Background
SHANK3 is a master scaffolding protein enriched at the postsynaptic density of excitatory glutamatergic synapses in the brain that has critical roles in synaptogenesis and synaptic function [
1‐
6].
SHANK3 is the key gene implicated in the neurobehavioral features of individuals with chromosome 22q13.3 deletion syndrome or Phelan-McDermid syndrome (PMS) [
7,
8]. Moreover, genetic studies have identified point mutations in the
SHANK3 gene in cases of autism spectrum disorder (ASD) that establish the causal role of
SHANK3 mutations in ~ 1% of individuals with ASD [
9‐
11].
Animal models of ASD that mimic
SHANK3 genetic detects have facilitated a better understanding of the underlying molecular mechanisms and development of more effective treatments [
2,
12]. More than a dozen different lines of
Shank3 mutant mice have been generated and characterized [
4,
13‐
15]. Almost all
Shank3 mutant mice exhibit some of the core behavioral features of ASD [
4,
13,
14]. Despite significant advantages, there are clear disadvantages associated with the use of rodent models. For example, it remains difficult to scale up for high-throughput drug screening in rodent models [
12]. Compared to rodent models, zebrafish (
Danio rerio) exhibit much more efficient reproduction, rapid external development [
12,
16,
17], and optical transparency [
17]. Previous studies have shown that the gene orthologous to human
SHANK3 is duplicated in zebrafish as
shank3a (in chromosome 18) and
shank3b (in chromosome 4) [
18,
19]. Transient knockdown of both
shank3a and
shank3b expressions by morpholino method has been reported [
19,
20]. However, previously, the analysis of developmental and behavioral characteristics was only conducted within 5 days of post-fertilization (dpf), an early stage of development [
19]. In the present study, we generated and characterized the first CRISPR/Cas9 engineered
shank3b loss-of-function mutation that is stably transmitted in zebrafish. This model will enable a comprehensive study of a mechanistic link between
shank3 loss-of-function and ASD and provide a new experimental platform for high throughput drug screening in the future.
Methods
Generation of shank3b mutant zebrafish
The detailed procedure for CRISPR/Cas9 editing in zebrafish was described previously [
21,
22]. The
shank3b target in this study was 5′-GGGCGTGTTGTTGCCACGGCCGG-3′ (Additional file
1: Table S1). Injection mixtures included 500 pg of Cas9 mRNA and 120 pg of gRNA. Eighty zebrafish were screened to identify a founder, and the germline mutation frequency was approximately 35%. Mutant sites were verified by comparison to the WT unaffected sequences (chimerism). Chimeric zebrafish were mated onto a Tu background for three generations to obtain
shank3b
+/−
zebrafish. We crossed
shank3b
+/−
males and
shank3b
+/−
females to obtain
shank3b
+/+
,
shank3b
+/−
, and
shank3b
−/−
littermates for all experiments of phenotypic analyses.
Tg (HuC: RFP) transgenic line and zebrafish maintenance
The wild-type (WT) Tu zebrafish strain was acquired from the Institute of Zebrafish, Children’s Hospital of Fudan University. The zebrafish were raised and maintained in a standard laboratory environment (28.5 °C) and a 14 h light/10 h dark cycle according to a standard protocol [
17,
23]. The
Tg (
shank3b
+/+
-
HuC: RFP
+/−) transgenic line, kindly provided by Dr. Xu Wang (Fudan University), was made via plasmid injection with tol2 mRNA at single-cell stage followed by screening for germline transmission. The vector was generated by inserting the
HuC promoter [
24] upstream of RFP cDNA followed by polyA sequence in a Tol2 destination vector, using multisite Gateway cloning [
25]. In order to collect enough eggs efficiently for the RFP imaging experiments, we crossed
Tg (
shank3b
+/−
-
HuC: RFP
+/−) with
Tg (
shank3b
+/−
-HuC: RFP
+/−) to obtain
Tg (
shank3b
−/−
-HuC: RFP
+/+) for the experimental group. We crossed
Tg (
shank3b
+/+
-
HuC: RFP
+/−) and
Tg (
shank3b
+/+
-
HuC: RFP
+/−) to obtain the control group,
Tg (
shank3b
+/+
-
HuC: RFP
+/+).
