BACKGROUND
rett syndrome  is an x-linked dominant neurological disorder affecting predominantly females  <cit>  and has an estimated prevalence of  <dig> in  <dig> - <dig>  females  <cit> .
in patients with rtt, symptoms are generally recognized around  <dig> to  <dig> months of age following an apparently normal period of early development.
regression of previously acquired skills and deceleration of head growth are key features of the disease in conjunction with stereotypic hand movements.
ultimately, affected individuals frequently display cognitive, social, and language impairments as well as a variety of serious motor and breathing abnormalities, seizures, microcephaly, gastrointestinal dysfunction, scoliosis, kyphosis, and rigidity  <cit> .
pathological studies of human rtt cases have shown a reduction in brain size, and an increase in cell packing density  <cit> .
these studies have also noted a significant reduction in the dendritic branching of neurons in specific layers of the frontal, motor, temporal and limbic cortex  <cit> .
most cases of rtt are caused by point mutations, insertions, duplications, or deletions in the mecp <dig>  gene  <cit> .
over  <dig> distinct mecp <dig> mutations have been described and certain mutations occur with high frequency  .
at least two important functional domains within the mecp <dig> protein have been identified.
the first is a methyl-cpg binding domain  which binds specifically to methylated cpg- dinucleotides and the second is a transcriptional repressor domain , which interacts with a transcriptional repressor complex  <cit> .
while widely described in the past as a transcriptional repressor, a recent report indicates that mecp <dig> may also be a transcriptional activator, and may actually function more often as an activator than a repressor of transcription  <cit> .
it is also believed to participate in recruiting chromatin remodeling and co-repressor proteins  <cit> , and  <cit> .
mecp <dig> acts to modulate the transcription of many genes, one of which, bdnf  <cit> , is particularly relevant to diseases causing mental retardation as it is heavily involved in synaptic plasticity  <cit>  and influences disease progression in mecp <dig> mutant mice  <cit> .
in addition to the classic or variant rtt phenotypes in females, mecp <dig> gene mutation has also been described in males  <cit> , sometimes causing a severe and fatal early onset encephalopathy  <cit> .
one specific mutation of mecp <dig>  a140v, does not result in a severe phenotype or in early lethality.
while not resulting in classical rtt features, this mutation has been reported to cause x-linked mental retardation  and neuropsychiatric syndromes as well as ppm-x syndrome   <cit> .
the a140v mutation is a recurring mutation within the mbd of mecp <dig> accounting for about  <dig> % of all reported mecp <dig> mutations .
this mutation was first reported in a female with mild mental retardation, her daughter with mild mental retardation, and her four sons with severe mental retardation  <cit> .
in a cohort of mentally retarded patients negative for expansion of the cgg repeat at the fmr <dig> locus, couvert et al found two sporadic cases of a140v mutation  <cit> .
a three-generation family with five affected males  was shown to carry the a140v mutation  <cit> .
the a140v mutation has also been reported in a young boy with language disorder and schizophrenia  <cit> .
in addition to the previously listed symptoms, dysarthric speech, mild dysmorphic features, spasticity, gait abnormalities, mild microcephaly, trophic changes of the legs, distal atrophy in the limbs, kyphoscoliosis, ankle clonus, and abnormal deep tendon reflexes are also symptoms associated with this mutation  <cit> .
thus, the a140v mecp <dig> mutation is an important cause of xlmr and developmental disorders and is somewhat distinct clinically from classic rtt.
it has been predicted based on structural data that the a140v mutation, located in the α <dig> helix of the mbd, could potentially affect the structure of the mecp <dig> mbd and therefore the methyl-cpg dna binding function.
ohki, et al.
<cit>  speculate that due to its bulky side chain, the substituted valine may cause reduced dna binding affinity and orrico, et al.,  <cit>  using secondary structure prediction programs, proposed that the a140v mutation shortens the α <dig> helix by half, potentially resulting in an alteration of function.
although many mecp <dig> gene mutations do affect the methyl-cpg dna binding function of the mecp <dig> protein, the a140v mutation has been reported not to, despite predictions based on its location within that functional domain  <cit> .
indeed, mecp <dig> a140v protein localizes to heterochromatic foci, containing abundant methyl- cpg sites, as efficiently as wild type  mecp <dig>  in addition, dna binding assays in cultured cells have demonstrated that the a140v mutation does not affect binding to methylated dna  <cit> .
nevertheless, another interesting property of mecp <dig> a140v protein was discovered in studies of transcriptional activity in transfected cells.
mecp <dig> a140v repressed transcriptional activity from methylated promoters at levels comparable to wt and from unmethylated promoters at a level approximately 40% higher than wt mecp <dig>  <cit> .
this raises the possibility that higher repressive activity by mecp <dig> a140v may affect the normal expression of unmethylated genes.
the a140v mutation is also reported to directly affect the ability of mecp <dig> to bind to the atrx protein  <cit> , a helicase/atpase believed to be involved in chromatin remodeling  <cit> .
alpha-thalassemia x-linked mental retardation syndrome  is caused by mutation of the atrx gene.
in addition to the hematologic abnormality alpha-thalassemia, atrx gene mutations also result in mental retardation, microcephaly, dysmorphic features, genital and renal abnormalities, growth deficiency, seizures, spasticity, gastrointestinal dysfunction, and kyphoscoliosis and other skeletal abnormalities  <cit> .
many of the symptoms of atrx overlap those seen in patients with a classic rtt mutation of the mecp <dig> gene.
