Gene expression analysis on sections of zebrafish regenerating fins reveals limitations in the whole‐mount in situ hybridization method

Smith, Amanda; Zhang, J.; Guay, D.; Quint, Elizabeth; Johnson, Amanda M.; Akimenko, Marie‐Andrée

doi:10.1002/dvdy.21417

Cited by 74 publications

(67 citation statements)

References 28 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We detected msxb, a marker of de-differentiated cells and of the distal blastema (Nechiporuk and Keating, 2002), in the entire blastema and in the differentiation zone of the 3 dpa regenerating fin (supplementary material Fig. S1B), consistent with the reported expression of msxb in osteoblasts (Smith et al, 2008). A second…”

Section: Notch Pathway Genes Are Expressed In Blastema Cellssupporting

confidence: 87%

Notch regulates blastema proliferation and prevents differentiation during adult zebrafish fin regeneration

2013

View full text Add to dashboard Cite

SUMMARYZebrafish have the capacity to regenerate several organs, including the heart and fins. Fin regeneration is epimorphic, involving the formation at the amputation plane of a mass of undifferentiated, proliferating mesenchymal progenitor-like cells, called blastema. This tissue provides all the cell types that form the fin, so that after damage or amputation the fin pattern and structure are fully restored. How blastema cells remain in this progenitor-like state is poorly understood. Here, we show that the Notch pathway plays an essential role during fin regeneration. Notch signalling is activated during blastema formation and remains active throughout the regeneration process. Chemical inhibition or morpholino-mediated knockdown of Notch signalling impairs fin regeneration via decreased proliferation accompanied by reduced expression of Notch target genes in the blastema. Conversely, overexpression of a constitutively active form of the Notch1 receptor (N1ICD) in the regenerating fin leads to increased proliferation and to the expansion of the blastema cell markers msxe and msxb, as well as increased expression of the proliferation regulator aldh1a2. This blastema expansion prevents regenerative fin outgrowth, as indicated by the reduction in differentiating osteoblasts and the inhibition of bone regeneration. We conclude that Notch signalling maintains blastema cells in a plastic, undifferentiated and proliferative state, an essential requirement for fin regeneration.

show abstract

Section: Notch Pathway Genes Are Expressed In Blastema Cellssupporting

confidence: 87%

Notch regulates blastema proliferation and prevents differentiation during adult zebrafish fin regeneration

2013

View full text Add to dashboard Cite

show abstract

“…from 72 hpa onwards (Smith et al, 2006;Smith et al, 2008;Brown et al, 2009). The gene expression analysis at early stages of fin regeneration presented here reveals that some of the genes that specify skeletal lineages are present as early as 24 hpa.…”

Section: Lepidotrichia Regeneration Is Initiated As Early As 24 Hpa Imentioning

confidence: 70%

“…For cross sections, fins were embedded in gelatin, processed using standard conditions and sectioned (10-14 m) using a cryostat (Leica). In situ hybridisation on cryosections was performed as described by Smith et al (Smith et al, 2008). Details for RNA probes for sox9a, osterix (sp7 -Zebrafish Information Network), col1a2 and col10a1 genes were described by Li et al (Li et al, 2009).…”

Section: Whole-mount In Situ Hybridisationmentioning

confidence: 99%

“…From expression studies of skeletogenesis genes at 72 and 96 hpa (Smith et al, 2006;Smith et al, 2008;Brown et al, 2009) it is thought that during caudal fin regeneration lepidotrichia regeneration starts after the blastema formation. However, gene expression analysis at earlier phases has never been done systematically.…”

Section: Prompt Activation Of Skeletogenesis Genes Reveals An Early Omentioning

confidence: 99%

See 1 more Smart Citation

Differentiated skeletal cells contribute to blastema formation during zebrafish fin regeneration

et al. 2011

View full text Add to dashboard Cite

SUMMARYThe origin of cells that generate the blastema following appendage amputation has been a long-standing question in epimorphic regeneration studies. The blastema is thought to originate from either stem (or progenitor) cells or differentiated cells of various tissues that undergo dedifferentiation. Here, we investigate the origin of cells that contribute to the regeneration of zebrafish caudal fin skeletal elements. We provide evidence that the process of lepidotrichia (bony rays) regeneration is initiated as early as 24 hours post-amputation and that differentiated scleroblasts acquire a proliferative state, detach from the lepidotrichia surface, migrate distally, integrate into the blastema and dedifferentiate. These findings provide novel insights into the origin of cells in epimorphic appendage regeneration in zebrafish and suggest conservation of regeneration mechanisms between fish and amphibians.

show abstract

“…In situ hybridization of whole fins was performed as described (Poss et al, 2000b) with 15 minute proteinase K (20 mg/ml, Sigma-Aldrich) treatment. In situ hybridization on fin cryosections was according to (Smith et al, 2008).…”

Section: In Situ Hybridizationmentioning

confidence: 99%

IGF signaling between blastema and wound epidermis is required for fin regeneration

2010

View full text Add to dashboard Cite

In mammals, the loss of a limb is irreversible. By contrast, urodele amphibians and teleost fish are capable of nearly perfect regeneration of lost appendages. This ability depends on direct interaction between the wound epithelium and mesenchymal progenitor cells of the blastema. It has been known for decades that contact between the wound epithelium and the underlying blastema is essential for successful regeneration. However, the underlying mechanisms are poorly understood. Here, we show that upon amputation the blastema induces expression of the ligand Igf2b, which then activates IGF signaling specifically in cells of the adjacent apical epithelium. Inhibition of IGF signaling by either morpholino antisense technology, or by specific chemical inhibitors of Igf1 receptor function NVP-AEW541 and NVP-ADW742, impairs fin regeneration. At the cellular level, this block in regeneration is reflected by a lack of the distinctive basal epithelium, increased apoptosis in the wound epidermis and reduced proliferation of blastema cells. Furthermore, induction of the blastemal and wound epidermal markers cannot be supported in the absence of IGF signaling. These data provide evidence that Igf2b expressed in the blastema promotes the properties of the adjacent wound epidermis, which subsequently are necessary for blastema function. Thus, IGF signaling upregulated upon fin amputation represents a signal from the blastema to the wound epithelium, a crucial step in appendage regeneration.

show abstract

Gene expression analysis on sections of zebrafish regenerating fins reveals limitations in the whole‐mount in situ hybridization method

Cited by 74 publications

References 28 publications

Notch regulates blastema proliferation and prevents differentiation during adult zebrafish fin regeneration

Notch regulates blastema proliferation and prevents differentiation during adult zebrafish fin regeneration

Differentiated skeletal cells contribute to blastema formation during zebrafish fin regeneration

IGF signaling between blastema and wound epidermis is required for fin regeneration

Contact Info

Product

Resources

About