RT-qPCR
Real-time quantitative polymerase chain reaction (RT-qPCR) was performed in triplicate, with 4–10 zebrafish per sample. Total RNA was extracted from the larval or adult brains using TRIzol reagent (Ambion, USA). Reverse transcription was performed with a PrimeScript™ RT Reagent Kit (RR037A, TaKaRa, Japan), according to the manufacturer’s protocol. Oligo dT primer (25 pmol) and random 6 mers (50 pmol) were added in 10 μl mixture to efficiently obtain full-length cDNA. RT-qPCR was performed using a LightCycler® 480 apparatus (Roche, Germany) and SuperRealPreMix Plus (Tiangen, China), according to the manufacturers’ instructions. Finally, we used the delta delta CT method to calculate the expression levels. The primers used in this study are described in Table S1 in Additional file
1.
Larval activity and light/dark tests
A ViewPoint setup combined with an automated computer recording system equipped with VideoTrack software was used to measure locomotor activity. The camera was a Point Grey black-and-white camera with a resolution of 1024 × 768. Videos were recorded for 60 min at 25 fps and were pooled into 1-min time bins. The detection threshold was set to 25. Activity was quantified using Zebralab software. The distance traveled by the larvae in the well was measured to analyze general locomotor activity. For all behavioral analyses, we used a commercial Viewpoint tracking system and custom software written in C++. All behavioral assays were analyzed by experimenters who were blinded to the genotypes. To further analyze the variances of different activity intensity scales among WT,
shank3b
+/−
, and
shank3b
−/−
zebrafish, we divided the activity equally into five levels (10, 20, 30, 40, and 50) (Additional file
1: Figure S6). Next, we calculated the activity frequency of different activity intensity scales.
Larvae were habituated in 48-well plates, with one animal per well, in our behavioral assessment room, and videos were recorded for 60 min. The diameter of each well was 1.2 cm. After 30 min of habituation, each larva was recorded for a total of 30 min with three light/dark cycles (each consisting of 5 min of light and 5 min of dark). The light intensity for photo motor response (PMR) was 100 lx and the frame rate was 25/s.
Open-field test
Behavioral experiments were conducted between 10 a.m. and 4 p.m. Each tank was 30 × 30 × 30 cm, with walls made of opaque partitions, and a video camera was suspended above the tank. Adult male zebrafish were allowed to freely swim inside the tank, and videos were recorded for 30 min. The timing of all supplementary videos began at approximately the 10th min.
The thigmotaxis test was performed in the tank divided into two equal zones, a peripheral and a central zone. Adult zebrafish swam freely in the tank. The longer the zebrafish stayed in the peripheral zone, the greater their awareness of danger [
12]. The time ratio was the time the zebrafish spent in the peripheral zone divided by the total time spent in tank, and the distance ratio was the distance the zebrafish traveled in the peripheral zone divided by the total distance traveled.
Shoaling test
Adult male zebrafish were acclimated to the novel tank apparatus for 1–2 min before the test [
26]. Videos were recorded for 30 min. The shoaling assessment was performed by measuring the inter-fish distance that represents the average of all distance between each zebrafish in a shoal [
27,
28].
Social preference test
Social preference testing was performed in a standard mating tank (inner dimensions 21 × 10 × 7.5 cm). The tank was separated into two halves by a Plexiglas transparent barrier that allowed the zebrafish to swim freely and was provided sufficient visual information to allow the zebrafish to form a social preference. Behavioral recordings typically started after an acclimation period (1–2 min), when zebrafish usually explored the tank. Videos were recorded for 30 min. The zebrafish behaviors were quantified as a distance distribution or as presence in a zone adjacent to the group or conspecifics. The time ratio was the time spent in the conspecific sector divided by the total time. The distance ratio was the distance traveled in the conspecific sector divided by the total distance traveled. The zebrafish tested were all adult males.
