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

Abstract

Summary Zebrafish regenerate fin rays following amputation through epimorphic regeneration, a process that has been proposed to involve the epithelial-to-mesenchymal transition (EMT). We performed single-cell RNA sequencing (scRNA-seq) to elucidate osteoblastic transcriptional programs during zebrafish caudal fin regeneration. We show that osteoprogenitors are enriched with components associated with EMT and its reverse, mesenchymal-to-epithelial transition (MET), and provide evidence that the EMT markers cdh11 and twist2 are co-expressed in dedifferentiating cells at the amputation stump at 1 dpa, and in differentiating osteoblastic cells in the regenerate, the latter of which are enriched in EMT signatures. We also show that esrp1 , a regulator of alternative splicing in epithelial cells that is associated with MET, is expressed in a subset of osteoprogenitors during outgrowth. This study provides a single cell resource for the study of osteoblastic cells during zebrafish fin regeneration, and supports the contribution of MET- and EMT-associated components to this process.

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

“… 63 Lmx1b is also expressed in the joint mesenchyme surrounding the bone during mouse limb development and zebrafish fin regeneration. 33 , 64 While our results seem contrary to these findings, it is possible that Lmx1b has a similar anti-osteogenic function in mouse digit tip regeneration. Loss of Lmx1b in blastema cells could cause premature osteogenic differentiation during regeneration, thereby disrupting regeneration and causing loss of regenerated bone length and volume.…”

Regeneration in calcareous sponge relies on ‘purse-string’ mechanism and the rearrangements of actin cytoskeleton

“…The former type is based on transformations of epithelial cell layers in an injured area with minor or no contribution of cell proliferation. Mesenchymal morphogenesis, on the other hand, assumes blastema formation through intensive proliferation of mesenchymal cells undergoing the EMT and MET (Hay and Zuk, 1995; Vervoort, 2011; Borisenko et al ., 2015; Alibardi, 2022; Tang et al ., 2022).…”

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 [36] and mesenchymal-like osteoblastic cells in zebrafish [37] Staining for wnt16 was detected in notochord (star) and was mostly negative for pax7a. Staining for wnt16 was also detected in cells in the lateral portion of the myotome (inset, long arrow), within or adjacent to presumptive myosepta (inset, short arrow), and sporadically within the myotome (inset, arrowhead).…”

“…RNA-ISH using RNAScope was performed as we have previously described [37]. 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.…”

“…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 [37]. Whole mount RNA in situ hybridization was performed as previously described The cDNA probes used were: myog [88], ntla/tbxta [89], and pax7a [90].…”