Autism-relevant social abnormalities and cognitive deficits in engrailed-2 knockout mice

Abstract

ENGRAILED 2 (En2), a homeobox transcription factor, functions as a patterning gene in the early development and connectivity of rodent hindbrain and cerebellum, and regulates neurogenesis and development of monoaminergic pathways. To further understand the neurobiological functions of En2, we conducted neuroanatomical expression profiling of En2 wildtype mice. RTQPCR assays demonstrated that En2 is expressed in adult brain structures including the somatosensory cortex, hippocampus, striatum, thalamus, hypothalamus and brainstem. Human genetic studies indicate that EN2 is associated with autism. To determine the consequences of En2 mutations on mouse behaviors, including outcomes potentially relevant to autism, we conducted comprehensive phenotyping of social, communication, repetitive, and cognitive behaviors. En2 null mutants exhibited robust deficits in reciprocal social interactions as juveniles and adults, and absence of sociability in adults, replicated in two independent cohorts. Fear conditioning and water maze learning were impaired in En2 null mutants. High immobility in the forced swim test, reduced prepulse inhibition, mild motor coordination impairments and reduced grip strength were detected in En2 null mutants. No genotype differences were found on measures of ultrasonic vocalizations in social contexts, and no stereotyped or repetitive behaviors were observed. Developmental milestones, general health, olfactory abilities, exploratory locomotor activity, anxiety-like behaviors and pain responses did not differ across genotypes, indicating that the behavioral abnormalities detected in En2 null mutants were not attributable to physical or procedural confounds. Our findings provide new insight into the role of En2 in complex behaviors and suggest that disturbances in En2 signaling may contribute to neuropsychiatric disorders marked by social and cognitive deficits, including autism spectrum disorders.

Figure 1. RTQPCR analysis demonstrates that En2 is expressed in multiple adult brain structures.

Average ▵Ct values with standard error are shown for the following brain structures and tissue: somatosensory cortex (ssctx), hippocampus (hippo), striatum, hypothalamus (hypoth), thalamus (thal), colliculi (coll), cerebellum (cereb), brainstem (bstem), amygdala (amyg), visual cortex (vctx), prefrontal cortex (pctx), olfactory bulb (ob), and hindlimb muscle (ms). Lower ▵Ct values indicate high gene expression, whereas higher values reflect lower levels. nd  =  none detected.

Figure 2. Juvenile En2 mutant mice display fewer reciprocal social interactions, as replicated in two cohorts.

Cohort 1: As compared to wildtype littermates (+/+), En2 null mutant mice (−/−) exhibited fewer bouts of (A) nose-to-nose sniffing. En2+/− and −/− mice displayed fewer bouts of (B) anogenital sniffing, and (C) following as compared to +/+ controls. No significant genotype differences were detected in (D) front approach, (E) self-grooming, and (F) exploration. Cohort 2: As compared to +/+, +/− and −/− exhibited fewer bouts of (G) nose-to-nose sniffing and (H) anogenital sniffing. En2−/− mice exhibited fewer bouts of (J) front approach as compared to +/+. No significant genotype differences were detected for (I) following behaviors, (K) self-grooming, or (L) arena exploration. Cohort 1: N = 15+/+; N = 15+/−; N = 16−/−; Cohort 2: N = 14+/+; N = 15+/−; N = 10−/−. *p<05 vs. +/+.

Figure 3. Adult En2−/− exhibit absence of sociability and deficits in male-female social interactions.

Social approach was tested in two separate cohorts using our automated three-chambered apparatus. Cohort 1: (A) En2+/+ and +/− displayed sociability, defined as spending more time in the chamber with the novel mouse than in the chamber with the novel object. En2−/− did not spend more time in the novel mouse chamber as compared to the novel object chamber, meeting the definition of lack of sociability for this task. (B) En2+/+ and +/− spent more time sniffing the novel mouse than the novel object. En2−/− did not spend more time sniffing the novel mouse than the novel object, meeting the definition of lack of sociability on this more sensitive parameter of social interaction, and confirming results from the chamber time parameter investigation. (C) No genotype differences were found for time spent in each chamber during the habituation phase. Cohort 2: (D) Similar lack of sociability was seen in En2−/− mice for time spent in the novel mouse chamber vs. the novel object chamber. (E) En2−/− mice again failed to spend more time sniffing the novel mouse vs. the novel object. (F) Time spent in each chamber during the habituation phase was not different between genotypes. Cohort 1: N = 16+/+, N = 16+/−, N = 15−/−; Cohort 2: N = 10+/+, N = 13+/−, N = 14−/−. *p<05 vs. novel object. Reciprocal social interactions and ultrasonic vocalizations (USVs) were measured in male En2 mice during interaction with an unfamiliar estrus female mouse. (G) En2−/− males spent less time engaged in sniffing the body and anogenital regions of the female as compared to +/+ males. (H) The total number of USVs emitted during the test session did not differ between genotypes. (I) No genotype differences were found for bouts of test cage exploration during the 5-minute test session. N = 10+/+, N = 13+/−, N = 13−/−. *p<05 vs. +/+.

