Myosin phosphatase Fine-tunes Zebrafish Motoneuron Position during Axonogenesis

Bremer, Juliane; Granato, Michael

doi:10.1371/journal.pgen.1006440

Cited by 12 publications

(9 citation statements)

References 59 publications

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“…To generate Tg(hb9 : slc5a7a , hb9 : mKate) p418 , slc5a7a was cloned from 5 dpf larval cDNA into PENTR-D-TOPO (Invitrogen). Gateway cloning was performed with a double hb9 promoter pDest plasmid that includes I-SceI sites [ 92 ] to make hb9 : slc5a7a , hb9 : mKate . The construct and I-SceI were injected, as described previously [ 93 ], into one-cell stage embryos from an outcross of slc5a7a p417/+ .…”

Section: Methodsmentioning

confidence: 99%

A forward genetic screen identifies Dolk as a regulator of startle magnitude through the potassium channel subunit Kv1.1

et al. 2021

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The acoustic startle response is an evolutionarily conserved avoidance behavior. Disruptions in startle behavior, particularly startle magnitude, are a hallmark of several human neurological disorders. While the neural circuitry underlying startle behavior has been studied extensively, the repertoire of genes and genetic pathways that regulate this locomotor behavior has not been explored using an unbiased genetic approach. To identify such genes, we took advantage of the stereotypic startle behavior in zebrafish larvae and performed a forward genetic screen coupled with whole genome analysis. We uncovered mutations in eight genes critical for startle behavior, including two genes encoding proteins associated with human neurological disorders, Dolichol kinase (Dolk), a broadly expressed regulator of the glycoprotein biosynthesis pathway, and the potassium Shaker-like channel subunit Kv1.1. We demonstrate that Kv1.1 and Dolk play critical roles in the spinal cord to regulate movement magnitude during the startle response and spontaneous swim movements. Moreover, we show that Kv1.1 protein is mislocalized in dolk mutants, suggesting they act in a common genetic pathway. Combined, our results identify a diverse set of eight genes, all associated with human disorders, that regulate zebrafish startle behavior and reveal a previously unappreciated role for Dolk and Kv1.1 in regulating movement magnitude via a common genetic pathway.

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Section: Methodsmentioning

confidence: 99%

A forward genetic screen identifies Dolk as a regulator of startle magnitude through the potassium channel subunit Kv1.1

et al. 2021

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“…Zebrafish has emerged as an excellent model to study the role of MP during early vertebrate development. Mutation or knockdown of zygotic Mypt1 ( ppp1r12a ) is embryonic lethal, resulting in the failure of liver development, disorganized somites, excessive cellular contractility of the neural epithelium, and motor axonogenesis [ 32 , 33 , 34 , 35 ]. If both maternal and zygotic ppp1r12a are knocked down, embryos develop hypercontractile mesodermal cells that fail to undergo proper morphogenetic cell movement, including the convergent extension (CE) of the presomitic mesoderm during gastrulation [ 36 , 37 ].…”

Section: Introductionmentioning

confidence: 99%

The Evolution of Duplicated Genes of the Cpi-17/Phi-1 (ppp1r14) Family of Protein Phosphatase 1 Inhibitors in Teleosts

Lang

Virk

Zheng

et al. 2020

IJMS

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The Cpi-17 (ppp1r14) gene family is an evolutionarily conserved, vertebrate specific group of protein phosphatase 1 (PP1) inhibitors. When phosphorylated, Cpi-17 is a potent inhibitor of myosin phosphatase (MP), a holoenzyme complex of the regulatory subunit Mypt1 and the catalytic subunit PP1. Myosin phosphatase dephosphorylates the regulatory myosin light chain (Mlc2) and promotes actomyosin relaxation, which in turn, regulates numerous cellular processes including smooth muscle contraction, cytokinesis, cell motility, and tumor cell invasion. We analyzed zebrafish homologs of the Cpi-17 family, to better understand the mechanisms of myosin phosphatase regulation. We found single homologs of both Kepi (ppp1r14c) and Gbpi (ppp1r14d) in silico, but we detected no expression of these genes during early embryonic development. Cpi-17 (ppp1r14a) and Phi-1 (ppp1r14b) each had two duplicate paralogs, (ppp1r14aa and ppp1r14ab) and (ppp1r14ba and ppp1r14bb), which were each expressed during early development. The spatial expression pattern of these genes has diverged, with ppp1r14aa and ppp1r14bb expressed primarily in smooth muscle and skeletal muscle, respectively, while ppp1r14ab and ppp1r14ba are primarily expressed in neural tissue. We observed that, in in vitro and heterologous cellular systems, the Cpi-17 paralogs both acted as potent myosin phosphatase inhibitors, and were indistinguishable from one another. In contrast, the two Phi-1 paralogs displayed weak myosin phosphatase inhibitory activity in vitro, and did not alter myosin phosphorylation in cells. Through deletion and chimeric analysis, we identified that the difference in specificity for myosin phosphatase between Cpi-17 and Phi-1 was encoded by the highly conserved PHIN (phosphatase holoenzyme inhibitory) domain, and not the more divergent N- and C- termini. We also showed that either Cpi-17 paralog can rescue the knockdown phenotype, but neither Phi-1 paralog could do so. Thus, we provide new evidence about the biochemical and developmental distinctions of the zebrafish Cpi-17 protein family.