Kin preference test
The specifications of the mating cylinder were the same as those in the social preference test. Two opaque separators divided the cylinder into three compartments. Videos were recorded for 30 min. Kin preference was represented by the ratio of time spent in the kin sector divided by the total time. The zebrafish tested were all adult males.
Western blot and antibodies
WT and shank3b
−/−
zebrafish brains were prepared for western blotting by dissociating the tissues in lysis buffer (RIPA, Beyotime Biotechnology, China) and 1% protease inhibitor mixture Set I (Calbiochem, San Diego, CA, USA). The lysates were then centrifuged at 12,000 rpm for 5 min, and the supernatant was collected and denatured. 20 μg of total protein were separated on an SDS-PAGE gel (12%) and were blotted onto a polyvinylidene difluoride membrane (Bio-Rad Laboratories, Hercules, CA, USA). Next, the membrane was blocked with 5% bovine serum albumin for 1–2 h at room temperature and was incubated with primary antibodies overnight at 4 °C. The membrane was rinsed and incubated with HRP-conjugated secondary antibodies for 2 h. Finally, chemiluminescent detection was performed with an ECL kit (Rockford, IL, USA). ImageJ software was used for the densitometric analysis (N = 3 for each group).
The synaptophysin (1:2000; ab32594) and homer1 (1:1000; ARP40181_P050) antibodies were purchased from Abcam (Cambridge, UK) and Aviva Systems Biology (San Diego, USA), respectively. The β-actin antibody was obtained from Biotech Well (1:2000; code No. WB0196, Shanghai, China).
Statistical analysis
Statistical analyses were performed using GraphPad Prism software. Simple comparisons between adult shank3b
+/+
and shank3b
−/−
zebrafish were performed with two-sided unpaired Student’s t tests. Analysis of variance (ANOVA) tests were used to compare three genotypes. All the experiments were conducted in triplicate using different samples. P values < 0.05 were considered as statistically significant. Values are presented as mean ± SEM.
Discussion
In this study, we generated the first
shank3b loss-of-function mutation in zebrafish using the CRISPR/Cas9 gene editing method and reported the morphological, behavioral and neurological characterizations of
shank3b zebrafish mutants at both early developmental stage and adulthood. The
shank3b deficiency caused partial lethality during early development as well as defective and delayed neurodevelopment at the larval stage. The brain volume of
shank3b−/− zebrafish is enlarged but the brain weight is comparable to
shank3b+/+, which may indicate the ventricles in
shank3b−/− are larger than in WT zebrafish. This observation is reminiscent of the enlarged ventricular size frequently reported in human PMS patients [
32,
33]. However, it is interesting to note that the defective and delayed neurodevelopment in
shank3b−/− larvae becomes less noticeable later in development. The exact reason for the finding is not immediately clear but may support a different functional role of shank3b protein at different developmental stages.
s
hank3b−/− zebrafish in adulthood display significantly abnormal behaviors while
shank3b
+/−
zebrafish showed intermediate phenotypes compared to those of
shank3b
−/−
and
shank3b+/+ zebrafish. The phenotypes observed in
shank3b
+/−
zebrafish are analogous to the haploinsufficiency of
SHANK3 seen in PMS and
SHANK3-related disorders [
9,
34]. The observed early-stage developmental defects and abnormal behaviors in both
shank3b
+/−
and
shank3b
−/−
zebrafish larvae are different from
Shank3 rodent models, in which early developmental defects have not been reported, and phenotypes in heterozygous mutants are generally not significant [
4,
35,
36]. The reason for these differences between the two species is not clear. Considering that zebrafish have both
shank3a and
shank3b homologs to human
SHANK3, it is somewhat unexpected or counterintuitive that
shank3b mutant zebrafish have more prominent phenotypes for survival and behavior. An alternative explanation for the behavioral phenotypes is that the more significant abnormal behaviors in
shank3
+/−
zebrafish are because behavioral assays in zebrafish are more sensitive than that in rodents.
