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A forward genetic screen identifies Dolk as a regulator of startle magnitude through the potassium channel subunit Kv1.1
['Joy H. Meserve', 'Department Of Cell', 'Developmental Biology', 'Perelman School Of Medicine', 'University Of Pennsylvania', 'Philadelphia', 'Pennsylvania', 'United States Of America', 'Jessica C. Nelson', 'Kurt C. Marsden']
Date: None

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.

Underlying all animal behaviors are neural circuits, which are controlled by numerous molecular pathways that direct neuron development and activity. To identify and study the 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 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 subunit Kv1.1, respectively. We demonstrate that loss of dolk or kcna1a causes larval zebrafish to perform exaggerated swim movements, and 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.

Funding: This work was supported by NIH (R01MH109498 and R01NS118921 to MG, F32MH115434 to JHM, and R01DC011099 to AEP). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

The second group of mutants perform high amplitude bends, resulting in an “exaggerated” response. For one of the exaggerated startle mutant lines, we identified a causative mutation in PR domain containing 12b (prdm12b), a transcription factor that controls development of inhibitory neurons in the spinal cord and has previously been shown to be important for regulation of movement in fish [ 27 ]. The remaining two exaggerated mutant lines harbor mutations in dolichol kinase (dolk), which encodes a glycosylation pathway enzyme, and in potassium voltage-gated channel, shaker-related subfamily, member 1a (kcna1a), which encodes the potassium channel subunit Kv1.1. We demonstrate that dolk and kcna1a likely act in a common pathway to control startle movement magnitude as Dolk is required for Kv1.1 protein localization. Additionally, we demonstrate that Kv1.1 and Dolk act in the spinal cord to control the magnitude of body bends. Thus, through our forward genetic screen we identified a number of genes that are essential to regulate body movement. Furthermore, we demonstrate how a broadly expressed protein, Dolk, selectively regulates behavior through the Kv1.1 protein.

We previously performed a forward genetic screen to identify functional regulators of the acoustic startle response. This approach identified a distinct set of genes with previously unrecognized roles critical for startle habituation [ 23 , 24 ], startle sensitivity [ 25 ], and sensorimotor decision making [ 26 ]. Here, we describe kinematic mutants identified from this screen that display defects in executing swim movements of the startle response. These mutants fall into two general categories. One group of mutants display a “weak” acoustic startle response characterized by shallow bends and minimal displacement. Using high-throughput sequencing, we have identified causative mutations in five weak startle mutants. In all five lines, the affected genes control muscle or neuromuscular junction (NMJ) function and are associated with locomotor disorders in humans.

( A) The acoustic startle response is driven by an action potential from the Mauthner neuron (green), which activates motor neurons in the spinal cord to drive a contralateral body bend. Excitatory and inhibitory neurons in the hindbrain and in the spinal cord impinge upon the Mauthner cells to ensure motor neurons fire on only one side. (B-F) A representative acoustic startle response in a 5 dpf (days post fertilization) wild type larva. An acoustic stimulus is delivered at 0 ms (B), which elicits a rapid turn (C) followed by a counter bend (D) and swimming away (projection of 90 ms response in E, color coded by time). Automated tracking of the curvature of the larva throughout the behavior (F) reveals the response latency (blue bar = latency in F,K,P). (G-K) ryr1b mutants display a weak startle response with reduced bend and counter bend angles (H,I) and reduced displacement (J), largely due to minimal swimming after the counter bend (K). (L-P) prdm12b mutants display an exaggerated startle response with increased bend and counter bend angles (M,N). The duration of each bend is longer than in wild type as well (P). (Q-T) Quantification of response latency (Q; manual measurement); max C1 head angle (R; automated measurement); distance traveled along escape trajectory (S; automated measurement); and displacement from initial head position to final head position (T; automated measurement). Each point is the average response over ten trials for an individual larva. n≥10 larvae, *p = 0.002, **p<0.0001 (one-way ANOVA with Tukey correction for multiple comparisons).

Over the past several decades, the larval zebrafish has emerged as a powerful vertebrate model organism for unbiased genetic screens to identify genes critical for basic locomotion [ 11 – 13 ] and more recently for more complex behaviors, including visual behaviors and sleep [ 14 , 15 ]. However, an unbiased genetic screen to identify the genes critical for the execution of the startle response has been absent. By five days post fertilization (dpf), in response to an acoustic stimulus, zebrafish larvae undergo a characteristic short latency C-start (SLC), consisting of a sharp C-shaped turn and swimming away from the stimulus [ 16 ] ( Fig 1B–1F and S1 Video ). The behavioral circuit for the acoustic startle response is well characterized (reviewed in [ 9 , 17 ], Fig 1A ) and is functionally similar to the human startle circuit [ 5 ]. Central to the zebrafish acoustic startle circuit are the Mauthner cells, a bilateral pair of reticulospinal neurons in the hindbrain. The Mauthner cells are necessary and sufficient for this short latency escape behavior [ 16 , 18 , 19 ]. Hair cell activation following an acoustic stimulus leads to activation of the eighth cranial nerve, which, along with the spiral fiber neurons, provides excitatory input to the Mauthner cell. The Mauthner cell directly activates contralateral primary motor neurons and excitatory interneurons to drive unilateral body contraction and turning away from the acoustic stimulus [ 20 , 21 ]. Inhibitory input prevents Mauthner cell firing at subthreshold stimuli or when the other Mauthner cell has already fired [ 22 ]. Because the circuit is well defined and the behavior is robust, this system is ideal for investigating how genes regulate behavior.