there are also clear differences, the presence of dysmorphic features in atrx mutations for example.
interestingly, the a140v mutation reproduces symptoms of both conditions while still being somewhat distinct from each of them.
this may provide a glimpse into the mechanism underlying each of these conditions.
currently there are no complete atrx knock out mouse models due likely to the described embryonic lethality in males  <cit> .
tissue specific conditional inactivation of atrx results in enhanced cell death during corticogenesis with early lethality   <cit>  and interneuron defects in the retina   <cit> .
a variety of mouse models of rtt have been previously developed and tested.
one of the first mecp <dig> mutant mouse models generated lacked exons  <dig> and  <dig>  this model demonstrated that mecp2-null animals were viable and did not have a specific initial phenotype.
between the ages of  <dig> and  <dig> weeks these animals  developed abnormalities of gait and breathing, ultimately leading to early death at around  <dig> months of age  <cit> .
a second mecp2-null mouse was generated by deletion of exon  <dig>  male mice appeared healthy for the first few weeks of age but developed abnormal movements, motor control, and breathing by  <dig> weeks of age with death occurring around  <dig> weeks.
female mice in this study exhibited similar symptoms but with a time course delayed by months  <cit> .
shahbazian et al.
<cit>  generated mice with truncation of the c-terminal third of the coding region  by introducing a stop codon following amino acid  <dig>  which is located at the c terminal end of the transcriptional repressor domain.
male mutant mice survived to at least  <dig> year of age and developed a variety of symptoms including tremor, myoclonic seizures, kyphosis and stereotyped forelimb movements.
the motor performance of these mice declined with age.
histological studies of the brain were normal and fear conditioning and spatial learning were normal, however, the mice did display enhanced anxiety and an abnormal corticosterone stress response  <cit> .
recently, a knock-in model of rtt  was generated, reproducing a common non-sense mutation of mecp <dig> found in human cases.
as with other rtt model mice, male r168x mice die early at around  <dig> weeks of age, but display symptoms suggestive of rtt such as breathing irregularities, hypoactivity, and hind limb atrophy and clasping.
female heterozygous mice show similar symptoms but not until around  <dig> months of age  <cit> .
as reported in pathological studies of human rtt brain tissues  <cit> , abnormalities of brain size  <cit>  and cell packing density have been observed in mouse models of rtt  <cit> .
abnormalities of dendrite branching have also been reported in some rett syndrome mouse models  <cit> .
much of the testing in mouse models has been done using hemizygous males to avoid the phenotype variability resulting from random x inactivation in females, which is considered to influence phenotype in human rtt patients.
we have performed our initial tests in males for this reason and because a140v is more commonly recognized in longer surviving male patients.
the pathogenesis of the neurological phenotype in rtt remains unclear, and continued study of mouse models will improve our understanding.
the various mouse models recapitulate the symptoms of rtt in many ways but are often distinct from each other.
although much has been learned about rtt through the study of existing mouse models, the a140v model we have created is unique.
it reproduces a recurring missense mutation seen in humans and, in males, results in an xlmr phenotype .
in addition, due to the long life span of mutant male mice, developmental studies can be performed.
we expect that by a careful analysis of the a140v mouse, we will better understand the important roles that mecp <dig> plays in the pathogenesis of developmental brain disorders that result in mental retardation.
here we describe the construction and initial characterization of brain abnormalities in the mecp <dig> a140v mutant mouse model of rtt/xlmr.
in addition to being a model for these two diseases, the a140v mutant mouse permits the study of a novel mechanism by which mecp <dig> mutation might affect neural development; its interaction with the atrx chromatin remodeling protein.
RESULTS
generation of the mecp <dig> a140v mutant mouse
the a140v mouse model of rtt/xlmr was generated by introducing a point mutation  that results in the a140v missense mutation of mecp <dig> protein.
the wt locus, targeting vector, and recombined locus, are diagrammed in figure 1a; also shown in figure 1a is the location of probe a  used for southern blot analysis as well as the southern blot bam hi fragments.
in addition, the location of the genotyping primers  is indicated.
results of a pcr based test of es cell dna to detect homologous recombination at the 3'-end of the targeting vector are shown in figure 1b .
the wt allele yields a  <dig> bp fragment while the mutant allele yields a  <dig> bp fragment.
as a confirmatory test these fragments were digested with acl i which cuts the mutant allele but not the wt allele, yielding a double band of  <dig> bp and  <dig> bp .
southern blot analysis of bam hi digested es cell dnas detects a  <dig>  kbp band, confirming homologous recombination at the 5'-end of the targeting vector .
es cell clones that were positive by pcr  and southern blot  were then sequenced to verify that they were hemizygous for the a140v mutation  and two of these clones were used for blastocyst injection.
shown in figure 1d are pcr genotyping results of f <dig> females: heterozygous females produce two bands and wt females produce a single  band.
plasmid controls show either the single smaller band  or single larger band .
a chimeric male created from the 177u <dig> es cell clone was used to produce heterozygous female founders for the mecp <dig> a140v mouse colony.
mutant mecp <dig> mrna is expressed in brain tissue
we confirmed that only mutant mecp <dig> mrna was present in brain tissue of male hemizygous animals by reverse transcription pcr .
cdna was prepared from cerebellar total rna extracted from male animals , and tested by pcr .
as shown in figure 2a  mutant animals display only the larger fragment while wt animals display only the smaller fragment.
this result also confirms the presence of the mecp <dig> 3'-utr.
