DNA hypomethylation restricted to the murine forebrain induces cortical degeneration and impairs postnatal neuronal maturation

Abstract

DNA methylation is a major epigenetic factor regulating genome reprogramming, cell differentiation and developmental gene expression. To understand the role of DNA methylation in central nervous system (CNS) neurons, we generated conditional Dnmt1 mutant mice that possess approximately 90% hypomethylated cortical and hippocampal cells in the dorsal forebrain from E13.5 on. The mutant mice were viable with a normal lifespan, but displayed severe neuronal cell death between E14.5 and three weeks postnatally. Accompanied with the striking cortical and hippocampal degeneration, adult mutant mice exhibited neurobehavioral defects in learning and memory in adulthood. Unexpectedly, a fraction of Dnmt1(-/-) cortical neurons survived throughout postnatal development, so that the residual cortex in mutant mice contained 20-30% of hypomethylated neurons across the lifespan. Hypomethylated excitatory neurons exhibited multiple defects in postnatal maturation including abnormal dendritic arborization and impaired neuronal excitability. The mutant phenotypes are coupled with deregulation of those genes involved in neuronal layer-specification, cell death and the function of ion channels. Our results suggest that DNA methylation, through its role in modulating neuronal gene expression, plays multiple roles in regulating cell survival and neuronal maturation in the CNS.

Figure 1.

Hypomethylated cortical cells are detected in Emx1-cre; Dnmt1^2lox/2lox mutants throughout the normal life span (A) LacZ histology pattern shows brain regions with Dnmt1 gene deletion via the R26R reporter (ref. 20). (B) The recombination efficiency was analyzed by Southern blotting analysis. The percentages of Dnmt1 gene deletion are derived from the ratio of recombined null allele (1lox) over the sum of functional Dnmt1^2lox (2lox) and 1lox alleles. (C) The graph view of the efficiency of Dnmt1 gene deletion in control and mutant mice from E11.5 to adulthood at 1.5 years old. (D) Southern blot analysis of DNA methylation in dorsal cortex. DNA was digested with methyl-sensitive enzyme HpaII (CCGG) and was hybridized to an IAP probe. CON, Emx1-cre; Dnmt1^210x/+ control; MUT, Emx1-cre; Dnmt1^210x/210x mutant; Ctx, cortex; str, striatum.

Figure 2.

Cortical degeneration in dorsal forebrain of Emx1-cre; Dnmt1^2lox/2lox mutants (A and B) Microscopic images of cresyl-violet staining in control and mutant brain sections at newborn, early postnatal P7, and adult stages. (C) NeuN immunohistochemistry at P7 reveals a decrease in the density of neurons in the cortex of the mutant. (D and E). Immunostaining for GFAP in control and mutant cortex. Ctx, cortex; vz, ventricular zone; str, striatum; hipp, hippocampus; lv, lateral ventricle; fx, fornix; ic, internal capsule; I–IV, cortical layers; wm, white matter; mz, marginal zone; dcp, dense cortical plate. Con, Emx1-cre; Dnmt1^2lox/+ control; mut, Emx1-cre; Dnmt1^2lox/2lox mutant.

Figure 3.

Localization of demethylated cells in mutant brains by IAP immunostaining. (A) Western blotting of IAP protein in the dorsal cortex of wild type (con) and Emx1-cre; Dnmt1^2lox/2lox mutants mice (mut) and Nestin-cre; Dnmt1^2lox/2lox mutant mice (Nestin Dnmt1, see ref. 20). (B) Immunohistochemical detection of IAP in the dorsal cortex at 1 month of age in coronal sections. (scale bar = 100 microns) (C) Co-staining with NeuN (a pan-neuronal marker) reveals an IAP-positive staining pattern in mutant cortex only. (scale bar = 25 microns) (D) Cell counts show that approximately 28–32% of the total cells are IAP-positive in 1 or 2 months (mo) old mutant mice. Among those IAP+ cells, 70.67 ± 1.8% are NeuN+ neurons.

Figure 4.