Defects in initiating or executing movements are associated with a range of disorders. While some neurological disorders are primarily defined by motor impairments, several disorders defined primarily by cognitive deficits include motor features. For example, the eyeblink response, in which a patient reacts to a startling stimulus, is disrupted in a variety of neurodevelopmental and psychiatric disorders, including obsessive compulsive disorder, schizophrenia, posttraumatic stress disorder, and autism spectrum disorder [ 1 – 4 ]. The eyeblink response is one component of the startle response, which in humans is a whole-body defensive maneuver to shield the upper body from impact and in aquatic vertebrates, including zebrafish, is critical to evade avoid predators [ 5 , 6 ]. A combination of electrophysiological, lesion, and imaging studies have uncovered the core neural circuity underlying startle behavior in human and various vertebrate animal models [ 7 – 10 ]. Yet despite its critical role in animal survival and its link to several neurological disorders, the repertoire of genes and genetic pathways that regulate startle behavior has not been explored using an unbiased genetic approach.

Results

A forward genetic screen for regulators of the larval startle response We previously performed a forward genetic screen for regulators of the acoustic startle response [23–26]. In brief, using a high-speed camera and automatic tracking, individual F3 larvae were exposed to a series of startling stimuli (for details on mutagenesis and the breeding scheme to obtain F3 larvae see [23]). This portion of the screen focused on kinematic mutants, characterized by significant changes in any of the stereotypic parameters characteristic of the startle response (Table 1). Putative mutant lines were retested in the subsequent generation to confirm genetic inheritance. This screen identified a group of eight mutants defective in kinematic parameters (including response latency, turn angle, distance, and displacement). These mutants fall into two categories: five mutant lines display shallow bends of decreased turning angle compared to their siblings (one representative mutant line shown in in Fig 1G–1K and 1Q–1T, S2 Video); we refer to these as “weak” startle mutants. Three mutant lines perform numerous high amplitude bends in response to an acoustic stimulus. These turns often result in larvae swimming in a figure eight pattern (one representative mutant line shown in Fig 1L–1T and S3 Video); we refer to these as “exaggerated” startle mutants. Combined, these eight mutant lines offer an opportunity to reveal genetic regulators of movement kinematics. PPT PowerPoint slide

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larger image TIFF original image Download: Table 1. Genes identified from a forward genetic screen that regulate locomotor behaviors in larval zebrafish. https://doi.org/10.1371/journal.pgen.1008943.t001 To identify the molecular mechanisms underlying these behavioral phenotypes, we first set out to identify the causative mutations. In all kinematic mutant lines, approximately 25% of progeny from carrier incrosses display the mutant behavioral phenotype, consistent with the causative mutations being recessive, monoallelic, and causing highly penetrant phenotypes. In addition, when behaviorally mutant larvae were raised for each of seven lines, no animals survived past two weeks, indicating these lines harbor homozygous lethal mutations. For the eighth line (kcna1ap181), we found rare escapers (~5%) that lived >two months, but these fish did not grow past a juvenile stage (<1 cm in length). This lethality can likely be attributed to defects with swimming and hence deficits in prey capture and feeding. For each mutant line, after behavioral testing, pools of behaviorally mutant larvae and behaviorally wild type siblings were collected for high-throughput DNA sequencing. We performed whole genome sequencing (WGS) and homozygosity analysis on mutant and sibling pools for four of the lines, as described in more detail in [23]. Using known single nucleotide polymorphisms (SNPs) present in our wild type background, we identified regions of homozygosity in the mutant pools that were heterozygous in the sibling pool. Within these regions of homozygosity, potentially detrimental exonic SNPs not observed in wild type fish were identified. Nonsense mutations, particularly in genes known to function in neurons or muscle, were prioritized. Individual larvae displaying mutant or wild type behavior were then sequenced for potentially causative SNPs. If a potential SNP was observed as homozygous in 100% of mutant larvae (>20 individuals) and heterozygous or homozygous wild type in all siblings (>20 individuals), we considered the SNP to likely be causative. For the remaining four mutant lines, we performed whole exome sequencing (WES) [28]. To identify linkage (SNPs with allelic frequencies ~100% in mutants and ~33% in siblings, based on ratio of heterozygous to homozygous wild type larvae) and potentially causative mutations, we used the online tool SNPTrack [29]. Confirmation of potentially causative mutations was performed as described above for candidates from WGS. Five lines (neb, cacna1ab, ryr1b, rapsn, and slc5a7a) contain nonsense mutations (or a splice mutation resulting in nonsense mutation) in genes known to regulate neuron or muscle function (see Table 1), strongly indicating we have identified the correct mutation (see also discussion of identified genes below). Two of the mutant lines (prdm12b and kcna1a) have missense mutations, and we or others have generated nonsense alleles that display the same phenotype. For the remaining mutant line (dolk) containing a nonsense mutation, we generated an independent second mutant allele to confirm that mutations in dolk are causative for the exaggerated locomotor phenotype (see below). Based on these data, we are confident we have identified mutant alleles of eight genes critical for regulating proper kinematic behavior during the acoustic startle response. We note that all genes have a human disease associated counterpart and that six of the eight genes are associated with human movement disorders, further underscoring conservation of disease associated genes in zebrafish [30] (Table 1). Based on their molecular identities, the affected genes can be subdivided by their likely site of action. Below, we report on several genes that act in skeletal muscle, at the neuromuscular junction, or in inhibitory spinal neurons, and we then focus on two genes likely to act in the same pathway to regulate movement magnitude.

