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The accessible genetics and extensive skeletal musculature of the zebrafish make it a versatile and increasingly used model for studying muscle contraction. We here describe the development of an in vivo assay for measuring the contractile force of intact zebrafish at the larval stage. In addition, as proof of applicability, we have used this assay to quantify contractile strength of zebrafish larvae in a morphant model of deranged rbfox function. Average maximum tetanic (180 Hz) whole body forces produced by wild-type larvae at 2, 3, 4, and 5 days postfertilization amounted to 3.0, 7.2, 9.1, and 10.8 mN, respectively. To compare at potentially different stages of muscle development, we developed an immunohistological assay for empirically determining the cross-sectional area of larval trunk skeletal muscle to quantify muscle-specific force per cross-sectional area. At 4-5 days postfertilization, specific force amounts to ~300 mN/mm², which is similar to fully developed adult mammalian skeletal muscle. We used these assays to measure contractile strength in zebrafish singly or doubly deficient for two rbfox paralogs, rbfox1l and rbfox2, which encode RNA-binding factors shown previously to modulate muscle function and muscle-specific splicing. We found rbfox2 morphants produce maximal tetanic forces similar to wild-type larvae, whereas rbfox1l morphants demonstrate significantly impaired function. rbfox1l/rbfox2 morphants are paralyzed, and their lack of contractile force production in our assay suggests that paralysis is a muscle-autonomous defect. These quantitative functional results allow measurement of musclespecific phenotypes independent of neural input. [ABSTRACT FROM AUTHOR]

Unlabelled: NRAS mutation at codons 12, 13, or 61 is associated with transformation; yet, in melanoma, such alterations are nearly exclusive to codon 61. Here, we compared the melanoma susceptibility of an NrasQ61R knock-in allele to similarly designed KrasG12D and NrasG12D alleles. With concomitant p16INK4a inactivation, KrasG12D or NrasQ61R expression efficiently promoted melanoma in vivo, whereas NrasG12D did not. In addition, NrasQ61R mutation potently cooperated with Lkb1/Stk11 loss to induce highly metastatic disease. Functional comparisons of NrasQ61R and NrasG12D revealed little difference in the ability of these proteins to engage PI3K or RAF. Instead, NrasQ61R showed enhanced nucleotide binding, decreased intrinsic GTPase activity, and increased stability when compared with NrasG12D. This work identifies a faithful model of human NRAS-mutant melanoma, and suggests that the increased melanomagenecity of NrasQ61R over NrasG12D is due to heightened abundance of the active, GTP-bound form rather than differences in the engagement of downstream effector pathways., Significance: This work explains the curious predominance in human melanoma of mutations of codon 61 of NRAS over other oncogenic NRAS mutations. Using conditional "knock-in" mouse models, we show that physiologic expression of NRASQ61R, but not NRASG12D, drives melanoma formation., (©2014 American Association for Cancer Research.)