Biology subjects

Le Bleu, H. K.

Publications and source records attributed to Le Bleu, H. K..

4 recordsLinked to original sources

bioRxiv2024

Voltage-gated calcium channels generate blastema Ca2+ fluxes restraining zebrafish fin regenerative outgrowth

Adult zebrafish fins regenerate to their original size regardless of damage extent, providing a tractable model of organ size and scale control. Gain-of-function of voltage-gated K+ channels expressed in fibroblast-lineage blastema cells promotes excessive fin outgrowth, leading to a long-finned phenotype. Similarly, inhibition of the Ca2+-dependent phosphatase calcineurin during regeneration causes dramatic fin overgrowth. However, Ca2+ fluxes and their potential origins from dynamic membrane voltages have not been explored or linked to fin size restoration. We used fibroblast-lineage GCaMP imaging of regenerating adult fins to identify widespread, heterogeneous Ca2+ transients in distal blastema cells. Membrane depolarization of isolated regenerating fin fibroblasts triggered Ca2+ spikes dependent on voltage-gated Ca2+ channel activity. Single cell transcriptomics identified the voltage-gated Ca2+ channels cacna1c (L-type channel), cacna1ba (N-type), and cacna1g (T-type) as candidate mediators of fibroblast-lineage Ca2+ signaling. Small molecule inhibition revealed L- and/or N-type voltage-gated Ca2+ channels act during regenerative outgrowth to restore fins to their original scale. Strikingly, cacna1g homozygous mutant zebrafish regenerated extraordinarily long fins due to prolonged outgrowth. The regenerated fins far exceeded their original length but with otherwise normal ray skeletons. Therefore, cacna1g mutants uniquely provide a genetic loss-of-function long-finned model that decouples developmental and regenerative fin outgrowth. Live GCaMP imaging of regenerating fins showed T-type Cacna1g channels enable Ca2+ dynamics in distal fibroblast-lineage blastemal mesenchyme during the outgrowth phase. We conclude "bioelectricity" for fin size control likely entirely reflects voltage-modulated Ca2+ dynamics in fibroblast-lineage blastemal cells that specifically and steadily decelerates outgrowth at a rate tuned to restore the original fin size.

developmental biology↗

bioRxiv2022

Insulin-like growth factor receptor / mTOR signaling elevates global translation to accelerate zebrafish fin regenerative outgrowth

Zebrafish robustly regenerate fins, including their characteristic bony ray skeleton. Amputation activates intra-ray fibroblasts and dedifferentiates osteoblasts that migrate under a wound epidermis to establish an organized blastema. Coordinated proliferation and re-differentiation across lineages then sustains progressive outgrowth. We generate a single cell transcriptome dataset to characterize regenerative outgrowth and explore coordinated cell behaviors. We computationally identify sub-classes of fibroblast-lineage cells and describe novel markers of intra- and inter-ray fibroblasts and growth-promoting distal blastema cells. A pseudotemporal trajectory and in vivo photoconvertible lineage tracing indicate distal blastemal mesenchyme generates both re-differentiated intra- and inter-ray fibroblasts. Gene expression profiles across this trajectory suggest elevated protein translation in the blastemal mesenchyme state. O-propargyl-puromycin incorporation and small molecule inhibition identify insulin growth factor receptor (IGFR) / mechanistic target of rapamycin kinase (mTOR)-dependent elevated bulk translation in blastemal mesenchyme and differentiating osteoblasts. We test candidate cooperating differentiation factors identified from the osteoblast trajectory, finding IGFR/mTOR signaling expedites glucocorticoid-promoted osteoblast differentiation in vitro. Concordantly, mTOR inhibition slows but does not prevent fin regenerative outgrowth in vivo. IGFR/mTOR may elevate translation in both fibroblast- and osteoblast-lineage cells during the outgrowth phase as a tempo-coordinating rheostat.

developmental biology↗

bioRxiv2019

longfin causes cis-ectopic expression of the kcnh2a ether-a-go-go K+ channel to autonomously prolong fin outgrowth

Organs stop growing to achieve a characteristic size and shape in scale with the animals body. Likewise, regenerating organs sense injury extents to instruct appropriate replacement growth. Fish fins exemplify both phenomena through their tremendous diversity of form and remarkably robust regeneration. The classic zebrafish mutant longfint2 develops and regenerates dramatically elongated fins and underlying ray skeleton. We show longfint2 chromosome 2 overexpresses the ether-a-go-go-related voltage-gated potassium channel kcnh2a. Genetic disruption of kcnh2a in cis rescues longfint2, indicating longfint2 is a regulatory kcnh2a allele. We find longfint2 fin overgrowth originates from prolonged outgrowth periods including by showing Kcnh2a chemical inhibition during late stage regeneration fully suppresses overgrowth. Cell transplantations demonstrate longfint2-ectopic kcnh2a acts tissue autonomously within the fin intra-ray mesenchymal lineage. Temporal inhibition of the Ca2+-dependent phosphatase calcineurin indicates it likewise entirely acts late in regeneration to attenuate fin outgrowth. Epistasis experiments suggest longfint2-expressed Kcnh2a inhibits calcineurin output to supersede growth cessation signals. We conclude ion signaling within the growth-determining mesenchyme lineage controls fin size by tuning outgrowth periods rather than altering positional information or cell-level growth potency.

developmental biology↗

bioRxiv2019

Transpositional scaling and niche transitions restore organ size and shape during zebrafish fin regeneration

Regenerating fish fins return to their original size and shape regardless of the nature or extent of injury. Prevailing models for this longstanding mystery of appendage regeneration speculate fin cells maintain uncharacterized positional identities that instruct outgrowth after injury. Using zebrafish, we find differential Wnt production correlates with the extent of regeneration across the caudal fin. We identify Dachshund transcription factors as markers of distal blastema cells that produce Wnt and thereby promote a pro-progenitor and -proliferation environment. We show these Dach-expressing \"niche cells\" derive from mesenchyme populating cylindrical and progressively tapered fin rays. The niche pool, and consequently Wnt, steadily dissipates as regeneration proceeds; once exhausted, ray and fin growth stops. Supported by mathematical modeling, we show longfint2 zebrafish regenerate exceptionally long fins due to a perdurant niche, representing a \"broken countdown timer\". We propose regenerated fin size is dictated by the amount of niche formed upon damage, which simply depends on the availability of intra-ray mesenchyme defined by skeletal girth at the injury site. Likewise, the fin reestablishes a tapered ray skeleton because progenitor osteoblast output reflects diminishing niche size. This \"transpositional scaling\" model contends mesenchyme-niche state transitions and positional information provided by self-restoring skeletal geometry rather than cell memories determine a regenerated fins size and shape.

developmental biology↗