Genes controlling muscle function modulate acoustic startle kinematics Of the five weak startle mutant lines, two have mutations in genes that are required for muscle function. p413 mutants contain a nonsense mutation in the nebulin (neb) gene (Table 1), which encodes a protein necessary for sarcomere assembly and subsequent function [31]. Previous work in zebrafish demonstrated that a loss-of-function neb allele displays defects in sarcomere assembly, leading to reduced swim movement [32]. The second mutant line we identified, p414, contains a splice mutation in the ryanodine receptor 1b (ryr1b) gene (Table 1 and Fig 1G–1K and 1Q–1T). RyR1 is required for calcium release at the sarcoplasmic reticulum, which drives muscle contraction. A previously characterized zebrafish allele of ryr1b, called relatively relaxed, displays a decreased touch response and reduced Ca2+ transients in fast muscle [33]. Interestingly, RyR1b is expressed primarily in fast muscle while RyR1a is expressed primarily in slow muscle [33]. Consistent with this finding, ryr1bp414 mutants display a drastic startle response defect, which is dependent on fast muscle. However, spontaneous movement, which is dependent on slow muscle, is not significantly different from siblings (Table 1). This result emphasizes the importance of examining different behaviors, especially ones that utilize different circuitry and muscle groups [20,34,35], when characterizing locomotion in mutant animals.

Genes acting at the neuromuscular junction regulate acoustic startle kinematics Three genes identified in our screen are required at the neuromuscular junction (NMJ). This includes a new mutant allele, p416, of the gene receptor-associated protein of the synapse (rapsn), originally identified as twitch once mutants [11], and subsequently shown to be caused by mutation in the rapsn locus [36]. rapsn mutants display reduced spontaneous swimming and touch responses [11] caused by decreased acetylcholine receptor clustering at NMJs [36]. In addition, we identified a nonsense mutation (p415) in the calcium channel, voltage-dependent, P/Q type, alpha 1A subunit, b (cacna1ab) gene, which encodes Ca v 2.1. A previously identified mutant allele of cacna1ab, fakir, displays a weak touch response [11,37]. This weak touch response in fakir larvae can be attributed to defects in transmission at the NMJ [38]. This likely accounts for the reduction in startle response and spontaneous movement we observe in p415 (Table 1). Since rapsn and cacna1ab were both previously identified in a screen for escape response following a tail touch [11], which is driven by the Mauthner cells [39], we predicted they could be identified in the Mauthner-dependent acoustic startle screen described here. The fifth weak startle mutant, p417, demonstrates a unique behavior (S1 Fig). Unlike sibling larvae that initiate the startle response at the head (S1C–S1G Fig), in p417 mutants, the startle response often initiates with a turn in the tail instead (S1H–S1L Fig and S4 Video). Subsequent body bends are uncoordinated, rather than a smooth progression from head to tail. The C1 angle, distance, and displacement are also drastically reduced in these mutants, and mutants undergo very little spontaneous movement (Table 1). We identified the causative p417 mutation as a nonsense mutation in slc5a7a, which encodes the high-affinity choline transporter (CHT) (S1A and S1B Fig). Uptake of choline by the high-affinity choline transporter is the rate limiting step for acetylcholine synthesis [40], so Slc5a7 is essential for cholinergic transmission. There are a number of cholinergic neuronal populations in the acoustic startle circuit. Most notably, spinal motor neurons release acetylcholine at the NMJ to activate skeletal muscle in the trunk and tail [41]. We hypothesized the slc5a7a mutant phenotype of reduced turn angle and minimal displacement is caused by reduced muscle contractions due to reduced acetylcholine release at the NMJ. To test this, we generated a transgenic line in which slc5a7a is expressed under the control of a motor neuron specific promoter, mnx1/hb9 [42] (S1M Fig). Compared to slc5a7a mutants lacking the transgene (S1N–S1P Fig), mutant larvae expressing the transgene (slc5a7a-/-; Tg(hb9:slc5a7a), display a significantly increased turn angle and distance traveled following an acoustic stimulus. Thus, motor neuron specific expression of slc5a7a partially rescues the mutant phenotype, providing strong evidence that Slc5a7a plays a critical role in regulating coordinated swim movements, presumably by increasing acetylcholine level release at NMJs.

