Journal: bioRxiv

Article Title: Two opposing roles for Bmp signalling in the development of electrosensory lateral line organs

doi: 10.1101/2024.03.07.583945

Figure Lengend Snippet: Sterlet larvae after in situ hybridisation (ISH) for the hair cell and electroreceptor marker Cacna1d (also expressed in taste buds on barbels) or the electroreceptor-specific marker Kcnab3 . Black arrowheads indicate examples of neuromasts; white arrowheads indicate examples of ampullary organs. ( A-H ) Stage 45 larvae that had been treated for 20 hours from stage 36 (i.e., from hatching to approximately stage 38, just prior to the onset of ampullary organ development) with either DMH1 or DMSO as controls. Larvae are numbered for cross-referencing with ampullary organ counts in Supplementary Table S3. ISH for Cacna1d (A-D) or Kcnab3 (E-H) shows that, relative to DMSO-treated controls (A,B,E,F), DMH1-treated larvae have many more ampullary organs (C,D,G,H). This phenotype is particularly prominent in the three dorsal-most ampullary organ fields, where the dorsal supraorbital, dorsal otic and supratemporal fields - clearly separate in DMSO-treated larvae (A,B,E,F) - almost fuse together in DMH1-treated larvae (C,D,G,H). ( I,J ) A much older wild-type larva (2.8 cm in length, ∼65 dpf) after ISH for Cacna1d . The dorsal supraorbital, dorsal otic and supratemporal ampullary organ fields are clearly separated, suggesting the supernumerary ampullary organs in this region in DMH1-treated larvae (C,D,G,H) are ectopic, not precocious. ( K-N 1 ) Most DMH1-treated larvae also develop an ectopic offshoot from the supraorbital neuromast line. This is visible after ISH for Cacna1d (K,L; compare with DMSO control in A,B) and confirmed to represent neuromasts in DMH1-treated larvae via ISH for electroreceptor-specific Kcnab3 (M,N) followed by immunostaining for the supporting cell marker Sox2 to reveal neuromasts (M 1 ,N 1 ). Abbreviations: dot, dorsal otic ampullary organ field; ds, dorsal supraorbital ampullary organ field; e, eye; S, stage; so, supraorbital neuromast line; st, supratemporal ampullary organ field; WT, wild type. Scale bar: 250 μm.

Article Snippet: Stage 36 (post-hatching) yolksac larvae were incubated for 20 hours in 50 μM DMH1 (Cayman Chemical) in 1% dimethyl sulfoxide (DMSO) or in 1% DMSO as a control.

Techniques: In Situ, Hybridization, Marker, Immunostaining

Journal: bioRxiv

Article Title: Two opposing roles for Bmp signalling in the development of electrosensory lateral line organs

doi: 10.1101/2024.03.07.583945

Figure Lengend Snippet: ( A ) Scatter plot showing median and interquartile range for the total number of ampullary organs on one side of the head in stage 45 sterlet larvae that had been treated for 20 hours from stage 36 (i.e., from hatching to approximately stage 38, just prior to the onset of ampullary organ development) with DMH1 (n=17) or DMSO as controls (n=12). DMH1-treated larvae have significantly more ampullary organs (P<0.0001; two-tailed Mann-Whitney test). Ampullary organs were counted after in situ hybridisation [ISH] for Cacna1d or Kcnab3 ; raw counts are provided in Supplementary Table S3. ( B ) Scatter plots showing median and interquartile range for the number of ampullary organs in each individual ampullary organ field on one side of the head in stage 45 sterlet larvae that had been treated for 20 hours from stage 36 with DMH1 (n=17), versus with DMSO as controls (n=12). Raw counts are provided in Supplementary Table S3. For the location of each field, see schematic in panel C (reproduced from ). Scatter plots are grouped with differently coloured titles according to lateral line placode (LLp) origin, following : blue, anterodorsal LLp origin (supraorbital and infraorbital fields); orange, anteroventral LLp origin (preopercular fields); green, otic LLp origin (dorsal otic field); pink, supratemporal LLp origin (supratemporal field). All fields have significantly more ampullary organs in DMH1-treated larvae (n=17) than in DMSO controls (two-tailed Mann-Whitney tests). Asterisks on plots represent P values: **, P≤0.01; ***, P≤0.001; ****, P≤0.0001. P values for all fields are <0.0001 except for the ventral supraorbital field (P=0.0074), anterior preopercular field (P=0.0002) and posterior preopercular field (P=0.0003). ( C ) Schematic of a stage 45 sterlet larval head. Ampullary organ fields are represented by coloured patches flanking the neuromast lines, which are represented as dotted lines. The different field colours indicate their lateral line placode origin (consistent with scatter plot titles in B). Abbreviations for ampullary organ fields: app, anterior preopercular; di, dorsal infraorbital; dot, dorsal otic; ds, dorsal supraorbital; ppp, posterior preopercular; st, supratemporal; vi, ventral infraorbital; vs, ventral supraorbital. Abbreviations for neuromast lines: io, infraorbital; m, middle; ol, otic; pop, preopercular; so, supraorbital; st, supratemporal. Abbreviations for anatomical landmarks: an, anterior naris; b, barbel; e, eye; m, mouth; op, operculum; ot, otic vesicle; pn, posterior naris; s, spiracle (first gill cleft).

