My postdoctoral studies from Catherine Dulac’s laboratory at Harvard have finally been published as two companion papers in the August 6th issue of Science [Gregg et al., Science 2010; Gregg et al., Science 2010b]. For those who would like some different perspectives on the findings, the work has been reviewed in Nature [Keverene 2010], Science [Wilkinson 2010], and Neuron [Shah 2010]. In this blog entry I wish to provide a summary of the findings for a general audience. I hope you find it interesting.
In many ways, adult health and behavior is a reflection of developmental processes and early life experiences. Uncovering the early life processes and mechanisms of inheritance that influence adult behavior and health is fundamental to our understanding and treatment of neurological and psychiatric diseases, as well as to broader social issues related to diet, parenting, education, and socioeconomic policies. When we think of infant and childhood development and early life experiences, we can’t help but think of the paramount roles that parents play. Parents influence our brain development and behavior in many ways and so substantially that they can set us on a course for life.
Often when we consider the impact of parents on their offspring we think in terms of either parental behavior or genetic inheritance. Recall that each of us inherits 23 chromosomes from Mum and 23 chromosomes from Dad (19 in the case of mice). Importantly, maternally and paternally inherited chromosomes are not functionally equivalent, due to heritable epigenetic marks established on the chromosomal DNA in the parental gametes, called genomic imprints. Genomic imprinting is thought to be rare in the genome, affecting ~100 genes in mice, and yet examples of parental effects upon gene expression, brain function and the behavior of offspring are growing in number and becoming increasingly mysterious. For example, experiments in which chimeric mice were generated from a mix of wildtype cells and parthenogenetic cells (PG, cells from embryos with two mothers and no father) or androgenetic cells (AG, cells from embryos with two fathers and no mother) revealed the preferential contribution of cells with a maternally-derived genome (PG cells) to cortical and limbic brain regions, but cells with a paternally-derived genome (AG cells) contributed strictly to hypothalamic regions. Cortical brain regions have executive functions in the brain (top-down control), while hypothalamic regions control primary drive (feeding, sleeping, sex, etc). From these striking observations, Barry Keverne proposed the idea that mothers and fathers differentially influence the evolution and function of the cortical versus the hypothalamic brain regions. Several other transgenerational parental effects have also been uncovered. In a study of genetically identical uniparental mice, a complex paternal transmission pattern of anxiety-related behaviors and growth effects was found that suggests epigenetic and sex-specific transgenerational effects. Similarly complex effects have been described in humans. These studies all suggest that some information is passed through the germline from parents to offspring in a manner that is distinct from the genome sequence, and further, that this information can influence the behavior and physiology of the offspring. Scientists call this information “epigenetic”. My studies have worked to uncover forms of epigenetic regulation that are differentially inherited from mothers versus fathers, thereby resulting in genomic imprinting that influences gene expression in the brain.
“Epigenetics is the study of changes in phenotype (appearance) or gene expression caused by mechanisms other than changes in the underlying DNA sequence.”
One outstanding question in the field of genomic imprinting is why a mechanism that results in differential gene expression from maternally versus paternally inherited chromosomes would ever have evolved. The leading theoretical explanation is even more captivating than the phenomenon itself and is called the Parental Conflict Theory (or Kinship Theory). Imprinting has been identified in placental mammals and flowering plants only. What is unique about these species is the preferential investment of maternal resources in the growth, development and rearing of the offspring. In mammals, a second important point is that monogamy is extremely rare, and therefore, fathers not only invest comparatively few resources in their offspring, but they also can’t guarantee that they will father their partner’s future offspring. David Haig’s Parental Conflict Theory proposes that a conflict results from this situation, such that fathers effectively compete to have their offspring consume the maximum amount of maternal resources possible, even at the expense of the mother’s long term survival. To counter this genetic arms race, mothers evolve mechanisms that reduce offspring consumption of maternal resources, so they can distribute those resources to future litters. Thus, unique maternal and paternal gene expression programs evolve through this conflict to function antagonistically.Currently, there is good evidence to support this theory for many, but not all cases of imprinting.
“The Kinship Theory for the evolution of genomic imprinting proposes that maternal and paternal gene expression programs are in conflict and function antagonistically to each other.”
Genomic imprinting has been clearly linked to human brain function and behavior through studies of Prader-Willi (PWS) and Angelmen Syndrome (AS), which result from a paternally or maternally inherited deletion of an imprinted gene cluster on chromosome 15, respectively. PWS is associated with hyperphagia, stubbornness and compulsive traits, while AS is associated with absent speech, happy affect and inappropriate laughter. Importantly the only difference between PWS and AS at the level of the genome is that a mutation in a cluster of imprinted genes on chromosome 15 is inherited from the father, in the case of PWS, or the mother, in the case of AS. Recently, it has also been proposed that imprinted genes play a role in schizophrenia and autism. This theory, proposed by Crespi and Badcock, suggests that autism spectrum disorders are due to an imbalance in maternal and paternal gene expression programs in the brain such that the balance is too paternal. The same theory proposes that schizophrenia is an imbalance in the opposite direction, such that gene expression is too maternal. Currently, it is not yet clear whether these ideas are correct. Finally, disruptions in imprinting have also been identified in several forms of cancer, such as Wilms’ tumor and colorectal cancer. In sum, parental effects associated with imprinting influence the behavior and physiology of offspring and contribute to human disease, suggesting an underlying biology and epigenetic mode of inheritance that is clearly important, but poorly understood.
