Sexually dimorphic fin regeneration in zebrafish controlled by androgen/GSK3 signaling

Certain fish and amphibians regenerate entire fins and limbs after amputation, whereas such potential is absent in birds and limited in mammals to digit tips [1, 2]. Additionally, regenerative success can change during life stages. Anuran tadpoles gradually lose the capacity to regenerate limbs [3,?4], and digit regeneration occurs more effectively in fetal mice and human children than adults [5-8]. Little is known about mechanisms that control regenerative capacity. Here, we identify an unexpected difference between male and female zebrafish in the regenerative potential of a major appendage. Males display regenerative defects in amputated pectoral fins, caused by impaired blastemal proliferation. This regenerative failure emerges after sexual maturity, is mimicked in androgen-treated females, and is suppressed in males by androgen receptor antagonism. Androgen signaling maintains expression of dkk1b and igfbp2a, which encode secreted inhibitors of Wnt and Igf signaling, respectively. Furthermore, the regulatory target of Wnts and Igfs, GSK3β, is inefficiently inactivated in male fin regenerates compared with females. Pharmacological inhibition of GSK3 in males increases blastemal proliferation and restores regenerative pattern. Our findings identify a natural sex bias in appendage regenerative capacity and indicate an underlying regulatory circuit in which androgen locally restricts key morphogenetic programs after amputation.     

SAR studies on a series of N-benzyl-4-heteroaryl-1-(phenylsulfonyl)piperazine-2-carboxamides: potent inhibitors of the polymerase enzyme (NS5B) of the hepatitis C virus

Described herein is the initial optimization of (+/−) N-benzyl-4-heteroaryl-1-(phenylsulfonyl)piperazine-2-carboxamide (1), a hit discovered in a high throughput screen run against the NS5B polymerase enzyme of the hepatitis C virus. This effort resulted in the identification of (S)-N-sec-butyl-6-((R)-3-(4-(trifluoromethoxy)benzylcarbamoyl)-4-(4-(trifluoromethoxy)phenylsulfonyl)piperazin-1-yl)pyridazine-3-carboxamide (2), that displayed potent replicon activities against HCV genotypes 1b and 1a (EC₅₀ 1b/1a = 7/89 nM).     

Modulation of cofilin phosphorylation by inhibition of the Lim family kinases

A series of aminothiazoles that are potent inhibitors of LIM kinases 1 and 2 is described. Appropriate choice of substituents led to molecules with good selectivity for either enzyme. An advanced member of the series was shown to effectively interfere with the phosphorylation of the LIM kinases substrate cofilin. Consistent with the important role of the LIM kinases in regulating cytoskeletal structure, treated cells displayed dramatically reduced F-actin content.     

Regulation of zebrafish heart regeneration by miR-133

Zebrafish regenerate cardiac muscle after severe injuries through the activation and proliferation of spared cardiomyocytes. Little is known about factors that control these events. Here we investigated the extent to which miRNAs regulate zebrafish heart regeneration. Microarray analysis identified many miRNAs with increased or reduced levels during regeneration. miR-133, a miRNA with known roles in cardiac development and disease, showed diminished expression during regeneration. Induced transgenic elevation of miR-133 levels after injury inhibited myocardial regeneration, while transgenic miR-133 depletion enhanced cardiomyocyte proliferation. Expression analyses indicated that cell cycle factors mps1, cdc37, and PA2G4, and cell junction components cx43 and cldn5, are miR-133 targets during regeneration. Using pharmacological inhibition and EGFP sensor interaction studies, we found that cx43 is a new miR-133 target and regeneration gene. Our results reveal dynamic regulation of miRNAs during heart regeneration, and indicate that miR-133 restricts injury-induced cardiomyocyte proliferation.     

Genetic Reprogramming of Positional Memory in a Regenerating Appendage

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Getting to the heart of regeneration in zebrafish

A scientific and clinical prerogative of the 21st century is to stimulate the regenerative ability of the human heart. While the mammalian heart shows little or no natural regeneration in response to injury, certain non-mammalian vertebrates possess an elevated capacity for cardiac regeneration. Adult zebrafish restore ventricular muscle removed by surgical resection, events that involve little or no scarring. Recent studies have begun to reveal cellular and molecular mechanisms of this regenerative process that have exciting implications for human cardiac biology and disease.     

Specification of epibranchial placodes in zebrafish

In all vertebrates, the neurogenic placodes are transient ectodermal thickenings that give rise to sensory neurons of the cranial ganglia. Epibranchial (EB) placodes generate neurons of the distal facial, glossopharyngeal and vagal ganglia, which convey sensation from the viscera, including pharyngeal endoderm structures, to the CNS. Recent studies have implicated signals from pharyngeal endoderm in the initiation of neurogenesis from EB placodes; however, the signals underlying the formation of placodes are unknown. Here, we show that zebrafish embryos mutant for fgf3 and fgf8 do not express early EB placode markers, including foxi1 and pax2a. Mosaic analysis demonstrates that placodal cells must directly receive Fgf signals during a specific crucial period of development. Transplantation experiments and mutant analysis reveal that cephalic mesoderm is the source of Fgf signals. Finally, both Fgf3 and Fgf8 are sufficient to induce foxi1-positive placodal precursors in wild-type as well as Fgf3-plus Fgf8-depleted embryos. We propose a model in which mesoderm-derived Fgf3 and Fgf8 signals establish both the EB placodes and the development of the pharyngeal endoderm, the subsequent interaction of which promotes neurogenesis. The coordinated interplay between craniofacial tissues would thus assure proper spatial and temporal interactions in the shaping of the vertebrate head.