Developmental Delay And Behavioural Disorder In Pyramidal Neuron-targeted Nipbl Knock-out Mice

Context:Cornelia de Lange syndrome?CdLS?is a rare polymalformative syndrome,characterized by pre-and postnatal growth retardation,psychomotor and mental deficits.All of the causative mutant genes identified in the CdLS cases encode cohesin subunits or proteins that interact with the complex,including NIPBL?Nipped-B-like?,SMC1A,SMC3,RAD21,and HDAC8.Neurological defects including psychomotor development delay and behavioural disorders are universal findings of CdLS,but the pathological mechanisms are still unclear.Here,CaMKIIa-iCre mice were crossed with Nipblflox/flox mice to establish a pyramidal neuron-specific NIPBL knockout mouse model to study the effects and possible mechanisms of NIPBL on postnatal neurodevelopment.Methods:The levels of Nipbl expression in olfactory bulb,hippocampus,hypothalamus,cerebral cortex and cerebellum of mice were determined by fluorescent in situ hybridization?FISH?,in situ hybridization?ISH?and quantitative reverse transcription polymerase chain reaction?qRT-PCR?.Nipbl Floxed?Nipblflox/flox?mice were crossed with the CaMKIIa-iCre transgenic mice in which the iCre recombinase is expressed under the control of calcium-dependent calmodulin kinase II promoter to develop pyramidal neuron-specific NIPBL knock-out mice(CaMKlIa-iCre^+/-;Nipblflox/flox hereafter NipblcKO).Littermates carrying CaMKIIa-iCre^-/-;Nipblflox/+?Nipbl+/+?were used as controls.The weight gain and survival of mice were recorded.Mice were subjected to neurodevelopmental evaluations and behavioral tests that were videotaped.H&E staining was performed to reveal gross structure of the brain.TUNEL staining was used to identify the nerve cell apoptosis of central nervous system after NIPBL deletion.Immunohistochemistry using anti-PSD95 antibody and using anti-GFAP antibody were performed to further reveal synaptogenesis and gliosis respectively across the brain.Results:1.Nipbl was evident in olfactory bulb,mildly expressed in the hippocampus and hypothalamus,but not in the cerebral cortex of suckling mice.A small amount of Nipbl was expressed in the hypothalamus but not in the hippocampus,cerebellum and cerebral cortex of adult mice.2.NipblcKO mice showed overall postnatal growth retardation,neurobehavioral developmental delay,behavioural abnormality and early lethality.NipblcKO pups raised in pure CaMKlla-iCre^+/-:Nipblflox/flox litters showed the same phenotype as pups raised in litters with mixed genotype excluding competition with control mice as cause for the effect.The phenotypic severity varied in NipblcKO group.8/100 of NipblcKO pups had normal breast milk consumption and basal reflex in the first two weeks,but exhibited seizures after two weeks of age.31/100 of NipblcKO pups showed delayed transition from crawling to walking and death before two weeks of age.3.Histological examination of mutant brains revealed reduced volume but grossly normal morphology.Significant difference of TUNEL positive cells in cortex,hypothalamus and hippocampus was found between the two groups.Immunohistochemical staining showed lower PSD95?a selective marker for synapse?expression in hippocampus of NipblcKO mice.GFAP expression was significantly higher in the hypothalamus of NipblcKO mice compared with controls.Conclusion:1.Homozygous deletion of Nipbl in pyramidal neurons caused growth retardation,neurodevelopmental delay,behavioral disturbance and early lethality of mice.2.Nerve cell apoptosis in central nervous system,perturbed hippocampal synaptogenesis and hypothalamic gliosis caused by pyramidal neuron-specific Nipbl deficiency may involve in growth retardation,neurobehavioral developmental delay and early lethality of NipblcKO mice.