WDR62-deficiency Causes Autism-like Behaviors Independent of Microcephaly in Mice

Brain size abnormality is correlated with an increased frequency of autism spectrum disorder (ASD) in offspring. Genetic analysis indicates that heterozygous mutations of the WD repeat domain 62 (WDR62) are associated with ASD. However, biological evidence is still lacking. Our study showed that Wdr62 knockout (KO) led to reduced brain size with impaired learning and memory, as well as ASD-like behaviors in mice. Interestingly, Wdr62 Nex-cKO mice (depletion of WDR62 in differentiated neurons) had a largely normal brain size but with aberrant social interactions and repetitive behaviors. WDR62 regulated dendritic spinogenesis and excitatory synaptic transmission in cortical pyramidal neurons. Finally, we revealed that retinoic acid gavages significantly alleviated ASD-like behaviors in mice with WDR62 haploinsufficiency, probably by complementing the expression of ASD and synapse-related genes. Our findings provide a new perspective on the relationship between the microcephaly gene WDR62 and ASD etiology that will benefit clinical diagnosis and intervention of ASD. Supplementary Information The online version contains supplementary material available at 10.1007/s12264-022-00997-5.

).A Gene ontology (GO) analysis reveals the GO terms indicating the biological processes, cellular components, and molecular functions enriched in downregulated genes between WT mice receiving oral administration of vehicle (WT + Veh) and Wdr62 +/-mice receiving oral administration of vehicle (Wdr62 +/-+ Veh).B GO analysis reveals the GO terms indicating the biological processes, cellular components, and molecular functions enriched in upregulated genes between Wdr62 +/-mice receiving oral administration of vehicle (Wdr62 +/-+ Veh) and Wdr62 +/-mice receiving oral administration of ATRA (Wdr62 +/-+ RA).C GO analysis of the common regulatory gene in Figure 7B.Terms indicating the biological processes, cellular components, and molecular functions enriched in downregulated genes between WT mice receiving oral administration of vehicle (WT + Veh) and Wdr62 +/-mice receiving oral administration of vehicle (Wdr62 +/-+ Veh).

Animals
Mice were housed under standard laboratory conditions on a 12-h dark/light cycle, and all behavioral testing was carried out during the light phase.The generation of mice carried a Wdr62 floxed allele with a deletion of Wdr62 exon 2 and a reading frame shift in exon 3 [1].To generate mice carrying a universal null allele of Wdr62 (Wdr62-KO), Wdr62 floxed mice were crossed with ZP3-Cre mice.To conditionally inactivate the Wdr62 gene in mice, Wdr62 flox/flox mice were crossed with Nex cre/+ mice to obtain Wdr62 flox/flox ; Nex-Cre (Wdr62-Nex-cKO) mice.Mice were genotyped using the polymerase chain reaction (PCR) of DNA extracted from a neonatal toe biopsy.Primers are listed in Table S4.For most experiments, the resulting comparison littermates were analyzed.Male Wdr62 KO and littermate control mice were used for all of the experiment assays of this genotype.Male and female (male:female ratio 1:1) Wdr62 +/-and littermate control mice generated by in vitro fertilization were examined at 2 months for RA treatment, behavioral, morphological and RNA-seq analysis and no significant sexdependent differences were found.All available male and female Wdr62-Nex-cKO and littermate controls were used accordingly for experiments including behavioral, electrophysiological, and morphological studies, and no significant sex-dependent differences were found.

Open-field Test
The test mouse was first allowed to explore the open-field test (OFT) apparatus for 5 min.After washing the apparatus with 75% ethanol, the test mouse was introduced into the apparatus again and allowed to explore.The time spent in the center region (25%) of the apparatus and the distance traveled in the apparatus were calculated.

