dkk1 protein  (R&D Systems)

Journal: EvoDevo

Article Title: The evolution of cephalic fins in manta rays and their relatives: functional evidence for initiation of domain splitting and modulation of the Wnt signaling pathway in the pectoral fin AER of the little skate

doi: 10.1186/s13227-024-00233-3

Figure Lengend Snippet: DKK1 bead implantation interrupts fin ray outgrowth in the little skate. Compare the control bead ( A ) vs. DKK1-soaked beads ( B – F ). Note the reduced fin ray outgrowth in 100% embryos implanted with a DKK1-soaked bead ( n = 21), which is not observed with the control bead. The truncated fin rays observed in association with DKK1 bead implantations ( B – F ) resemble the shorter fin rays observed in the “notch” mirroring the early stages of pectoral fin domain splitting in the myliobatid phenotype (Fig. ). See text for explanation of why the phenotype could not be driven to completion due to limited activity of the ectopic DKK1 protein. Pictures show dorsal views of the anterior pectoral fins of six different little skate embryos that have been incubated for 4 weeks after bead implantation. Scale bar represents 2.5 mm

Article Snippet: O’Shaughnessy (personal communication) with the following modifications: Affigel-Blue (Bio-Rad, 1537302) beads were soaked overnight at 4 °C in DKK1 protein (mouse origin; R&D Systems, 5897-DK-010) reconstituted at 100 μg/mL in PBS containing 0.1% bovine serum albumin (BSA) or, as a control, in PBS containing 0.1% BSA.

Techniques: Control, Activity Assay, Incubation

Journal: EvoDevo

Article Title: The evolution of cephalic fins in manta rays and their relatives: functional evidence for initiation of domain splitting and modulation of the Wnt signaling pathway in the pectoral fin AER of the little skate

doi: 10.1186/s13227-024-00233-3

Figure Lengend Snippet: Ectopic DKK1 interrupts AER maintenance/ Axin2 expression in the little skate pectoral fin for up to 72 h. Compare left column with interrupted Axin2 expression near the Dkk1 soaked bead with the right column that shows stage-specific native expression with implantation of a control bead. Axin2 expression is depicted in the anterior pectoral fins of little skate embryos at stages 30 ( A , B ; G , H ) and 31 ( C – F ; I , J ) of development after implantation of agarose beads soaked in DKK1 protein in the left fin (left column) and PBS in the right fin (right column) with the following incubation times: 6 h ( A , B ), 12 h ( C , D ), 24 h ( E , F ), 36 h ( G , H ) and 72 h ( I , J ). Comparisons between the DKK1 (left pectoral fin) and PBS control (right pectoral fin) beads illustrate treatment effects from implantations in the same individual. In some individuals ( C , D and G , H ), beads came out during the ISH process. Former location of beads is visible and denoted by blue dashed circles. Solid purple circles denote regions with Axin2 expression and dashed purple circles denote regions where Axin2 expression is expected but has been interrupted. For all incubation times, PBS beads had no effect on Axin2 expression. After 6 h, interruption of AER-associated Axin2 expression in the DKK1 treatment is clearly visible near the bead ( A , B ). After 12, 24, and 36 h incubation times, Axin2 expression in the anterior pectoral fins was completely interrupted. After 72 h of incubation ( I , J ), the effect of DKK1 inhibition on Axin2 is diminished but still visible near the bead, suggesting that the temporal scale of DKK1 protein activity is approximately 1–3 days. Axin2 expression in the posterior pectoral fins ( L , M ) was not affected in any individual regardless of treatment or incubation time, suggesting a spatial scale of approximately 1 cm from the implanted bead. Posterior pectoral fins at 24 hpi are depicted as this is when DKK1 reaches peak penetrance. Sense probe (see B ) showed no staining. Since embryos with different incubation times were at different developmental stages, the expected expression domain of Axin2 varies accordingly. Scale bar represents 1 mm

Article Snippet: O’Shaughnessy (personal communication) with the following modifications: Affigel-Blue (Bio-Rad, 1537302) beads were soaked overnight at 4 °C in DKK1 protein (mouse origin; R&D Systems, 5897-DK-010) reconstituted at 100 μg/mL in PBS containing 0.1% bovine serum albumin (BSA) or, as a control, in PBS containing 0.1% BSA.

Techniques: Expressing, Control, Incubation, Inhibition, Activity Assay, Staining

Journal: EvoDevo

Article Title: The evolution of cephalic fins in manta rays and their relatives: functional evidence for initiation of domain splitting and modulation of the Wnt signaling pathway in the pectoral fin AER of the little skate

doi: 10.1186/s13227-024-00233-3

Figure Lengend Snippet: Dkk1 expression modifies AER outgrowth during pectoral fin development. Here, we demonstrate Dkk1 expression in cownose ray cephalic fins at stage 3 (stage 31 in skates), the only stage available for this experiment, which is after development of the “notch”. A No staining with the Dkk1 control sense probe. B , C Expression in the distal ridge of the AER indicated by purple staining with the Dkk1 antisense experimental probe on the left side ( B ), and right side ( C ). Purple arrows denote regions where Dkk1 expression occurs. While Dkk1 was expressed in the “notch” region at an earlier stage (based on ), it is still expressed in the cephalic fin AER where fin ray length is somewhat reduced. Scale bar represents 2.5 mm

Article Snippet: O’Shaughnessy (personal communication) with the following modifications: Affigel-Blue (Bio-Rad, 1537302) beads were soaked overnight at 4 °C in DKK1 protein (mouse origin; R&D Systems, 5897-DK-010) reconstituted at 100 μg/mL in PBS containing 0.1% bovine serum albumin (BSA) or, as a control, in PBS containing 0.1% BSA.

Techniques: Expressing, Staining, Control