a different segment of cerebellar mecp <dig> cdna  was then amplified to confirm that there was no genomic dna contamination of the cdna used in these tests.
samples from both wt and mutant animals showed only the cdna band  and not a larger genomic dna band , thus the cdna samples were free of genomic dna contamination .
sequencing performed on purified cdna from these bands confirmed the a140v mutation .
mecp <dig> protein is expressed in the brain of a140v mutant animals
as this is not a knock-out or truncating model, it was expected that the full length mecp <dig> protein would be present in the brains of hemizygous male mice.
immunostaining in brain slices using an anti-mecp <dig> antibody directed against the c-terminus of the protein demonstrated the presence of the mecp <dig> protein in the brain tissue of hemizygous mice as well as wild type control mice .
a peptide blocking assay confirmed the specificity of the antibody .
the mutation does not appear to have affected the nuclear or brain regional distribution of mecp <dig> protein.
mecp <dig> mrna levels in wild-type and a140v mouse cerebellum by qrt-pcr
total rna was extracted from the cerebellum of three a140v male mice and three wt male littermates .
quantitative real time pcr  reactions were performed using pcr assays for mecp <dig> and gapdh from sa biosciences .
the primer set for mecp <dig> detects both the e <dig> and e <dig> isoforms of mecp <dig>  figure  <dig> shows data graphs for mecp <dig> and gapdh   as well as a table of ct  values including mean and standard deviation , and Δct used in the calculation of fold change  in mrna levels, comparing a140v to wt .
fold change between mutant and wild-type is calculated using the ΔΔct method ]).
in the cerebellum, fold change in mecp <dig> mrna levels  was  <dig>  .
this demonstrated that in the cerebellum of wt and mutant  <dig> month old animals we were unable to detect a significant difference in the abundance of wt and mutant steady state mecp <dig> mrna .
this suggests that the a140v mutation does not significantly affect transcription or mrna stability.
there are no obvious phenotypic abnormalities of the a140v mice
no obvious phenotypic differences between a140v and wt male mice were noted.
somatic growth between  <dig> and  <dig> weeks of age in mutant animals was similar to wt controls .
we have not observed abnormalities in body morphology  in animals observed up to  <dig> months of age.
necropsy of mutant animals has not revealed any gross organ pathology.
no seizures or tremors have been observed in these animals.
female heterozygous mice displayed a normal level of fertility although attempted breedings of male hemizygous mice have thus far been unsuccessful.
both male hemizygous and female heterozygous a140v mice have lived to greater than  <dig> months of age with no unusual health issues.
increase in cell packing density in mutant animals
we compared histological sections of brain tissue from  <dig> month old wt and a140v male mice .
although the wt and a140v sections were processed together, there was an apparent increase in staining intensity in the a140v mutant tissues.
on closer examination, it became apparent that this was due to an increase in the packing density of cells in the mutant animals.
this increase in packing density was noted in the frontal cortex and olfactory bulbs , in the dentate gyrus and regions ca <dig>  ca <dig>  and ca <dig> of the hippocampus , and in the granule cell layer of the cerebellum .
the difference in cell packing density between a140v and wt male mice was quantified by counting all nuclei in a standard volume of tissue  from the cerebellar granule cell layer .
this analysis demonstrated that there is a significantly higher  density of cell nuclei in the a140v cerebellar granule layer than in wt controls.
abnormal dendritic branching in mutant cortical neurons
qualitative abnormalities of dendritic branching were noted upon initial visualization of various regions of golgi-cox stained cortical tissue from mecp <dig> a140v mice.
an extended focus  image of the visual cortex is shown as an example .
in order to begin to evaluate neuronal dendritic structure in the a140v mouse, scholl analysis  <cit>  was performed on layer ii/iii pyramidal neurons of the somatosensory cortex.
this analysis was done using scholl analysis on brightfield z stack images of golgi-cox stained neurons in order to analyze the full arbors of both the apical and basal dendrites.
a diagram of this adaptation of the scholl analysis technique is shown in figure  <dig>  male a140v and wild type mice 2- <dig> months old were used for this analysis.
this quantitative analysis demonstrated significant differences in the branching complexities of the dendritic arbors of these neurons.
apical dendrites showed very significant differences in branching at  <dig> and  <dig> μm distances from the cell body and basal dendrites showed significant differences at  <dig>   <dig>  and  <dig> μm .
discussion
the a140v mouse model was constructed in order to more closely examine the mental retardation associated with rtt.
this was expected to be a mental retardation model in mice due to the effects of this mutation in human male patients.
male patients with the a140v mutation survive into adulthood, indicating that this mutation is less likely to compromise functions essential for life.
however, significant mental retardation is common in males carrying this mutation while carrier females show mild or no symptoms  <cit> .
the a140v mutation is predicted to be non-truncating and this was verified by immunoflourescence staining of brain slices from wt and mecp <dig> a140v male mice.
the staining was performed with a c-terminal mecp <dig> antibody to demonstrate that the mecp <dig> protein was not truncated by the a140v mutation.
the initial characterization of the mecp <dig> a140v mouse has revealed some interesting abnormalities.
during casual observation the mice appear normal with no variation in weight or body morphology and no indications of seizures or tremors.
however, there are cellular abnormalities in the brain consistent with those seen in rtt syndrome and other diseases resulting in mental retardation.
an increase in cell packing density was apparent upon histological studies of brain tissue from the mutant animals.
qualitative differences in cell density in the cerebellum were confirmed by performing cell counts.
