μm (B,N,U); 2 μm (F); 20 μm (I); 3 μm (M); 5 μm (R,S,X,Y).

Article Snippet: Embryos were stained using mouse monoclonal anti-Myosin heavy chain (MHC) antibody (Developmental Studies Hybridoma Bank, A4.1025; RRID:AB_528356) at 1:200 dilution, rat monoclonal anti-mCherry antibody (Invitrogen, M11217 ; RRID:AB_2536611) at 1:500 dilution, rabbit polyclonal anti p-Myosin light chain (p-MLC) antibody (Cell Signaling Technology, 3671; RRID:AB_330248) at 1:500 dilution, rabbit anti-Thrombospondin4b antibody (Thbs4b) (GeneTex, GTX125869; RRID:AB_2885605) at 1:1000 dilution.

Techniques: Immunofluorescence, Staining, Expressing, Gene Expression, Marker

Journal: Development (Cambridge, England)

Article Title: Analysis of cranial tenocyte heterogeneity reveals a role for Wnt signaling in tendon attachments

doi: 10.1242/dev.205047

Figure Lengend Snippet: Subcluster and is HCR analyses showing spatial heterogeneity of extraocular tenocytes. (A) Diagram of ventral view of the cranial musculoskeletal tissues in a Tg(scxa:mCherry; sox10:eGFP) 72 hpf embryo showing tenocytes (magenta), ligamentocytes (red), muscle fibers (blue) and chondrocytes (green) with the ventral extraocular muscle tendons (EOMTs) highlighted. (B,H) Ventral views of 72 hpf embryo heads stained with anti-mCherry and anti-Phosphomyosin (P-myosin) and is HCR for klf2b (B) and col22a1 (H). (C-E) Oblique views of the region marked by dotted lines in B showing is HCR expression of klf2b (C), colocalization of anti-mCherry signal and klf2b (D) and a magnified view of the region marked by dotted lines in D, with orange arrowheads marking EOMT enthesis cells with colocalized anti-mCherry signal and klf2b expression (E). (F) Violin plots with overlayed box plots showing expression of scxa , ifitm5 , klf2a , and col22a1 across the re-clustering of cluster 30. P -values calculated using Wilcoxon rank sum test. ** P <0.01, *** P <0.001, **** P <0.0001. The box region comprises 50% of cells representing the interquartile range (IQR), while the bottom and top regions represent 25th and 75th percentile. The whiskers represent the spread of data outside the IQR within the normal variability of gene expression. (G) UMAP plot displaying tenocyte cluster 19 (EOMT tenocytes) re-clustered to show separation of distinct ocular regions. (I-K) Oblique views of the region marked by dotted lines in H showing is HCR expression of col22a1 (I), colocalization of anti-mCherry signal and col22a1 (J) and a magnified view of the region marked by dotted lines in J with cyan arrowheads marking EOMT MTJ cells with colocalization of anti-mCherry signal and col22a1 expression (K). (L) Feature plot of cluster 19 showing relative expression of scxa , klf2b and col22a1 . Scale bars: 20 μm (B,H); 7 μm (C); 5 μm (E,I); 4 μm (K).

Article Snippet: Embryos were stained using mouse monoclonal anti-Myosin heavy chain (MHC) antibody (Developmental Studies Hybridoma Bank, A4.1025; RRID:AB_528356) at 1:200 dilution, rat monoclonal anti-mCherry antibody (Invitrogen, M11217 ; RRID:AB_2536611) at 1:500 dilution, rabbit polyclonal anti p-Myosin light chain (p-MLC) antibody (Cell Signaling Technology, 3671; RRID:AB_330248) at 1:500 dilution, rabbit anti-Thrombospondin4b antibody (Thbs4b) (GeneTex, GTX125869; RRID:AB_2885605) at 1:1000 dilution.

Techniques: Staining, Expressing, Gene Expression