Journal: bioRxiv

Article Title: Developmental system drift in dorsoventral patterning is linked to transitions to autonomous development in Annelida

doi: 10.1101/2025.05.29.656861

Figure Lengend Snippet: ( a ) Whole mount in situ hybridisation of the secondary messenger smad1/5/8 and receptors bmpr1 and bmpr2 of the BMP pathway at the blastula (6 hpf), gastrula (9 hpf) and larval (24 hpf) stages in O. fusiformis . ( b ) Schematic diagram of a multiple protein alignment of the MH domains and linker region of SMAD1/5/8 across bilaterian and cnidarians highlighting the high conservation of the MH domains. ( c ) Multiple protein alignment in the linker region of SMAD1/5/8 showing the presumptive MAPK phosphorylation sites (highlighted in violet). Only Deuterostomes have all sites conserved. ( d ) Multiple protein alignment of the C-terminus of SMAD1/5/8 proteins showing the conserved residues recognised by the pSMAD1/5/8 antibody used in this study. In ( a ), the arrowheads point to the 4d organiser, and the asterisks mark the animal/apical pole. Scale bars are 50 µm. an anus, ao apical organ, bp blastopore, ch chaetae, cs chaetal sac, fg foregut, lat lateral, mo mouth, pt prototroch, veg vegetal.

Article Snippet: To detect the activation of SMAD1/5/8, the primary antibody rabbit anti-Phospho- Smad1/5 (Cell Signaling Technology; Cat No.: 9516) was used following a modified protocol used in other spiralians .

Techniques: In Situ, Hybridization, Phospho-proteomics

Journal: Nature Communications

Article Title: Distinct ossification trade-offs illuminate the shoulder girdle reconfiguration at the water-to-land transition

doi: 10.1038/s41467-025-60236-z

Figure Lengend Snippet: a The experimental scheme of the unbiased high-throughput genomic screening (see Methods). From CNE14:egfp; gli3 +/+ and CNE14:egfp; gli3 14ins/14ins embryos, EGFP-positive cells were separately isolated by FACS. The sorted cells were subjected to RNA-seq and ATAC-seq. Three biological replicates were conducted for RNA-sequencing and two were for ATAC-seq. b The heat map of top 2000 differentially expressed genes in CNE14:egfp; gli3 +/+ and CNE14:egfp; gli3 14ins/14ins embryos. Color code: red indicates high and blue indicates low enrichment. c The volcano plot of the RNA-seq result. The genes with p < 0.5 and >two-fold expression change are coded by red. The genes on the left side (Log2 fold change <0) are down-regulated genes in gli3 14ins/14ins embryos while the genes on the right side (Log2 fold change > 0) are up-regulated genes in the mutant embryos. Acvr11 is one of the prominent Gli3 candidate genes. The statistical analysis was conducted by EdgeR program. d The genome browser visualization of zebrafish RNA-seq (wildtype and gli3 14ins/14ins samples), ATAC-seq, and HiC results, and skate RNA-seq and ATAC-seq results at the acvr1l locus. In zebrafish, the transcript level is lower at the exons of acvr1l in gli3 14ins/14ins embryos than gli3 +/+ embryos. Zebrafish ATAC-seq and HiC showed that the promoter region of acvr1l is ACR and in a TAD. Zebrafish HiC result was produced from the previously published data . Skate RNA-seq and ATAC-seq data were generated from the previous paper . e , f The expression pattern of acvr1l in gli3 +/+ and gli3 14ins/14ins embryos. Expression in the pectoral fin is decreased in gli3 14ins/14ins embryos compared to gli3 +/+ embryos. n = 24. The scale is the same in ( e , f ). g – i Immunofluorescence of phosphorylated-Smad 1/5/8. The strong signal is observed in cells surrounding the cleithrum of wildtype embryos ( g , arrows), which decreases to gli3 14ins/14ins ( h ) and to gli2b 17ins/17ins ; gli3 14ins/14ins embryos ( i ). n = 6 for wildtype, 9 for gli3 14ins/14ins , and 5 for gli2b 17ins/17ins ; gli3 14ins/14ins embryos. j a bar graph of phosphorylated Smad1/5/8 staining level in gli3 +/+ , gli3 14ins/14ins and gli2b 17ins/17ins embryos. The fluorescence intensity in the cleithrum was quantified and standardized by the area size in the cleithrum (“Method”). All raw data points were plotted. Raw data are in Supplementary Table . The data were analyzed and compared among samples by two-tailed student’s t-test. * indicates p < 0.05. p -value between gli3 +/+ and gli3 14ins/14ins is 0.049 and between gli3 +/+ (WT) and gli3 14ins/14ins is 0.025. While gli3 14ins/14ins and gli2b 17ins/17ins ; gli3 14ins/14ins embryos exhibit statistically significant reduction of the PSmad1/5/8 signal from wildtype embryos, gli3 14ins/14ins and gli2b 17ins/17ins ; gli3 14ins/14ins embryos do not show statistically significant change ( p = 0.28). The unit of vertical axis is arbitrary unit. n = 6, 9, and 5 biologically independent samples for wildtype embryos, gli3 14ins/14ins and gli2b 17ins/17ins ; gli3 14ins/14ins embryos, respectively. Error bars show standard deviations. k-l ) LDN193189-treated pectoral fins stained by HCR with sp7 and col2a1a probes and DAPI staining. Sp7 expression was diminished by 10 μM of the inhibitor treatment. Arrows indicate sp7 expression, and arrowheads show its weak or no expression. sc scapula. n = 23 for 0 and 10 μM. The scale bar in ( e , g ) are 50 μm. g – i , k , l are the same scale.

Article Snippet: EGFP antibody (Abcam #ab290) and phosphorylated Smad 1/5/8 antibody (Cell Signaling #13820) were used at 1/1000 and 1/100 dilution, respectively.

Techniques: High Throughput Screening Assay, Isolation, RNA Sequencing, Expressing, Mutagenesis, Produced, Generated, Immunofluorescence, Staining, Fluorescence, Two Tailed Test