the results of these cell counts showed statistically significant differences in cell density.
increased cell packing density has been previously noted in both human rtt patients  <cit>  and in rtt mouse models  <cit> .
the mecp <dig> null mouse created by deletion of exon  <dig> revealed increases in cell density in the hippocampus, cerebral cortex, and cerebellum that closely parallel our own results  <cit> .
a study of the brains of three rtt patients found an increase in cell-packing density in multiple brain regions including the cerebral cortex, basal ganglia, thalamus, hippocampus, and amygdala.
abnormalities were also reported in the cerebellum although an increase in cell packing density was not reported  <cit> .
we find no previous descriptions of cell density abnormalities in the olfactory bulbs of rett mouse models.
we are uncertain if this is because cell density abnormalities have not previously been seen in olfactory bulbs or if the olfactory bulbs were not examined in these mice.
there have been reports of defects in neuronal development in olfactory neurons from the nasal epithelium of rtt patients  <cit> .
in addition, defects of olfactory neuron maturation and protein expression have been found in two previously described rtt mouse models  <cit> , indicating that olfactory defects are not unusual in rtt or in rtt mouse models.
in addition, a recent and very thorough study of brain morphology found changes in the volume and shape of olfactory bulbs in two mecp <dig> null mouse models  <cit> .
we have seen abnormalities in the shape of olfactory bulbs and the morphology of glomeruli in some the a140v male hemizygous mice although this has not been quantified and will be the subject of further study.
a lack of branching complexity in cortical pyramidal neurons in many brain regions has been reported in human cases of rtt  <cit>  as well as in mouse models  <cit> .
in fact, dendritic branching anomalies are often found in conjunction with mental retardation of many causes  <cit> .
the a140v mutation is strongly associated with mental retardation  <cit> ; therefore, we felt that abnormal dendritic branching patterns of neurons would likely occur in this new mouse model.
we explored the branching complexity of neurons in the a140v mouse model and found differences between wt and mecp <dig> a140v mice.
we noted significant differences in the branching complexity of both the apical and basal dendrites of layer ii/iii pyramidal neurons of the somatosensory cortex as measured by scholl analysis.
this correlates closely with other reports of decreases in branching complexity of cortical pyramidal neurons in tissue from both human rtt patients and in mecp <dig> mutant mouse models.
we also found apparent differences in branching complexity in the visual cortex although this has not yet been quantified.
the mecp <dig> a140v mouse has proven to be distinct from other mouse models of mecp <dig> mutation.
like its human counterpart, the a140v mutation in mice produces symptoms both correlating with and disparate from the classical symptoms of rtt.
in humans, the a140v mutation causes a mental retardation syndrome that has associated with it abnormalities of gait, kyphoscoliosis, mild microcephaly, spasticity, dysarthric speech, and occasionally mental illness such as schizophrenia  <cit> .
however certain features of classic rett syndrome such as stereotypic hand movements, breathing abnormalities and seizures  <cit>  are notably absent in humans with the a140v mutation.
these distinctions suggest that the a140v mutation affects certain functions of the mecp <dig> protein while preserving others.
there is for instance the report indicating that the mecp <dig> a140v protein has an increased binding affinity for unmethylated dna  <cit> , the functional consequences of which are unknown.
there is also the interesting finding that although the a140v mutation preserves the methyl-cpg binding function of mecp <dig> it does interfere with binding to the mental retardation associated protein atrx  <cit> .
this raises the possibility that disruption of the mecp2-atrx interaction is in someway related to the neurological symptoms that occur in both humans with the a140v mutation and in our engineered mice.
atrx is chromatin remodeling protein that is a swi2/snf <dig> dna helicase atpase that is the cause of atrx syndrome  <cit> .
this protein has only recently been identified as a binding partner of the mecp <dig> protein.
analysis of brain tissue from mecp2-null mice revealed that loss of mecp <dig> in mature neurons leads to delocalization of the atrx protein, suggesting that mecp <dig> may be required for the proper localization of atrx.
in vitro studies done using fusion proteins in mouse fibroblasts revealed that the mecp <dig> a140v protein, while properly localized to heterochromatin within the nucleus, fails to recruit atrx to the heterochromatin.
in contrast, wt mecp <dig> protein efficiently recruits atrx to heterochromatin  <cit> .
this failure to localize atrx to heterochromatin may be important in understanding the normal function of both the mecp <dig> and atrx proteins.
we believe that it is important to note that the mice used in these experiments belong to early generations  and are therefore in a mixed 129x1/s <dig> and c57bl/ <dig> background.
in addition, the neomycin resistance gene cassette used in the construction of these mice has not been excised from the experimental animals although we have begun production of a line of mice in which the neo cassette has been removed.
as genetic background and the presence of extra dna sequences such as the neo cassette can contribute to phenotype we will be repeating these experiments in later generation mice lacking the neo cassette in order to confirm our experimental results.
however, we believe that, given the close parallels of our data with that seen in other mouse models of mecp <dig> gene disruption, the phenotype we have observed is in fact due to the a140v mutation itself.
CONCLUSIONS
we believe that this new mouse model will provide insight into the functions of mecp <dig>  including its association with the atrx protein.
the a140v mouse has proven to reproduce some of the abnormalities of other mecp <dig> mouse models  while failing to reproduce others .
with this knowledge we can continue dissecting the functions of mecp <dig> in the hopes of explaining its relationship to mental retardation and other clinical symptoms.