My recently published companion studies, performed with collaborators at Harvard, are focused on understanding the nature and functions of paternal and maternal gene expression programs in the developing and adult brain. We initially mapped the expression pattern of 45 previously known imprinted genes across 118 adult brain regions. This study identified neural systems that are enriched for the expression of imprinted genes. We found that imprinted genes are preferentially expressed by the major monoaminergic nuclei of the brain (serotonin, dopamine, or noradrenaline expressing neurons). These areas of the brain have been implicated in a wide range of neurological and psychiatric disorders, including major depression, eating disorders, schizophrenia, and autism spectrum disorders. In addition, nuclei involved in feeding behavior, such as the arcuate nucleus, and social behaviors, such as the preoptic area, were also enriched for imprinted gene expression. This initial set of observations suggested that imprinted genes regulate neural systems of major interest to neuroscience, and prompted us to develop a genome-wide approach to study genomic imprinting in specific brain regions at different developmental stages.
“We found that imprinted genes are preferentially expressed in serotonergic, dopaminergic and noradrenergic brain nuclei. These are regions of the brain implicated in numerous psychiatric disorders and believed to be the sites of action for several anti-depressant drugs (ie. SSRIs).”
Our approach utilizes next generation sequencing technology. This is a new technology that just emerged when I began my postdoc in 2006 and, in collaboration with the company making the sequencing technology (Illumina Inc.), we were one of the first groups to begin using the system at Harvard. It allows one to sequence genome information with an incredibly high throughput. We sequenced the mRNA (rather than the genomic DNA) of RNA samples harvested from crosses of two distantly related subspecies of mice, called CASTEiJ and C57BL/6J. Our first step was to sequence the entire transcriptome (all the mRNA) of the individual CASTEiJ and C57BL/6J mice to identify all coding single nucleotide polymorphisms (SNPs, single base differences in the genome sequence) that distinguish the genes of the two strains. We then performed RNA-Seq on specific brain regions of F1 hybrid offspring generated by reciprocal crosses of CASTEiJ and C57BL/6J mothers and fathers. I could then distinguish gene expression levels from maternally versus paternally inherited alleles (gene copies) using the SNP base call information we had first generated.
Inspired by the chimera studies described above, which suggested preferential maternal control over cortical regions and preferential paternal control over hypothalamic regions, we compared parent specific gene expression programs in the brain in the adult medial prefrontal cortex (mPFC) and the preoptic area (POA) of the hypothalamus. The number of genes subject to parental effects that significantly altered maternal or paternal allele-specific expression in these regions is greater than expected (~372 genes) and involves complex isoform-specific parental effects. However, we did not find evidence for biased maternal control over the cortex. Instead, we found that in both the mPFC and POA, ~70% of autosomal genes (autosomes are all the chromosomes that are not X or Y chromosomes) exhibiting parental effects preferentially express the paternal allele. Thus, instead of mums controlling the cortex and dads controlling the hypothalamus, we found that dads appear to have strongly biased control over both regions. We do not currently know if there are any regions of the adult brain that mothers have biased control over. Interestingly, an analysis of gene expression on the X chromosome in females revealed preferential expression of the maternally inherited X chromosome in the adult female brain, especially in the cortex. This result was further confirmed with a transgenic approach. In males, the X is strictly maternally derived and since females have two X chromosomes, and the X therefore spends 2/3 of its time in a female body, David Haig has proposed that evolution will select for genes and mutations on the X that favour maternal interests. Remarkably, we know from previous work that the X chromosome has evolved a preferential role in the regulation of the brain and many forms of mental retardation are associated with X-linked genes. We therefore speculate that the autosomes and X chromosome give rise to paternal and maternal gene expression programs, respectively, which influence adult brain function and behavior.
The parental effects in the developing fetal brain differed from those found in the adult. We found ~553 genes subject to parental effects in the fetal brain, compared to 257 in the adult POA and 153 in the adult mPFC. Thus, parent specific gene expression programs dominate during development of the brain, rather than in the adult brain. Further, rather than a paternal expression bias, 61% of the imprinted genes in the developing brain exhibited preferential expression of the maternal allele. These results reveal maternal effects that are specifically associated with brain development.
“Our data suggests mothers have biased control over gene expression in the developing fetal brain. However, in the adult brain, fathers have biased control over autosomal gene expression, while the X chromosome appears to function as the nexus of maternal control.”
Finally, we analyzed males and females separately and uncovered evidence for sex specific parental effects. An important example is interleukin 18 (Il18), which exhibits preferential expression in the female, but not male, mPFC. Il18 has been linked to multiple sclerosis, a sexually dimorphic neurological disease that predominates in women and is associated with maternal parent-of-origin effects.In the POA of the hypothalamus, we also noted that females have 3 times the number of genes subject to sex specific parental effects as males. The POA plays a central role in regulating maternal behavior. Given that maternal behavior alone impacts offspring brain development and behavior, this result suggests a remarkable convergence of parental influences.
Our studies of parent-specific gene expression programs in the CNS suggest surprising and complex modes of parental influence over brain development and adult brain function in offspring. What are the mechanisms that regulate these effects? What are the functions of maternal and paternal gene expression programs in the brain? Do parental influences on gene expression adapt to environmental pressures? In what ways do imprinted genes influence the behavior and physiology of offspring? What is the nature of genomic imprinting in humans? Do maternal and/or paternal gene expression programs play a role in human diseases and disorders? These questions set a course for an exciting frontier and may shed new light on our understanding of brain evolution, function and disease.
Thank you for reading about my work. Please contact me with any questions through this blog site.