Hypomethylated cells in the dorsal cortex undergo apoptotic cell death perinatally TUNEL immunohistochemistry were performed with E11.5, E14.5, P0 and P7 brain samples. No difference in levels of apoptotic index at E11.5 even though demethylated cells IAP-positive at this time point. By E14.5, there is a dramatic increase in apoptosis in the ventricular zone and the emerging cortical plate of the mutant embryo. This region is positive for strong IAP staining and activated caspase-3 immunoreactivity. The neurotubulin marker TUJ1 (green) is used to show the cortical plate (cp) boundary from the ventricular zone (vz). Note high magnification images from boxed region shows co-labeling of TUNEL-positive cells with IAP reactivity (arrowhead). Apoptotic signals are also easily detected in mutant cortex at P0 and P7. con, Emx1-cre; Dnmt1^2lox/+ control; mut, Emx1-cre; Dnmt1^2lox/2lox mutant; Lv, lateral ventricle; str, striatum; cc, corpus callosum; ctx, cortex.

Figure 5.

Locomotion, thigmotaxis, and Morris Water Maze behavioral tests of control and mutant mice. (A) Open field test assesses animal exploratory behavior. Mutants exhibit hyperactivity as a function of increased locomotor activity; however, mutants do not appear to have altered anxiety levels as measured by thigmotatic behavior. (B) Hippocampal dependent contextual fear tasks show a severe reduction of long term memory in the mutants, consistent with the loss of the hippocampal structure in mutants. Re-exposure to the fear conditioning chamber (7 day) elicited no fear response in mutant animals, akin to the lack of freezing seen in hippocampal lesioned animals. (C) Morris water maze testing the ability of mice to recall spatial navigation is a hippocampal specific learning and memory task. Over continuous learning trial days, mutants do not improve in finding the hidden platform, nor do they exhibit a learning curve for the task of locating the platform. (D) Conditional mutants score below chance (25%) in probe trials after a 14-day learning protocol, indicative of poor spatial memory in the mutants. For each category of mice, more than 11 adult mice/genotype around the age of 6 months were used in behavioral tests.

Figure 6.

Hypomethylation alters neuronal gene expression in mutant cortex in both pre- and postnatal stages. (A and B) The expression of X-linked gene (Magea3, A) and apoptosis-related genes (Gadd45, Casp4, Ngfr, B) in the dorsal cortex of wild type (WT, open bar) or Emx1-cre; Dnmt1 mutant (mut, black bar) mice. (C and D) The expression of layer specific gene (Lhx2, C) and neuronal channels genes (Kcnh5, Kcnj9 and Scnn1a, D) in the dorsal cortex of wild type and mutant mice. Quantitative real-time PCR was performed in cDNAs derived from E14.5 and P5 dorsal cortex of each mouse by using the specific primers. Statistical significance was evaluated by the t-test. Mean ± SEM (n = 3). *P < 0.05.

Figure 7.

Hypomethylated cortical neurons exhibit defects in dendritic arborization (A–D). Morphology of M4-egfp-positive neurons in control and mutant cortices at P0 (A and B) and P7 (C and D). (E) A high percentage of M4-egfp+ cells colocalize with IAP reactivity in mutant cortices (94.98 ± 0.85%). (F) Camera lucida drawings of individual M4-egfp-positive neurons filled with biocytin from either control or mutant cortex. (G) Sholl analysis to analyze dendritic branching reveals an increase in basal and apical branching patterns (P < 0.01, student t-tests).

Figure 8.

Hypomethylated neurons exhibit defects in resting membrane and action potentials. (A) Significant changes in resting membrane potential (RMP) were graphed (*P < 0.05, student t-test). (B) Action potential wave forms in control and mutant cells including M4-eGFP-positive neurons. (C) Action potential half-width and maximum rate of repolarization in P1–4, 5–8, 9–12, 13–16, 17–20 and P21–adult regular spiking cortical neurons in control and mutant slices. Note the increased half-width resulting from decrease in the maximal rate of repolarization at all ages. (D) Voltage schematic and leak subtracted currents elicited by stepping from −110 mV to +40 mV, with or without a 50 ms pre-step to −40 mV in outside out patches pulled from control and mutant RS cortical neurons. Figures are averages of currents obtained from 23 control patches and 8 mutant patches. Subtraction of pulses with the pre-step from pulses without the pre-step result in isolation of the rapidly inactivating potassium conductance (I_A) in control neurons but not mutant neurons. Note that potassium currents in mutant somatic outside out patches inactivate much more slowly than those obtained from control neurons.