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

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

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

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

Adult <i>En2</i>−/− exhibit absence of sociability and deficits in male-female social interactions.

<p>Social approach was tested in two separate cohorts using our automated three-chambered apparatus. Cohort 1: (<b>A</b>) <i>En2</i>+/+ and +/− displayed sociability, defined as spending more time in the chamber with the novel mouse than in the chamber with the novel object. <i>En2</i>−/− 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>B</b>) <i>En2</i>+/+ and +/− spent more time sniffing the novel mouse than the novel object. <i>En2</i>−/− 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. (<b>C</b>) No genotype differences were found for time spent in each chamber during the habituation phase. Cohort 2: (<b>D</b>) Similar lack of sociability was seen in <i>En2</i>−/− mice for time spent in the novel mouse chamber vs. the novel object chamber. (<b>E</b>) <i>En2</i>−/− mice again failed to spend more time sniffing the novel mouse vs. the novel object. (<b>F</b>) 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 <i>En2</i> mice during interaction with an unfamiliar estrus female mouse. (<b>G</b>) <i>En2</i>−/− males spent less time engaged in sniffing the body and anogenital regions of the female as compared to +/+ males. (<b>H</b>) The total number of USVs emitted during the test session did not differ between genotypes. (<b>I</b>) 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. +/+.</p

Statistical results for olfactory habituation/dishabituation.

<p>Summary of statistical results of the olfactory habituation/dishabituation test. Data are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040914#pone-0040914-g010" target="_blank">Figure 10</a>.</p

Juvenile <i>En2</i> mutant mice display fewer reciprocal social interactions, as replicated in two cohorts.

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

Cognitive deficits in <i>En2</i> null mutants.

<p>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. (<b>A</b>) Despite normal postshock freezing during training, <i>En2</i>−/− exhibited significantly less freezing than <i>En2</i>+/+ and +/− mice upon testing of contextual and cued fear memory. N = 23+/+, N = 23+/−, N = 20−/−. *p<.005 vs. +/+ and +/−. (<b>B</b>) In the novel object recognition test, a lack of innate object preference was observed for <i>En2</i>+/+, +/− and −/− mice during the familiarization phase of the task. (<b>C</b>) <i>En2</i>+/+ displayed novel object recognition memory, defined as spending more time sniffing the novel object as compared to the familiar object. <i>En2</i>+/− 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. (<b>D</b>) In the Morris water maze, <i>En2</i>−/− showed longer latencies to reach the hidden platform during training trials as compared to +/+. *p<u><</u>.01 vs. +/+. (<b>E</b>) 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. (<b>F</b>) <i>En2</i>+/+ 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.</p

RTQPCR analysis demonstrates that <i>En2</i> is expressed in multiple adult brain structures.

<p>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.</p

Statistical results for selected parameters of open field locomotor activity.

<p>Summary of statistical results of selected parameters of open field locomotor activity. Data are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040914#pone-0040914-g009" target="_blank">Figure 9</a>.</p

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

<p>In cohort 1 (<b>A</b>) and cohort 2 (<b>B</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−/−. <i>En2</i>+/+, +/− and −/− mice in Cohort 1 (<b>C</b>) and Cohort 2 (<b>D</b>) 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−/−.</p