Article Snippet: Stage 36 (post-hatching) yolksac larvae were incubated for 20 hours in 50 μM DMH1 (Cayman Chemical) in 1% dimethyl sulfoxide (DMSO) or in 1% DMSO as a control.

Techniques: Two Tailed Test, MANN-WHITNEY, In Situ, Hybridization

Journal: bioRxiv

Article Title: Two opposing roles for Bmp signalling in the development of electrosensory lateral line organs

doi: 10.1101/2024.03.07.583945

Figure Lengend Snippet: ( A,B ) Scatter plots showing median and interquartile range for the number of ampullary organs on one side of the head in stage 45 sterlet larvae that had been treated for 20 hours from stage 36 with DMH1 (n=17) versus 2.0/2.8 cm wild-type larvae (∼50/65 dpf; n=10). Raw counts are provided in Supplementary Table S3. Two-tailed Mann-Whitney tests were used for statistical analysis. DMH1-treated larvae have significantly more ampullary organs overall at stage 45 than wild-type older larvae (P<0.0001; A). In B, scatter plots are grouped with differently coloured titles according to lateral line placode (LLp) origin, following : blue, anterodorsal LLp (supraorbital and infraorbital fields); orange, anteroventral LLp (preopercular fields); green, otic LLp (dorsal otic field); pink, supratemporal LLp (supratemporal field). DMH1-treated larvae have significantly more ampullary organs at stage 45 than older wild-type larvae in all fields except the ventral supraorbital and posterior preopercular fields. Symbols on plots represent P values: ns, not significant, P>0.05; *, P≤0.05; **, P≤0.01; ***, P≤0.001; ****, P≤0.0001). Dorsal supraorbital: P<0.0001. Ventral supraorbital: not significant (P=0.5109). Dorsal infraorbital: P=0.0123. Ventral infraorbital: P=0.0002. Anterior preopercular: P=0083. Posterior preopercular: not significant (P=0.1789). Dorsal otic: P<0.0001. Supratemporal: P<0.0001. ( C ) Schematic of a stage 45 sterlet larval head. Ampullary organ fields are represented by coloured patches flanking the neuromast lines, which are represented as dotted lines. The different field colours indicate their lateral line placode origin (consistent with scatter plot titles in B). Abbreviations for ampullary organ fields: app, anterior preopercular; di, dorsal infraorbital; dot, dorsal otic; ds, dorsal supraorbital; ppp, posterior preopercular; st, supratemporal; vi, ventral infraorbital; vs, ventral supraorbital. Abbreviations for neuromast lines: io, infraorbital; m, middle; ol, otic; pop, preopercular; so, supraorbital; st, supratemporal. Other abbreviations: an, anterior naris; b, barbel; e, eye; m, mouth; op, operculum; ot, otic vesicle; pn, posterior naris; s, spiracle (first gill cleft); WT, wild type.

Article Snippet: Stage 36 (post-hatching) yolksac larvae were incubated for 20 hours in 50 μM DMH1 (Cayman Chemical) in 1% dimethyl sulfoxide (DMSO) or in 1% DMSO as a control.

Techniques: Two Tailed Test, MANN-WHITNEY

Journal: Development (Cambridge, England)

Article Title: Wnt/β-catenin and FGF signalling direct the specification and maintenance of a neuromesodermal axial progenitor in ensembles of mouse embryonic stem cells

doi: 10.1242/dev.112979

Figure Lengend Snippet: Elongation process. (A) Examples of aggregates from Sox1::GFP mESCs aggregated for 2 days in N2B27 before further treatment with N2B27, which may include a 24 h pulse on day 3 with Chiron, 3 days of Chiron, or Chiron and the MEK inhibitor PD03, or 3 days with the BMP inhibitor DMH1. Aggregates were imaged on day 5 by wide-field epifluorescence microscopy. The phase-contrast and fluorescence images shown are representative examples. Aggregates exposed to a 24 h pulse of Chiron are able to show a single large extension containing Sox1::GFP-expressing cells with a region at the tip that is negative for the fluorescence reporter. (B) Wnt reporter TLC2 mESC aggregates differentiated in N2B27 with a 24 h pulse of Chiron on day 3 and imaged on day 5. (C) Aggregate as in B fixed and immunostained for brachyury (white) and Sox2 (green). The fluorescent reporter is expressed predominately in the aggregate extension corresponding to a Sox2 + Bra + region. The white arrowhead indicates the region of highest Sox2 expression. Hoechst was used to label the nuclei. Scale bars: 500 µm in A; 200 µm in B,C.

Article Snippet: Using Sox1::GFP mESCs and exposing them to continuous differentiation in N2B27, we observe the emergence of Sox1::GFP-positive cells throughout the aggregate ( A) and a similar pattern is observed if, on day 3, the aggregates are exposed to DMH1, a BMP inhibitor ( A; 2-5 DMH1), SB43, an activin/nodal inhibitor (data not shown) or PD03 ( A; 2-5 Chi+PD03).

Techniques: Epifluorescence Microscopy, Fluorescence, Expressing