Figure 4. Cognitive deficits in En2 null mutants.

Cumulative time spent freezing during the fear conditioning test sessions, as quantified by the VideoFreeze software, was converted to percent time freezing for data analysis and presentation. (A) Despite normal postshock freezing during training, En2−/− exhibited significantly less freezing than En2+/+ and +/− mice upon testing of contextual and cued fear memory. N = 23+/+, N = 23+/−, N = 20−/−. *p<.005 vs. +/+ and +/−. (B) In the novel object recognition test, a lack of innate object preference was observed for En2+/+, +/− and −/− mice during the familiarization phase of the task. (C) En2+/+ displayed novel object recognition memory, defined as spending more time sniffing the novel object as compared to the familiar object. En2+/− exhibited a trend towards significant preference for the novel object, whereas −/− failed to display a preference for the novel object. N = 16+/+, N = 17+/−, N = 16−/−. *p<0.05 vs. familiar object. (D) In the Morris water maze, En2−/− showed longer latencies to reach the hidden platform during training trials as compared to +/+. *p<.01 vs. +/+. (E) In the probe trial, +/+ and +/− mice showed selective quadrant search with a greater percentage of time spent in the training quadrant as compared to the non-trained quadrants, while −/− failed to show selective search. (F) En2+/+ displayed a greater proportion of platform crossings in the trained quadrant as compared to the analogous locations in the non-trained quadrants, whereas +/− and −/− did not. N = 16+/+, N = 13+/−, N = 13−/−. *p<05 vs. non-trained quadrant.

Figure 5. Increased depression-related behavior in En2 null mutants.

(A) Percentage of observations in which immobility was displayed, during the last 4 min of the forced swim test, was significantly greater for En2−/− as compared to +/+ and +/−. N = 16+/+; N = 14+/−; N = 13−/−. *p<.005 vs. +/+ and +/−. (B) No genotype differences in immobility were observed over the 6-min test session for the tail suspension test. N = 15+/+; N = 16+/−; N = 15−/−.

Figure 6. En2 null mutants display reduced startle reactivity and reduced prepulse inhibition of acoustic startle.

All three genotypes in both cohorts displayed graded startle reactivity as expected, and minimal reactivity at baseline. Cohort 1: (A) En2−/− displayed significantly lower startle responses to the 110 and 120 dB startle stimuli as compared to +/−. Cohort 2: (B) No genotype differences in startle reactivity were found. Cohort 1: N = 16+/+; N = 20+/−; N = 18−/−; Cohort 2: N = 12+/+, N = 14+/−, N = 15−/−. All three genotypes in both cohorts also displayed the expected increase in prepulse inhibition (PPI) of acoustic startle as a function of increasing prepulse intensity. Cohort 1: (C) En2−/− displayed significantly lower PPI as compared to +/− at the 78 dB prepulse intensity and significantly lower PPI as compared to +/+ and +/− mice at the 82, 86 and 92 dB prepulse intensities. N = 15+/+; N = 17+/−; N = 15−/−. *p<.05 vs. +/+ and +/−; #p<.05 vs. +/−. Cohort 2: (D) En2−/− displayed lower PPI as compared to +/+ at the 78 and 82 dB prepulse intensities. N = 13+/+, N = 13+/−, N = 12. *p<.05 vs. +/+.

Figure 7. En2 mice display deficits in forelimb grip strength and in rotarod motor learning and coordination under certain testing conditions.