 rett syndrome is an x-linked dominant neurological disorder affecting predominantly females and has an estimated prevalence of in - females. in patients with rtt, symptoms are generally recognized around to months of age following an apparently normal period of early development. regression of previously acquired skills and deceleration of head growth are key features of the disease in conjunction with stereotypic hand movements. ultimately, affected individuals frequently display cognitive, social, and language impairments as well as a variety of serious motor and breathing abnormalities, seizures, microcephaly, gastrointestinal dysfunction, scoliosis, kyphosis, and rigidity. pathological studies of human rtt cases have shown a reduction in brain size, and an increase in cell packing density. these studies have also noted a significant reduction in the dendritic branching of neurons in specific layers of the frontal, motor, temporal and limbic cortex. most cases of rtt are caused by point mutations, insertions, duplications, or deletions in the mecp gene. over distinct mecp mutations have been described and certain mutations occur with high frequency. at least two important functional domains within the mecp protein have been identified. the first is a methyl-cpg binding domain which binds specifically to methylated cpg- dinucleotides and the second is a transcriptional repressor domain, which interacts with a transcriptional repressor complex. while widely described in the past as a transcriptional repressor, a recent report indicates that mecp may also be a transcriptional activator, and may actually function more often as an activator than a repressor of transcription. it is also believed to participate in recruiting chromatin remodeling and co-repressor proteins, and. mecp acts to modulate the transcription of many genes, one of which, bdnf, is particularly relevant to diseases causing mental retardation as it is heavily involved in synaptic plasticity and influences disease progression in mecp mutant mice. in addition to the classic or variant rtt phenotypes in females, mecp gene mutation has also been described in males, sometimes causing a severe and fatal early onset encephalopathy. one specific mutation of mecp a140v, does not result in a severe phenotype or in early lethality. while not resulting in classical rtt features, this mutation has been reported to cause x-linked mental retardation and neuropsychiatric syndromes as well as ppm-x syndrome. the a140v mutation is a recurring mutation within the mbd of mecp accounting for about % of all reported mecp mutations. this mutation was first reported in a female with mild mental retardation, her daughter with mild mental retardation, and her four sons with severe mental retardation. in a cohort of mentally retarded patients negative for expansion of the cgg repeat at the fmr locus, couvert et al found two sporadic cases of a140v mutation. a three-generation family with five affected males was shown to carry the a140v mutation. the a140v mutation has also been reported in a young boy with language disorder and schizophrenia. in addition to the previously listed symptoms, dysarthric speech, mild dysmorphic features, spasticity, gait abnormalities, mild microcephaly, trophic changes of the legs, distal atrophy in the limbs, kyphoscoliosis, ankle clonus, and abnormal deep tendon reflexes are also symptoms associated with this mutation. thus, the a140v mecp mutation is an important cause of xlmr and developmental disorders and is somewhat distinct clinically from classic rtt. it has been predicted based on structural data that the a140v mutation, located in the α helix of the mbd, could potentially affect the structure of the mecp mbd and therefore the methyl-cpg dna binding function. ohki, et al. speculate that due to its bulky side chain, the substituted valine may cause reduced dna binding affinity and orrico, et al., using secondary structure prediction programs, proposed that the a140v mutation shortens the α helix by half, potentially resulting in an alteration of function. although many mecp gene mutations do affect the methyl-cpg dna binding function of the mecp protein, the a140v mutation has been reported not to, despite predictions based on its location within that functional domain. indeed, mecp a140v protein localizes to heterochromatic foci, containing abundant methyl- cpg sites, as efficiently as wild type mecp in addition, dna binding assays in cultured cells have demonstrated that the a140v mutation does not affect binding to methylated dna. nevertheless, another interesting property of mecp a140v protein was discovered in studies of transcriptional activity in transfected cells. mecp a140v repressed transcriptional activity from methylated promoters at levels comparable to wt and from unmethylated promoters at a level approximately 40% higher than wt mecp. this raises the possibility that higher repressive activity by mecp a140v may affect the normal expression of unmethylated genes. the a140v mutation is also reported to directly affect the ability of mecp to bind to the atrx protein, a helicase/atpase believed to be involved in chromatin remodeling. alpha-thalassemia x-linked mental retardation syndrome is caused by mutation of the atrx gene. in addition to the hematologic abnormality alpha-thalassemia, atrx gene mutations also result in mental retardation, microcephaly, dysmorphic features, genital and renal abnormalities, growth deficiency, seizures, spasticity, gastrointestinal dysfunction, and kyphoscoliosis and other skeletal abnormalities. many of the symptoms of atrx overlap those seen in patients with a classic rtt mutation of the mecp gene. there are also clear differences, the presence of dysmorphic features in atrx mutations for example. interestingly, the a140v mutation reproduces symptoms of both conditions while still being somewhat distinct from each of them. this may provide a glimpse into the mechanism underlying each of these conditions. currently there are no complete atrx knock out mouse models due likely to the described embryonic lethality in males. tissue specific conditional inactivation of atrx results in enhanced cell death during corticogenesis with early lethality and interneuron defects in the retina. a variety of mouse models of rtt have been previously developed and tested. one of the first mecp mutant mouse models generated lacked exons and this model demonstrated that mecp2-null animals were viable and did not have a specific initial phenotype. between the ages of and weeks these animals developed abnormalities of gait and breathing, ultimately leading to early death at around months of age. a second mecp2-null mouse was generated by deletion of exon male mice appeared healthy for the first few weeks of age but developed abnormal movements, motor control, and breathing by weeks of age with death occurring around