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-clusters representing most regenerative fin cell lineages, and define markers of osteoblasts, intra- and inter-ray fibroblasts and growth-promoting distal blastema cells. A pseudotemporal trajectory and in vivo photoconvertible lineage tracing indicate distal blastemal mesenchyme restores both intra- and inter-ray fibroblasts. Gene expression profiles across this trajectory suggest elevated protein production 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.

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 body of an animal. 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 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.

Histone demethylases Kdm6ba and Kdm6bb redundantly promote cardiomyocyte proliferation during zebrafish heart ventricle maturation.

Trimethylation of lysine 27 on histone 3 (H3K27me3) by the Polycomb repressive complex 2 (PRC2) contributes to localized and inherited transcriptional repression. Kdm6b (Jmjd3) is a H3K27me3 demethylase that can relieve repression-associated H3K27me3 marks, thereby supporting activation of previously silenced genes. Kdm6b is proposed to contribute to early developmental cell fate specification, cardiovascular differentiation, and/or later steps of organogenesis, including endochondral bone formation and lung development. We pursued loss-of-function studies in zebrafish to define the conserved developmental roles of Kdm6b. kdm6ba and kdm6bb homozygous deficient zebrafish are each viable and fertile. However, loss of both kdm6ba and kdm6bb shows Kdm6b proteins share redundant and pleiotropic roles in organogenesis without impacting initial cell fate specification. In the developing heart, co-expressed Kdm6b proteins promote cardiomyocyte proliferation coupled with the initial stages of cardiac trabeculation. While newly formed trabecular cardiomyocytes display a striking transient decrease in bulk cellular H3K27me3 levels, this demethylation is independent of collective Kdm6b. Our results indicate a restricted and likely locus-specific role for Kdm6b demethylases during heart ventricle maturation rather than initial cardiogenesis.

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

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 re-form a now branched skeletal structure.

Sequential and opposing activities of Wnt and BMP coordinate zebrafish bone regeneration.

Zebrafish fully regenerate lost bone, including after fin amputation, through a process mediated by dedifferentiated, lineage-restricted osteoblasts. Mechanisms controlling the osteoblast regenerative program from its initiation through reossification are poorly understood. We show that fin amputation induces a Wnt/ß-catenin-dependent epithelial to mesenchymal transformation (EMT) of osteoblasts in order to generate proliferative Runx2(+) preosteoblasts. Localized Wnt/ß-catenin signaling maintains this progenitor population toward the distal tip of the regenerative blastema. As they become proximally displaced, preosteoblasts upregulate sp7 and subsequently mature into re-epithelialized Runx2(-)/sp7(+) osteoblasts that extend preexisting bone. Autocrine bone morphogenetic protein (BMP) signaling promotes osteoblast differentiation by activating sp7 expression and counters Wnt by inducing Dickkopf-related Wnt antagonists. As such, opposing activities of Wnt and BMP coordinate the simultaneous demand for growth and differentiation during bone regeneration. This hierarchical signaling network model provides a conceptual framework for understanding innate bone repair and regeneration mechanisms and rationally designing regenerative therapeutics.

MADM-ML, a mouse genetic mosaic system with increased clonal efficiency.

Mosaic Analysis with Double Markers (MADM) is a mouse genetic system that allows simultaneous gene knockout and fluorescent labeling of sparse, clonally-related cells within an otherwise normal mouse, thereby circumventing embryonic lethality problems and providing single-cell resolution for phenotypic analysis in vivo. The clonal efficiency of MADM is intrinsically low because it relies on Cre/loxP-mediated mitotic recombination between two homologous chromosomes rather than within the same chromosome, as in the case of conditional knockout (CKO). Although sparse labeling enhances in vivo resolution, the original MADM labels too few or even no cells when a low-expressing Cre transgene is used or a small population of cells is studied. Recently, we described the usage of a new system, MADM-ML, which contains three mutually exclusive, self-recognizing loxP variant sites as opposed to a single loxP site present in the original MADM system (referred to as MADM-SL in this paper). Here we carefully compared the recombination efficiency between MADM-SL and MADM-ML using the same Cre transgene, and found that the new system labels significantly more cells than the original system does. When we established mouse medulloblastoma models with both the original and the new MADM systems, we found that, while the MADM-SL model suffered from varied tumor progression and incomplete penetrance, the MADM-ML model had consistent tumor progression and full penetrance of tumor formation. Therefore MADM-ML, with its higher recombination efficiency, will broaden the applicability of MADM for studying many biological questions including normal development and disease modeling at cellular resolution in vivo.

Polarity and intracellular compartmentalization of Drosophila neurons.

BACKGROUND: Proper neuronal function depends on forming three primary subcellular compartments: axons, dendrites, and soma. Each compartment has a specialized function (the axon to send information, dendrites to receive information, and the soma is where most cellular components are produced). In mammalian neurons, each primary compartment has distinctive molecular and morphological features, as well as smaller domains, such as the axon initial segment, that have more specialized functions. How neuronal subcellular compartments are established and maintained is not well understood. Genetic studies in Drosophila have provided insight into other areas of neurobiology, but it is not known whether flies are a good system in which to study neuronal polarity as a comprehensive analysis of Drosophila neuronal subcellular organization has not been performed. RESULTS: Here we use new and previously characterized markers to examine Drosophila neuronal compartments. We find that: axons and dendrites can accumulate different microtubule-binding proteins; protein synthesis machinery is concentrated in the cell body; pre- and post-synaptic sites localize to distinct regions of the neuron; and specializations similar to the initial segment are present. In addition, we track EB1-GFP dynamics and determine microtubules in axons and dendrites have opposite polarity. CONCLUSION: We conclude that Drosophila will be a powerful system to study the establishment and maintenance of neuronal compartments.