Cohort 1: (A) Grip strength was reduced in En2−/− as compared to +/+ and +/−. N = 19+/+, N = 15+/−, N = 15−/−; *p<.005 vs. +/+ and +/−. Cohort 2: (B) En2−/− displayed reduced grip strength as compared to +/+ only. N = 11+/+, N = 13+/−, N = 16−/−. *p<005 vs. +/+. Mice were tested for rotarod motor coordination and learning over a total of 6 trials. Cohort 1: (C) Mice were given two trials per day for three days, with a 60 minute intertrial interval. No genotype differences were observed in latency to fall from the rotarod. Mean latency to fall for the standard C57BL6/J (B6) strain is shown as an illustrative comparison. Cohort 2: (D) Mice were given three trials per day for two days, with a 30 minute intertrial interval. En2−/− displayed lower latencies to fall as compared to +/+ on trials 3 and 6. Cohort 1: N = 14+/+, N = 18+/−, N = 17−/−, N = 7 C57BL6/J; Cohort 2: N = 13+/+, N = 14+/−, N = 13−/−. *p<.05 vs. +/+.

Figure 8. Anxiety-like behaviors are normal in En2 null mutants.

On the elevated plus-maze, no genotype differences were seen in (A–B) percent open arm time, (C–D) number of open arm entries, or (E–F) total number of arm entries. Cohort 1: N = 16+/+; N = 15+/−; N = 15−/−; Cohort 2: N = 16+/+, N = 15+/−, N = 13−/−. In the light ↔ dark exploration task, no genotype differences were observed in (G–H) the number of transitions between the light and dark chambers or (I–J) time spent in the dark chamber. In Cohort 1 (K) and Cohort 2 (L), En2−/− mice displayed a higher mean latency to enter the dark chamber as compared to +/− and −/− mice. Latency to enter the dark chamber is not a standard parameter for anxiety-like traits or responses to anxiolytic drugs in this task, but may instead reflect the somewhat lower exploratory activity in −/−, as shown in Figure 8. Cohort 1: N = 16+/+; N = 15+/−; N = 15−/−; Cohort 2: N = 16+/+, N = 16+/−, N = 14−/−. *p<.005 vs. +/+ and +/−.

Figure 9. Genotype differences in selected parameters of open field activity in adult En2 mice.

Cohort 1: En2−/− mice (A) traversed less total distance as compared to +/− and (G) exhibited fewer bouts of vertical activity as compared to +/+ and +/− mice. No genotype differences were detected for (C) horizontal activity or (E) center time. N = 15+/+; N = 17+/−; N = 15−/−. *p<.05 vs. +/+ and +/−; #p<.005 vs. +/−. Cohort 2: En2+/− and −/− mice exhibited greater (D) horizontal activity as compared to +/+ mice during minutes 6–10 of the test session. No genotype differences were found for (B) total distance traveled, (F) center time or (H) vertical activity. N = 15+/+, N = 19+/−, N = 15−/−. *p<05 vs. +/+.

Figure 10. No genotype differences in olfactory habituation/dishabituation to social and non-social odors or repetitive self-grooming.

In cohort 1 (A) and cohort 2 (B), a significant decline in sniffing (habituation) to repeated presentations of water, two non-social odors and two social odors was observed all three genotypes. A significant increase in sniffing upon the first presentation of a novel odor (dishabituation) was also observed across genotypes. Cohort 1: N = 15+/+, N = 15+/−, N = 15−/−; Cohort 2: N = 12+/+, N = 10+/−, N = 10−/−. En2+/+, +/− and −/− mice in Cohort 1 (C) and Cohort 2 (D) spent a similar amount of cumulative time engaged in self-grooming during a 10 min test session. Cohort 1: N = 15+/+, N = 16+/−, N = 15−/−; Cohort 2: N = 11+/+, N = 13+/−, N = 15−/−.

Figure 11. Developmental milestones and pup ultrasonic vocalizations are generally normal in En2 null mutants.

Developmental milestones were measured every other day from postnatal day 2 to 14, using a modified version of the Fox battery. En2−/− pups exhibited reduced (A) body length as compared to +/+ pups on postnatal day 6 only. No genotype differences were observed in (B) body weight, (C) eye opening, (D) pinnae detachment, (E) righting reflex, and (F) negative geotaxis. N = 13+/+, N = 15+/−, N = 15. *p<05 vs. +/+. Pup ultrasonic vocalizations to separation from the mother and siblings were measured over postnatal days 4, 6, 8 and 11. (G) Number of calls emitted during the separation period did not differ across genotypes on any the four days of testing. (H) Body temperature did not differ between genotypes. N = 13+/+, N = 15+/−, N = 11−/−.