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“…To generate Tg(hb9:slc5a7a,hb9:mKate) p418 , slc5a7a was cloned from 5 dpf larval cDNA into PENTR-D-TOPO (Invitrogen). Gateway cloning was performed with a double hb9 promoter pDest plasmid that includes I-SceI sites [90] to make hb9:slc5a7a,hb9:mKate. The construct and I-SceI were injected, as described previously [91], into one-cell stage embryos from an outcross of slc5a7a p417/+ .…”

Section: Mutagenesis Wgs/wes and Transgenesismentioning

confidence: 99%

A forward genetic screen identifies Dolk as a regulator of startle magnitude through the potassium channel subunit Kv1.1

Meserve

Nelson

Marsden

et al. 2020

Preprint

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The acoustic startle response is an evolutionary conserved avoidance behavior. Disruptions in startle behavior, in particular startle magnitude, are a hallmark of several human neurological disorders. While the neural circuitry underlying startle behavior has been studied extensively, the repertoire of genes and genetic pathways that regulate this locomotor behavior has not been explored using an unbiased genetic approach. To identify such genes, we took advantage of the stereotypic startle behavior in zebrafish larvae and performed a forward genetic screen coupled with whole genome analysis. This identified mutants in eight genes critical for startle behavior, including two genes encoding proteins associated with human neurological disorders, Dolichol kinase (Dolk), a broadly expressed regulator of the glycoprotein biosynthesis pathway, and the potassium Shaker-like channel subunit Kv1.1. We demonstrate that Kv1.1 acts independently of supraspinal inputs to regulate locomotion, suggesting its site of action is within spinal circuitry.Moreover, we show that Kv1.1 protein is mis-localized in dolk mutants, suggesting they act in a common genetic pathway to regulate movement magnitude. Combined, our results identify a diverse set of eight genes all associated with human disorders that regulate zebrafish startle behavior and reveal a previously unappreciated role for Dolk and Kv1.1 in regulating movement magnitude via a common genetic pathway. Author summaryUnderlying all animal behaviors are neural circuits, which are controlled by numerous molecular pathways that direct neuron development and activity. To identify and study these molecular pathways that control behavior, we use a simple vertebrate behavior, the acoustic startle response, in the larval zebrafish. In response to an intense noise, larval zebrafish will quickly turn 3 and swim away to escape. From a genetic screen, we have identified a number of mutants that behave in abnormal ways in response to an acoustic stimulus. We cloned these mutants and identified eight genes that regulate startle behavior. All eight genes are associated with human disorders, and here we focus on two genes, dolk and kcna1a, encoding Dolk, a key regulator of protein glycosylation, and the potassium channel Kv1.1, respectively. We demonstrate that loss of dolk or kcna1a causes larval zebrafish to perform exaggerated swim movements and that Dolk is required for Kv1.1 protein localization to axons of neurons throughout the nervous system, providing strong evidence that dolk and kcna1a act in a common molecular pathway. Combined, our studies provide new insights into the genetic regulation of startle behavior.

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Myosin phosphatase Fine-tunes Zebrafish Motoneuron Position during Axonogenesis

Cited by 12 publications

References 59 publications

A forward genetic screen identifies Dolk as a regulator of startle magnitude through the potassium channel subunit Kv1.1

A forward genetic screen identifies Dolk as a regulator of startle magnitude through the potassium channel subunit Kv1.1

The Evolution of Duplicated Genes of the Cpi-17/Phi-1 (ppp1r14) Family of Protein Phosphatase 1 Inhibitors in Teleosts

A forward genetic screen identifies Dolk as a regulator of startle magnitude through the potassium channel subunit Kv1.1

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