Sonic hedgehog signaling promotes basal epidermal fibrillin 3 expression for zebrafish fin ray branching

Zebrafish robustly regenerate amputated fins, including restoring branched bony ray skeletons. During fin outgrowth, sonic hedgehog a (shha)-expressing basal epidermal cells (bEps) signal to themselves and adjacent progenitor osteoblasts (pObs) to progressively split pObs into daughter ray pools. Collectively moving bEps pass through a shha-expressing state to define distal shha-positive bEp domains that themselves separate laterally ahead of ray branching. As such, Shh/Smo signaling may promote transient associations between bEps and pObs that gradually splits pOb pools. We used bulk RNA sequencing of caudal fin regenerates after Smoothened (Smo) inhibition followed by a crispant screen to identify fibrillin 3 (fbn3) as a candidate Shh/Smo target gene for ray branching. Homozygous fbn3 mutant zebrafish regenerated caudal fins with fewer branched rays and increased distances to branch points. Shh/Smo signaling was required specifically for fbn3 induction in bEps but not pObs. Double homozygous mutants showed that fibrillin 2 (fbn2; FBN2 ortholog) redundantly supports fbn3-promoted ray branching. Fbn1-containing microfibrils accumulated at the bEp/pOb interface in fbn3 and, more so, fbn3/2 mutant regenerating caudal fins. Shh/Smo signaling may activate bEp fbn3 expression to locally modify microfibril structure and/or abundance, facilitating bEp-pOb interactions and/or movements underlying ray branching morphogenesis. Overall design: Samples were collected from shha:GFP zebrafish caudal fin regenerates at 4 days post amputation 5 hours after injection with injection buffer (50% PEG-400, 5% Propylene Glycol, 0.5% Tween-80) or 40 mg/kg of BMS-833923 from Cayman Chemical suspended in injection buffer. Only tissue distal to and including the shha:GFP domains were collected. Each sample is comprised of pools of 4 fin regenerates. This was done to compare transcript levels between controls (buffer injected) and Smoothened-inhibited (BMS-833923) regenerates to identify downstream targets of Sonic hedgehog signaling. All datasets were aligned to Danio rerio GRCz11.