Embryogenesis

The embryo is not protected from pharmaceuticals and environmental pollutants. Intended and unintended pharmacological effects of drugs are often exerted in the conceptus as well. They can be reversible, but have occasionally detrimental morphological and functional downstream effects. We aim to improve the mechanistic understanding of teratogenic processes and develop improved in vitro methodologies in developmental toxicology.
One project is to develop improved image analysis software for the characterization and scoring of rodent embryos undergoing organogenesis in whole embryo culture (WEC). This is performed in collaboration with Professor Ewert Bengtsson at the Centre of Image Analysis, Uppsala University. By combining image analysis with multivariate analysis to assess adverse effects of embryonic development in vitro, we believe that the objectivity and the sensitivity of the method will increase. One aspect of the image analysis software project is to develop better ways of analyzing the developmental toxicity effect of drugs on heart rate in WEC conditions. In addition, a more specific developmental heart rate associated project concentrated on the properties of potassium (IKr)-channels reached its conclusion during 2009. This latter research was lead by adjunct professor Bengt Danielssson.
A second approach concerns the proteomic characterization of the WEC culture medium (rat serum based). Increased knowledge of the culture medium properties will be the first step for the establishment of a defined culture medium, which will both reduce variability in the WEC method and the number of required animals. This is done in collaboration with Professor Peter Bergsten at the department of Medical Cell Biology, Uppsala University.
To improve scoring and data handling, we are also developing and anatomical ontology and controlled vocabulary that incorporates the different possible malformation phenotypes observed during organogenesis (in vivo and in vitro). These approaches will, when appropriate, be combined with our molecular biology data (functional genomics) and enable an improved monitoring of the “symphony” and its different tunes concerted by the collected expression of genes and proteins governing embryonic development and brain maturation.

Cell studies

In addition, we use the information from embryos (cultured in vitro, or exposed in vivo), and apply it on murine and human embryonic stem cells, to develop mechanism-based in vitro cell test systems to reveal the teratogenic potential of substances. Using the antiepileptic and teratogenic drug valproic acid, an Histone deacetylase inhibitor (HDACi), and some less teratogenic analogues of valproic acid, we try to visualize which categories of genes may be representative for the teratogenic action (such as neural tube defects). Presently, a battery of teratogenic and non-teratogenic compounds are tested with respect to their gene (de)regulation in murine embryonic stem cells. A third cell type, mesenchymal stem cell-like pericytes isolated from human full term placentas are also being evaluated for cellular and molecular VPA effects. The later project was performed in the context of a EU FP6 project (ReproTect) and in collaboration with Dr Christian Sundberg at IMBIM, Uppsala University and ended during 2009. We are at the same time exploring the global epigenomic effects of VPA:s HDACi capacity in collaboration with Professor Claes Wadelius at Rudbeck laboratory. Here, we have used chromatin immunoprecipitation (ChIP) on chip (ChIP-chip) to study histone modification changes in the model system (human hepatoma cell lines) used by Wadelius group as a precursor for later studies in a more embryonic context. These studies have shown us that VPA as an HDAC inhibitor has unsuspected complex genome wide effects outside the prediction of the literature so far by removing large regions of histone acetylation instead of promoting it. This project was ended during 2009.

Neurogenesis

Several chemicals exert estrogenicity, being a potential problem esp. during development (reproductive organs and sex-specific behaviour). There is relatively little known about the mechanisms behind their sex specific brain development in general, especially regarding the impact of the sex chromosomes, on future sex specific behaviors. We have been using Chicken and Japanese quail as models, to study basic sex differences (with and without estrogen exposure) in gene expression in the developing brain. We were the first to report that there is strong gonadal hormone independent sex chromosome based component in avian sex specific brain development. Although there were no clear-cut effects of estrogens in gene expression, there were some estrogen induced differences in the developing neuropeptidome, implicating posttranscriptional regulation. There was an overall up-regulation of peptides (about 60 identified) in diencephalons with embryonic age. One of the most interesting candidates for estrogenic effects on the developing diencephalon was the GnIH-RP2 peptide which is speculated to be involved in the establishment of the HPG axis during development.
Many chemicals (incl. drugs) given to newborn mice disrupts normal brain (growth spurt) development, resulting in disturbed spontaneous behavior in adulthood. Polybrominated diphenyl ethers (PBDEs) are environmental contaminants found in human and animal tissues worldwide. We have investigated their short-term effects on protein expression in hippocampus, striatum and cortex by using two-dimensional difference gel electrophoresis (2D-DIGE). We determined the identity of 111 differentially expressed proteins in cortex, 39 (35%) of which are known to be cytoskeleton-related. As in striatum, we found elevated levels of the neuron growth-associated protein Gap43 in the cortex. A more recent in vitro approach has generated some new insights to developmental PBDE neurotoxicity. Based on studies using fetal rat cortical cells, we have further strengthened our hypothesis regarding direct effects of PBDEs on cytoskeletal organization. Although not conclusive, the data indicates that behavioral alterations induced by neonatal exposure to PBDE occur at non-cytotoxic levels. In addition, we are evaluating the effects of PBDE-99 and DE-71 (a commercial mixture of PBDE congeners) exposure on neuritogenesis. Although much work is required to make a complete picture of these early changes, it is a beginning of a mechanistic approach to a potentially important general health problem caused by environmental chemicals as well as drugs.

Other projects

Members of the group have been involved in projects carried out on the BMMS in collaboration with groups in France and Great Britain (funded by the Michael J Fox foundation). This has resulted in publications on L-Dopa induced dyskinesia in a non-human primate parkinsonian model, were we report the proteomic changes in the striatum induced by both neurotoxin induced Parkinsonism (PD) and the effects of de-novo L-Dopa treatment and long-term treatment leading to dyskinesia. Our data points to a before now unprecedented and long term impact of the first de-novo L-Dopa dose in PD individuals. There are also ongoing studies that look closer of the impact of sample handling and the postmortem interval time on protein sample quality and tissue specific degradomes. The later is of clear importance for interpreting proteomics and biomarker data and the establishment of sample handling procedures in clinical biobanks.