The ortholog of human
SHANK3 is duplicated in the zebrafish genome as
shank3a and
shank3b during teleost evolution [
12,
17]. The duplicated and conserved shank3a and shank3b share high identity at the amino acid level and are expected to have a similar function in zebrafish [
17]. In a previous study, Kozol et al. reported the knock down of
shank3a and
shank3b by morpholino and observed embryonic defects in both morphants and impaired touch-induced startle responses in
shank3a morphants [
19]. However, abnormal ASD-like behaviors were not detected due to the limitations of morpholino technology. It would be interesting to compare the phenotypes of
shank3a and
shank3b mutants engineered by CRISPR/Cas9 in parallel or even the phenotypes of
shank3a and
shank3b double mutants in the future.
In recent years, the zebrafish has become an attractive alternative model for ASD researchers [
19,
27,
37]. Many behavioral assays have been developed in zebrafish models, including the assessment of social interaction, novelty seeking, courtship, inhibitory avoidance, fear and anxiety responses, repetitive/stereotyped behaviors, seizures, and aggression [
12,
38‐
41]. We employed some of the behavioral assays in the analyses of
shank3b mutant zebrafish and found striking differences in social and repetitive behavioral domains between
shank3b−/− and
shank3b+/+ zebrafish. For instance, in shoaling and kin-preference assays,
shank3b−/− zebrafish preferred to swim in loose schools and showed significantly decreased preference for conspecifics. These abnormal behaviors are reminiscent of reduced social interaction in the home cage or abnormal social novelty and preference using the three chamber paradigm reported in several lines of
Shank3 mutant mice [
35,
36,
42,
43]. In the open field,
shank3b−/− zebrafish displayed abnormal locomotor activity, such as figure “8” and “circling” movements that are apparently repetitive. Similarly, repetitive behavior measured by increased self-grooming has been observed in several lines of
Shank3 mutant mice [
4,
42]. However, like many other behavioral findings observed in animal models, the challenge remains to determine whether the abnormal behaviors observed in
shank3b-deficient zebrafish can be directly translated to human
SHANK3-related ASD. The study of the predictive validity of these abnormal behaviors to ASD may be warranted in the future, when feasible. Positive results could potentially provide further support for the translational value of these behavioral phenotypes. It also remains to be seen if these assays are universally valid and effective for ASD models caused by different genetic defects. Clinical and molecular heterogeneity have been well recognized in ASD in humans [
44]. Additional behavioral assays are certainly needed to assess face validity for ASD-like behaviors, and also for common comorbidities such as seizures and cognitive impairments.
Our finding of reduced postsynaptic homer1 protein levels in
shank3b-deficient zebrafish is consistent with the known function of SHANK3 as a scaffolding protein at the postsynaptic density from studies of
Shank3 mutant mice [
4,
45]. This finding, although limited, would suggest that the molecular mechanism-associated SHANK3 deficiency may be conserved between different species. It would be interesting to examine if the same defect occurs in
shank3a-deficient zebrafish. The finding of significantly reduced synaptophysin protein levels in the brain of
shank3b−/− zebrafish is novel, as synaptophysin is a known presynaptic protein [
31]. This observation implies that
shank3b deficiency may affect presynaptic function directly or via a trans-synaptic mechanism in zebrafish. Several recent studies have suggested that SHANK3 protein is located at the presynaptic terminus in the brain as well as in dorsal root ganglion neurons in rodents [
46]. Our finding in zebrafish also potentially suggests a role of shank3 protein in the presynaptic terminus. Future studies on the presynaptic function of
shank3b
−/−
are warranted and may shed additional insight in this direction.
The amenability to high-throughput drug screening is a tremendous advantage of the zebrafish model. The list of confirmed ASD-causing genes continues to grow, but the development of targeted molecular treatments significantly lags behind. A validated experimental platform that can translate the genetic discoveries to drug screening at a fast pace is urgently needed. We believe that the shank3b
−/−
model described in this study and other similar ASD zebrafish models will lay an important foundation for the development of a productive drug screening program for ASD and may ultimately lead to the discovery of an effective intervention.