weeks. female mice in this study exhibited similar symptoms but with a time course delayed by months. shahbazian et al. generated mice with truncation of the c-terminal third of the coding region by introducing a stop codon following amino acid which is located at the c terminal end of the transcriptional repressor domain. male mutant mice survived to at least year of age and developed a variety of symptoms including tremor, myoclonic seizures, kyphosis and stereotyped forelimb movements. the motor performance of these mice declined with age. histological studies of the brain were normal and fear conditioning and spatial learning were normal, however, the mice did display enhanced anxiety and an abnormal corticosterone stress response. recently, a knock-in model of rtt was generated, reproducing a common non-sense mutation of mecp found in human cases. as with other rtt model mice, male r168x mice die early at around weeks of age, but display symptoms suggestive of rtt such as breathing irregularities, hypoactivity, and hind limb atrophy and clasping. female heterozygous mice show similar symptoms but not until around months of age. as reported in pathological studies of human rtt brain tissues, abnormalities of brain size and cell packing density have been observed in mouse models of rtt. abnormalities of dendrite branching have also been reported in some rett syndrome mouse models. much of the testing in mouse models has been done using hemizygous males to avoid the phenotype variability resulting from random x inactivation in females, which is considered to influence phenotype in human rtt patients. we have performed our initial tests in males for this reason and because a140v is more commonly recognized in longer surviving male patients. the pathogenesis of the neurological phenotype in rtt remains unclear, and continued study of mouse models will improve our understanding. the various mouse models recapitulate the symptoms of rtt in many ways but are often distinct from each other. although much has been learned about rtt through the study of existing mouse models, the a140v model we have created is unique. it reproduces a recurring missense mutation seen in humans and, in males, results in an xlmr phenotype. in addition, due to the long life span of mutant male mice, developmental studies can be performed. we expect that by a careful analysis of the a140v mouse, we will better understand the important roles that mecp plays in the pathogenesis of developmental brain disorders that result in mental retardation. here we describe the construction and initial characterization of brain abnormalities in the mecp a140v mutant mouse model of rtt/xlmr. in addition to being a model for these two diseases, the a140v mutant mouse permits the study of a novel mechanism by which mecp mutation might affect neural development; its interaction with the atrx chromatin remodeling protein.  generation of the mecp a140v mutant mouse the a140v mouse model of rtt/xlmr was generated by introducing a point mutation that results in the a140v missense mutation of mecp protein. the wt locus, targeting vector, and recombined locus, are diagrammed in figure 1a; also shown in figure 1a is the location of probe a used for southern blot analysis as well as the southern blot bam hi fragments. in addition, the location of the genotyping primers is indicated. results of a pcr based test of es cell dna to detect homologous recombination at the 3'-end of the targeting vector are shown in figure 1b. the wt allele yields a bp fragment while the mutant allele yields a bp fragment. as a confirmatory test these fragments were digested with acl i which cuts the mutant allele but not the wt allele, yielding a double band of bp and bp. southern blot analysis of bam hi digested es cell dnas detects a kbp band, confirming homologous recombination at the 5'-end of the targeting vector. es cell clones that were positive by pcr and southern blot were then sequenced to verify that they were hemizygous for the a140v mutation and two of these clones were used for blastocyst injection. shown in figure 1d are pcr genotyping results of f females: heterozygous females produce two bands and wt females produce a single band. plasmid controls show either the single smaller band or single larger band. a chimeric male created from the 177u es cell clone was used to produce heterozygous female founders for the mecp a140v mouse colony. mutant mecp mrna is expressed in brain tissue we confirmed that only mutant mecp mrna was present in brain tissue of male hemizygous animals by reverse transcription pcr. cdna was prepared from cerebellar total rna extracted from male animals, and tested by pcr. as shown in figure 2a mutant animals display only the larger fragment while wt animals display only the smaller fragment. this result also confirms the presence of the mecp 3'-utr. a different segment of cerebellar mecp cdna was then amplified to confirm that there was no genomic dna contamination of the cdna used in these tests. samples from both wt and mutant animals showed only the cdna band and not a larger genomic dna band, thus the cdna samples were free of genomic dna contamination. sequencing performed on purified cdna from these bands confirmed the a140v mutation. mecp protein is expressed in the brain of a140v mutant animals as this is not a knock-out or truncating model, it was expected that the full length mecp protein would be present in the brains of hemizygous male mice. immunostaining in brain slices using an anti-mecp antibody directed against the c-terminus of the protein demonstrated the presence of the mecp protein in the brain tissue of hemizygous mice as well as wild type control mice. a peptide blocking assay confirmed the specificity of the antibody. the mutation does not appear to have affected the nuclear or brain regional distribution of mecp protein. mecp mrna levels in wild-type and a140v mouse cerebellum by qrt-pcr total rna was extracted from the cerebellum of three a140v male mice and three wt male littermates. quantitative real time pcr reactions were performed using pcr assays for mecp and gapdh from sa biosciences. the primer set for mecp detects both the e and e isoforms of mecp figure shows data graphs for mecp and gapdh as well as a table of ct values including mean and standard deviation, and Δct used in the calculation of fold change in mrna levels, comparing a140v to wt. fold change between mutant and wild-type is calculated using the ΔΔct method ]). in the cerebellum, fold change in mecp mrna levels was. this demonstrated that in the cerebellum of wt and mutant month old animals we were unable to detect a significant difference in the abundance of wt and mutant steady state mecp mrna. this suggests that the a140v mutation does not significantly affect transcription or mrna stability. there are no obvious phenotypic abnormalities of the a140v mice no obvious