Y Maze Test
The Y maze test is a rapid and easily-administered test to assess spatial working memory.A Y maze with a three-armed runway (36 × 6 × 12 cm; at a 120_ angle from each other) was placed in the test room.Different visual cues were located on the wall at the end of each arm.Activity was recorded using a camera, and behavior was assessed using automatic video tracking software, Smart 3.0 (Panlab Co.).Each mouse was randomly introduced into the end of one arm, facing the center, and allowed to freely explore the apparatus for 5 min.The equipment was cleaned with 70% ethanol and dried before a new trial started.Mice entered a different arm when they remembered which arm it had entered on a previous trial.A point was given when the mouse entered each of the three arms without repetition (e.g., CAB, BCA, and ABC).The number of points was then divided by the total arm entries minus one.

Radial Arm Maze Task
The radial arm maze is a paradigm that is used to assess working and reference memory in rats and mice.Before the task, mice were allowed 1 week to habituate to the testing room and fasted for 24 h.After every training day, restricted food was provided.For the working memory assessment, all arms contained a reward (food), and the animal was required to visit each arm once only.To assess reference memory, only some of the arms contained a reward, and the animal was required to visit only the baited arms.Visits to the same or non-baited arms counted as working memory or reference memory errors, respectively.

Novel Object Recognition
Novel object recognition is a relatively sensitive test for assessing cognitively-enhanced activity [1].
An open-field box (36 × 36 × 25 cm 3 ) was used for this test.During the training period, two identical novel objects were placed in the box, and mice freely explored the box for 5 min.After 1 day, one of the familiar objects used during the training session was replaced with a new object, and the mouse was placed back into the open-field box for 5 min.The time each mouse spent exploring objects during the training and test phases was recorded using automatic video tracking software.The ratio of time spent exploring the novel object to that spent exploring both objects was used as a measure of recognition memory.

Three-chamber Test
The three-chamber test is a simple social interaction test, in which behaviors are video-recorded and analyzed to assess the active interaction time between test and novel mice [2].It is a sensitive method for examining autism-related behavioral deficits and has been used to measure autism-like behaviors in mutant mouse models [3][4][5].Briefly, the arena consisted of a transparent box, which was divided into three chambers of the same size with removable doors in each chamber.The test mouse and the age/sex-matched C57BL/6 WT mouse (not littermates) were kept in the test room individually 1 h before the experiment.Two wire cups were placed in the corner of the left and right chambers.The test mouse was introduced into the central chamber and was allowed access to the three chambers for 10 min.During the first stage, a novel age/gender-matched C57BL/6 unfamiliar mouse (Stranger 1-'S1') was introduced into the wire cup of the left or right chamber, and an empty wire cup (Empty, 'E').The test mouse was allowed access to the three chambers and both wire cups for 10 min.The time the test mouse spent interacting with S1 or E was recorded.The chamber was cleaned with 75% ethanol after each test.During the second stage, the empty wire cage was replaced with another novel age/gender-matched C57BL/6 WT mouse (Stranger 2-'S2').The test mouse was allowed access to the three chambers and both wire cups for 10 min.The time the test mouse spent interacting with the S1 or S2 was recorded.No position bias was found.The preference index for each animal was calculated as: S1/(S1 + E) × 100% or S2/(S1 + S2) × 100% accordingly [6].

Self-grooming Test
The mice were first habituated in a clean chamber individually for 10 min.The time spent selfgrooming was recorded for 10 min.Self-grooming included actions of face-wiping, scratching of the head, neck, or ears, and full-body grooming.

Stranger-intruder Test
The stranger-intruder test is a simple method to directly assess sociability.Each test mouse was placed into a new cage with clean bedding.A C57 male mouse, matched for weight and age, was placed in the same cage and left for 10 min.The time the test mouse actively sniffed the C57 mice was recorded.

Fear-conditioning Task
In this test, a freezing response, which is considered a reliable measure of fear in mice, was elicited by unconditioned electric shock stimuli that were matched with conditioned stimuli of sounds or the surrounding context [7].The apparatus consisted of a conditional shock chamber (25 × 25 × 25 cm 3 ), surrounded by a large metal lockable box to minimize noise from the environment and control the recording software, PACKWIN 2.0 (Panlab).The conditional stimulus (CS) was an 80-dB sound at 2000 Hz for 28 s, and the unconditional stimulus (US) was a foot shock at 0.5 mA that lasted 2 s.
During the test, the background white noise was 60 dB.On day 1, mice were allowed to freely explore the shock chamber for 2 min.Then, the CS was presented, which was followed by the US.The CS and US were repeated three times.There was a 30-s interval after each CS-US pairing.Mice were introduced into the same contextual chamber 24 h later.The same procedure was applied, except the CS paired with the US was turned off.After 48 h, mice were placed into the same chamber with a novel context (i.e., the chamber was changed to a different color and shape).The same procedure was applied, except the US was turned off.Animals were observed for freezing behavior every 30 s in each condition.The percentage of freezing bouts was recorded.

