Shh promotes direct interactions between epidermal cells and osteoblast progenitors to shape regenerated zebrafish bone

Abstract

Zebrafish innately regenerate amputated fins by mechanisms that expand and precisely position injury-induced progenitor cells to re-form tissue of the original size and pattern. For example, cell signaling networks direct osteoblast progenitors (pObs) to rebuild thin cylindrical bony rays with a stereotypical branched morphology. Hedgehog/ Smoothened (Hh/Smo) signaling has been variably proposed to stimulate overall fin regenerative outgrowth or promote ray branching. Using a photoconvertible patched2 reporter, we resolve active Hh/Smo output to a narrow distal regenerate zone comprising pObs and adjacent motile basal epidermal cells. This Hh/Smo activity is driven by epidermal Sonic hedgehog a (Shha) rather than Ob-derived Indian hedgehog a (Ihha), which nevertheless functions atypically to support bone maturation. Using BMS-833923, a uniquely effective Smo inhibitor, and high-resolution imaging, we show that Shha/Smo is functionally dedicated to ray branching during fin regeneration. Hh/ Smo activation enables transiently divided clusters of Shha-expressing epidermis to escort pObs into similarly split groups. This co-movement likely depends on epidermal cellular protrusions that directly contact pObs only where an otherwise occluding basement membrane remains incompletely assembled. Progressively separated pObs pools then continue regenerating independently to collectively reform a now branched skeletal structure.

Widening control of fin inter‐rays in zebrafish and inferences about actinopterygian fins

“…Although we have used an inhibitor with off‐target effects on cell proliferation (Armstrong et al. ), our results also support a regulation of initial inter‐ray widening by this pathway, as they agree with narrow inter‐ray phenotypes observed after laser ablation of shh ‐expressing (co‐expressing fgfr1 , Lee et al. ) cells (Zhang et al.…”

“…Growth arrest is an off‐target effect of this inhibitor (Armstrong et al. ), precluding any conclusion on SHH/IHH regulation. Nevertheless, these results suggest independent regulation of rays and inter‐rays.…”

“…; Armstrong et al. ). Although we have used an inhibitor with off‐target effects on cell proliferation (Armstrong et al.…”

“…) hedgehog signalling pathway (Armstrong et al. ), which are activated by several other regulators from the distal fibroblast/epidermal domain (Laforest et al. ; Lee et al.…”

Inhibition of mmp13a during zebrafish fin regeneration disrupts fin growth, osteoblasts differentiation, and Laminin organization

“…As the MO-injected regenerates were shorter than the controls, we examined Laminin pattern in 2 dpa noninjected regenerates which have a similar size as the 3 dpa MO-injected regenerate ( Figure 6C). 35 We observed that shha expression was not affected by mmp13a knockdown ( Figure 6E). Therefore, the defect in mmp13a MO-injected regenerates was not due to a delay in Laminin processing/distribution.…”