phenotypic differences between a140v and wt male mice were noted. somatic growth between and weeks of age in mutant animals was similar to wt controls. we have not observed abnormalities in body morphology in animals observed up to months of age. necropsy of mutant animals has not revealed any gross organ pathology. no seizures or tremors have been observed in these animals. female heterozygous mice displayed a normal level of fertility although attempted breedings of male hemizygous mice have thus far been unsuccessful. both male hemizygous and female heterozygous a140v mice have lived to greater than months of age with no unusual health issues. increase in cell packing density in mutant animals we compared histological sections of brain tissue from month old wt and a140v male mice. although the wt and a140v sections were processed together, there was an apparent increase in staining intensity in the a140v mutant tissues. on closer examination, it became apparent that this was due to an increase in the packing density of cells in the mutant animals. this increase in packing density was noted in the frontal cortex and olfactory bulbs, in the dentate gyrus and regions ca ca and ca of the hippocampus, and in the granule cell layer of the cerebellum. the difference in cell packing density between a140v and wt male mice was quantified by counting all nuclei in a standard volume of tissue from the cerebellar granule cell layer. this analysis demonstrated that there is a significantly higher density of cell nuclei in the a140v cerebellar granule layer than in wt controls. abnormal dendritic branching in mutant cortical neurons qualitative abnormalities of dendritic branching were noted upon initial visualization of various regions of golgi-cox stained cortical tissue from mecp a140v mice. an extended focus image of the visual cortex is shown as an example. in order to begin to evaluate neuronal dendritic structure in the a140v mouse, scholl analysis was performed on layer ii/iii pyramidal neurons of the somatosensory cortex. this analysis was done using scholl analysis on brightfield z stack images of golgi-cox stained neurons in order to analyze the full arbors of both the apical and basal dendrites. a diagram of this adaptation of the scholl analysis technique is shown in figure male a140v and wild type mice 2- months old were used for this analysis. this quantitative analysis demonstrated significant differences in the branching complexities of the dendritic arbors of these neurons. apical dendrites showed very significant differences in branching at and μm distances from the cell body and basal dendrites showed significant differences at and μm. discussion the a140v mouse model was constructed in order to more closely examine the mental retardation associated with rtt. this was expected to be a mental retardation model in mice due to the effects of this mutation in human male patients. male patients with the a140v mutation survive into adulthood, indicating that this mutation is less likely to compromise functions essential for life. however, significant mental retardation is common in males carrying this mutation while carrier females show mild or no symptoms. the a140v mutation is predicted to be non-truncating and this was verified by immunoflourescence staining of brain slices from wt and mecp a140v male mice. the staining was performed with a c-terminal mecp antibody to demonstrate that the mecp protein was not truncated by the a140v mutation. the initial characterization of the mecp a140v mouse has revealed some interesting abnormalities. during casual observation the mice appear normal with no variation in weight or body morphology and no indications of seizures or tremors. however, there are cellular abnormalities in the brain consistent with those seen in rtt syndrome and other diseases resulting in mental retardation. an increase in cell packing density was apparent upon histological studies of brain tissue from the mutant animals. qualitative differences in cell density in the cerebellum were confirmed by performing cell counts. the results of these cell counts showed statistically significant differences in cell density. increased cell packing density has been previously noted in both human rtt patients and in rtt mouse models. the mecp null mouse created by deletion of exon revealed increases in cell density in the hippocampus, cerebral cortex, and cerebellum that closely parallel our own results. a study of the brains of three rtt patients found an increase in cell-packing density in multiple brain regions including the cerebral cortex, basal ganglia, thalamus, hippocampus, and amygdala. abnormalities were also reported in the cerebellum although an increase in cell packing density was not reported. we find no previous descriptions of cell density abnormalities in the olfactory bulbs of rett mouse models. we are uncertain if this is because cell density abnormalities have not previously been seen in olfactory bulbs or if the olfactory bulbs were not examined in these mice. there have been reports of defects in neuronal development in olfactory neurons from the nasal epithelium of rtt patients. in addition, defects of olfactory neuron maturation and protein expression have been found in two previously described rtt mouse models, indicating that olfactory defects are not unusual in rtt or in rtt mouse models. in addition, a recent and very thorough study of brain morphology found changes in the volume and shape of olfactory bulbs in two mecp null mouse models. we have seen abnormalities in the shape of olfactory bulbs and the morphology of glomeruli in some the a140v male hemizygous mice although this has not been quantified and will be the subject of further study. a lack of branching complexity in cortical pyramidal neurons in many brain regions has been reported in human cases of rtt as well as in mouse models. in fact, dendritic branching anomalies are often found in conjunction with mental retardation of many causes. the a140v mutation is strongly associated with mental retardation; therefore, we felt that abnormal dendritic branching patterns of neurons would likely occur in this new mouse model. we explored the branching complexity of neurons in the a140v mouse model and found differences between wt and mecp a140v mice. we noted significant differences in the branching complexity of both the apical and basal dendrites of layer ii/iii pyramidal neurons of the somatosensory cortex as measured by scholl analysis. this correlates closely with other reports of decreases in branching complexity of cortical pyramidal neurons in tissue from both human rtt patients and in mecp mutant mouse models. we also found apparent differences in branching complexity in the visual cortex although this has not yet been quantified. the mecp a140v mouse has proven to be distinct from other mouse models of mecp mutation. like its human