Elevated Plus-maze
The elevated plus-maze was used to investigate the anxiety level of mice and comprised two open arms (30 × 6 cm 2 ), two closed arms (30 × 6 cm 2 ), and a center section (6 × 6 cm 2 ).The same arms were on the opposite sides of the center section.The mouse was placed in the center section of the maze with its head facing the same closed arm at the start of the experiment.The behavior of mice in the elevated plus-maze was recorded and analyzed for 10 min.The time the mouse spent in open or closed arms was exported by the software.

Morris Water Maze Test
The water maze test consisted of a circular pool (120 cm in diameter and 50 cm in height), a platform (6 cm in diameter), a camera fixed above the pool, and a computer connected to the camera.The pool was filled with water at ~22°C and 25 cm deep.The platform was placed 1 cm underwater, and a layer of non-toxic titanium dioxide floated on the water surface to prevent animals from seeing the underwater platform.Brightly colored cardboard or plastic panels of different shapes were attached to the walls of the pool as markers for the spatial orientation of the animals.The experiment consisted of two phases: the place navigation and spatial probe experiments.During the training period (5 days), the platform was placed in one quadrant of the pool, and mice were placed into the water facing the wall from four entry points at a fixed time each day.The mice were then quickly dried with towels and placed under a 37°C heat lamp to maintain body temperature.On day 6, the spatial exploration test was conducted by removing the platform after the place navigation test, placing the mouse in the pool in the quadrant opposite to the previous platform, and recording their swimming trajectory for 60 s to examine the mouse's memory of the original platform.A video tracking system was used to record the swimming path of each mouse in the pool, and the ratio of the time spent within the quadrant where the original platform was located for 60 s to the time spent in the other three quadrants was calculated.

Nest Building Test
The mice were separated and kept in single cages 1 h before lights out.A 3.0 g square (10 × 10 cm 2 ) of skimmed cotton was placed in the cage.After 12 h, mice were observed for nesting, and their genotypes were photographed and scored according to scoring criteria: 0-1: 90% of the skimmed cotton remained intact; 1-2: a small portion of the skimmed cotton was torn but most remained intact (50-90%); 2-3: most of the skimmed cotton was torn into pieces (50-90%) but no nesting; 3-4: >90% of the skimmed cotton was torn into pieces and formed a flat nest (<50% of the height of the mice); 4-5: >90% of the skimmed cotton was torn into pieces, forming a high-quality nest (>50% of the height of the mice).Because of the complexity of the experiments, scores could be decimals.For example, if the nest was made perfectly, but 10% of the skimmed cotton remained unshredded, the score could be 4.5 points.

Rotarod Test
The rotarod apparatus was used to examine motor coordination.Each mouse was tested over four trials once a day for 2 days, with the rod rotation increasing from 4 to 40 rpm in 5 min.The interval between each trial was 30 min.The rotarod apparatus was stopped when the test mouse fell, and the latency to fall off the rod was determined.The average latency of the four trials on day 2 was calculated.

Underground Food Sniffing Test
The mice were fasted for 24 h the day before the test.During the task, each mouse was placed in a clean rat cage (42.5 × 26.6 × 15.5 cm 3 ) with 4-cm-deep bedding that contained a hidden food pellet

Fig. S5
Fig.S5 Morphology of layer 5 pyramidal neurons in the cortex (related to Figure 5).A Representative

Fig. S7 (
Fig.S7 (related to Figure 7).A Gene ontology (GO) analysis reveals the GO terms indicating the