Single-cell resolution of MET- and EMT-like programs in osteoblasts during zebrafish fin regeneration

Abstract

While humans have limited potential for limb regeneration, some vertebrates can regenerate bony appendages following amputation. During zebrafish fin regeneration, mature osteoblasts at the amputation stump dedifferentiate and migrate to the blastema, where they re-enter the cell cycle and then re-differentiate to form new bone. Osteoblastic cells exhibit dual mesenchymal and epithelial characteristics during fin regeneration, however little is known about why or how this occurs. Using single-cell RNA-sequencing, we found osteoprogenitors are enriched with components associated with the epithelial-to-mesenchymal transition (EMT) and its reverse, mesenchymal-to-epithelial transition (MET). In trajectory analyses, osteoblastic cells solely expressed EMT components, or transiently expressed MET components prior to expressing those for EMT. We found that cdh11, a cancer EMT marker, is expressed during osteoblast dedifferentiation. We also found that esrp1, a regulator of alternative splicing in epithelial cells whose expression is important for MET, is expressed in a subset of osteoprogenitors during outgrowth. This study provides a valuable single cell resource for the study of osteoblast differentiation during zebrafish fin regeneration, and identifies MET- and EMT-associated components which may be important for successful appendage regeneration.

En1 and Lmx1b do not recapitulate embryonic dorsal-ventral limb patterning functions during mouse digit tip regeneration

“…In contrast, Lmx1b has previously been characterized as anti-osteogenic during calvarial development, where Lmx1b is expressed in a population of mesenchymal cells that reside in the suture and loss of Lmx1b results in premature suture closure (Cesario et al, 2018). Lmx1b is also expressed in the joint mesenchyme surrounding the bone during mouse limb development and zebrafish fin regeneration (Dreyer et al, 2004; Tang et al, 2022). While our results seem contrary to these findings, it is possible that Lmx1b has a similar anti-osteogenic function in mouse digit tip regeneration.…”

wnt16 regulates spine and muscle morphogenesis through parallel signals from notochord and dermomyotome

“…To help distinguish whether wnt16 + cells were notochord sheath cells or osteoblastic cells condensing around the notochord, we performed co-staining for cdh11 , a marker of sclerotome in mouse embryos (Kimura et al, 1995) and mesenchymal-like osteoblastic cells in zebrafish (Tang et al, 2022) (Fig 5B). Staining for cdh11 was apparent in several locations associated with sclerotome domains, including in cells surrounding the notochord (Fig 5B, inset), the dorsal myotome (Fig 5B, long arrow), presumptive myosepta (Fig 5B, short arrow), and the medial portion of the ventral myotome (Fig 5B, arrowhead) (Ma et al, 2018).…”

“…Probes were purchased for wnt16, pax7a , and cdh11 from the manufacturer (ACD Bio). In situ hybridization was performed using the RNAScope 2.5 HD Duplex Detection kit essentially as per manufacturer instructions, with modifications described in (Tang et al, 2022). Whole mount RNA in situ hybridization was performed as previously described (Maves et al, 2007).…”

“…RNA in situ hybridization RNA-ISH using RNAScope was performed as we have previously described (Tang et al, 2022). Briefly, fixed tissues were washed in 1X PBS/0.05%Tween20 (PBS-T), then dehydrated in increasing graded concentrations of methanol and stored at −20°C.…”

“…To help distinguish whether wnt16+ cells were notochord sheath cells or osteoblastic cells condensing around the notochord, we performed co-staining for cdh11, a marker of sclerotome in mouse embryos (Kimura et al, 1995) and mesenchymal-like osteoblastic cells in zebrafish (Tang et al, 2022) (Ma et al, 2018). Notably, staining for wnt16 was localized within a single layer of cells surrounded by cells staining positively for cdh11 Fig 5B, inset).…”