counterpart, the a140v mutation in mice produces symptoms both correlating with and disparate from the classical symptoms of rtt. in humans, the a140v mutation causes a mental retardation syndrome that has associated with it abnormalities of gait, kyphoscoliosis, mild microcephaly, spasticity, dysarthric speech, and occasionally mental illness such as schizophrenia. however certain features of classic rett syndrome such as stereotypic hand movements, breathing abnormalities and seizures are notably absent in humans with the a140v mutation. these distinctions suggest that the a140v mutation affects certain functions of the mecp protein while preserving others. there is for instance the report indicating that the mecp a140v protein has an increased binding affinity for unmethylated dna, the functional consequences of which are unknown. there is also the interesting finding that although the a140v mutation preserves the methyl-cpg binding function of mecp it does interfere with binding to the mental retardation associated protein atrx. this raises the possibility that disruption of the mecp2-atrx interaction is in someway related to the neurological symptoms that occur in both humans with the a140v mutation and in our engineered mice. atrx is chromatin remodeling protein that is a swi2/snf dna helicase atpase that is the cause of atrx syndrome. this protein has only recently been identified as a binding partner of the mecp protein. analysis of brain tissue from mecp2-null mice revealed that loss of mecp in mature neurons leads to delocalization of the atrx protein, suggesting that mecp may be required for the proper localization of atrx. in vitro studies done using fusion proteins in mouse fibroblasts revealed that the mecp a140v protein, while properly localized to heterochromatin within the nucleus, fails to recruit atrx to the heterochromatin. in contrast, wt mecp protein efficiently recruits atrx to heterochromatin. this failure to localize atrx to heterochromatin may be important in understanding the normal function of both the mecp and atrx proteins. we believe that it is important to note that the mice used in these experiments belong to early generations and are therefore in a mixed 129x1/s and c57bl/ background. in addition, the neomycin resistance gene cassette used in the construction of these mice has not been excised from the experimental animals although we have begun production of a line of mice in which the neo cassette has been removed. as genetic background and the presence of extra dna sequences such as the neo cassette can contribute to phenotype we will be repeating these experiments in later generation mice lacking the neo cassette in order to confirm our experimental results. however, we believe that, given the close parallels of our data with that seen in other mouse models of mecp gene disruption, the phenotype we have observed is in fact due to the a140v mutation itself.  we believe that this new mouse model will provide insight into the functions of mecp including its association with the atrx protein. the a140v mouse has proven to reproduce some of the abnormalities of other mecp mouse models while failing to reproduce others. with this knowledge we can continue dissecting the functions of mecp in the hopes of explaining its relationship to mental retardation and other clinical symptoms. 
we report the construction and initial characterization of a mouse model expressing the a140v mecp mutation. these initial descriptive studies in male hemizygous mice have revealed brain abnormalities seen in both rtt and mental retardation. the abnormalities found include increases in cell packing density in the brain and a significant reduction in the complexity of neuronal dendritic branching. in contrast to some mecp mutation mouse models, the a140v mouse has an apparently normal lifespan and normal weight gain patterns with no obvious seizures, tremors, breathing difficulties or kyphosis. rett syndrome, a common cause of mental retardation in girls, is associated with mutations in the mecp gene. however, some mecp mutations can also lead to diseases characterized as mental retardation syndromes, particularly in boys. it has been described in familial x-linked mental retardation, ppm- x syndrome and in other neuropsychiatric syndromes. interestingly, this mutation has been reported to preserve the methyl-cpg binding function of the mecp protein while compromising its ability to bind to the mental retardation associated protein atrx. we have identified various neurological abnormalities in this mouse model of rett syndrome/x-linked mental retardation which may help to elucidate the manner in which mecp mutations cause neuronal changes resulting in mental retardation without the confounding effects of seizures, chronic hypoventilation, or other rett syndrome associated symptoms. one of these mutations, a140v, is a common, recurring missense mutation accounting for about % of all mecp mutations and ranking 21st by frequency. most human cases of mecp mutation in girls result in classical or variant forms of rtt.
 rett syndrome, a common cause of mental retardation in girls, is associated with mutations in the mecp gene. most human cases of mecp mutation in girls result in classical or variant forms of rtt. when these same mutations occur in males, they often present as severe neonatal encephalopathy. however, some mecp mutations can also lead to diseases characterized as mental retardation syndromes, particularly in boys. one of these mutations, a140v, is a common, recurring missense mutation accounting for about % of all mecp mutations and ranking 21st by frequency. it has been described in familial x-linked mental retardation, ppm- x syndrome and in other neuropsychiatric syndromes. interestingly, this mutation has been reported to preserve the methyl-cpg binding function of the mecp protein while compromising its ability to bind to the mental retardation associated protein atrx.  we report the construction and initial characterization of a mouse model expressing the a140v mecp mutation. these initial descriptive studies in male hemizygous mice have revealed brain abnormalities seen in both rtt and mental retardation. the abnormalities found include increases in cell packing density in the brain and a significant reduction in the complexity of neuronal dendritic branching. in contrast to some mecp mutation mouse models, the a140v mouse has an apparently normal lifespan and normal weight gain patterns with no obvious seizures, tremors, breathing difficulties or kyphosis.  we have identified various neurological abnormalities in this mouse model of rett syndrome/x-linked mental retardation which may help to elucidate the manner in which mecp mutations cause neuronal changes resulting in mental retardation without the confounding effects of seizures, chronic hypoventilation, or other rett syndrome associated symptoms. 