prdm12b regulates development of V1 interneurons and modulates acoustic startle kinematics In addition to the five weak startle mutants described above, we identified three exaggerated startle mutants. In the p419 mutant line in which the C1 turning angle of the startle response is dramatically increased (165% of wild type siblings; Table 1 and Fig 1L–1P and S3 Video), we identified a missense mutation in the coding sequence of PR domain containing 12b (prdm12b). Prdm proteins are transcription factors, and several family members have been shown to play roles in nervous system development [43]. Zebrafish prdm12b was previously identified in a screen for genes that express in the nervous system [44]. Morpholinos targeting prdm12b [27] and a prdm12b CRISPR mutation [45] result in larvae with aberrant, exaggerated touch behaviors, similar to the exaggerated acoustic startle response we observe in the p419 mutants. prdm12b is required for development of engrailed 1b positive, V1 interneurons in the spinal cord [27]. V1 interneurons are glycinergic and have been shown to regulate the speed of locomotor movements in mammals [46]. In zebrafish larvae, ablation of V1 interneurons results in an exaggerated touch response [47], reminiscent of the exaggerated startle response we observe in prdm12b mutants. Thus, Prdm12b is required during development for the specification of inhibitory spinal neurons utilized for the acoustic startle response to achieve coordinated and controlled movement.

The glycosylation pathway protein Dolk regulates acoustic startle magnitude In a second exaggerated movement mutant, p420, we identified a nonsense mutation in the dolichol kinase (dolk) gene (Fig 2A and 2B). Similar to prdm12b mutants, the dolk mutant C1 turn angle is dramatically increased (153% of wild type siblings; Table 1 and Fig 2C–2M). In addition to exaggerated C1 turn angles in response to an acoustic stimuli, dolk mutants swim in “figure eights,” resulting in reduced displacement (Fig 2K and 2O). Dolk catalyzes the CTP-dependent phosphorylation of dolichol to dolichol monophosphate. Dolichol monophosphate is an essential glycosyl carrier for C- and O-mannosylation and N-glycosylation of proteins [48]. To confirm that the dolk nonsense mutation identified in our screen (p420) is causative, we generated a second mutant dolk allele using CRISPR/Cas9. In this dolk p421 CRISPR allele, a 1 bp deletion results in a frameshift and premature stop codon at position 50. Both mutations occur early in the protein sequence (Fig 2B), and we predict they likely eliminate all protein function. Transheterozygous (p420/p421) dolk mutants display the same exaggerated phenotype as p420 homozygotes (Fig 2M–2O), confirming that mutations in dolk cause the behavioral deficits observed in the original p420 mutants. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 2. The glycosylation pathway enzyme Dolk regulates the magnitude of the startle response. (A) Gene structure for dolk with the nonsense mutation from the screen noted (p420). The CRISPR 1 bp deletion allele is also noted (p421). (B) Protein structure for Dolk on the endoplasmic reticulum [48], with the predicted amino acid change from the screen mutation indicated. The deletion in the CRISPR allele causes a frame shift in the sequence that results in a premature stop at amino acid 50. In contrast to siblings (C-G), dolk mutants (H-L) display an exaggerated startle response, with an increased bend and counter bend angle (I,J), resulting in larvae swimming in a “figure eight” (K). Blue bar = latency in G,L. (M-O) Kinematic parameters of the acoustic startle response in dolk siblings and mutants (homozygotes of the screen identified mutation, p420/p420, and transheterozygotes from the screen mutation and CRISPR, p420/p421). Each point represents average of ten trials for an individual fish. n≥6 larvae, **p<0.001, *p = 0.008 (one-way ANOVA with Tukey correction for multiple comparisons). https://doi.org/10.1371/journal.pgen.1008943.g002

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