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Group leader:
Sven Enerbäck
Members of the group: Zahra Arani, Michal Bell, Anna Cederberg, Mats Grände, Mikael Heglind, Daniel Nilsson, Sandra Rodrigo-Blomqvist, Gunilla Petersson, Rickard Westergren and Hilmar Vidarsson


Our research efforts are directed towards the analysis of the developmental programs in which winged helix genes contribute to correct pattern formation and organogenesis. Towards achieving these goals, we use a technology that specifically mutates any gene you choose in the mouse. This technology employs the exchange of DNA sequences, by homologous recombination, between exogenous, newly added DNA sequences and the cognate chromosomal DNA sequences in embryo-derived mouse stem (ES) cells. This process is referred to as "gene-targeting". The ES cells containing the desired targeting event are then used to generate mouse germ line chimeras, thereby transferring the alteration to subsequent mouse generations. We are now using this technology to analyze the function of winged helix genes believed to mediate important developmental decisions in the mouse. A similar approach is also being employed to study problems of more immediate relevance to human medicine. In particular, "gene-targeting" is being used to generate mouse models for human genetic diseases. Such animals will permit a deeper analysis of the pathogenesis of the genetic disease as well as provide appropriate subjects for testing new therapeutic protocols including somatic gene therapy. Eventually gene targeting should also become directly applicable toward correcting genetic defects in humans by somatic gene therapy.

An example of a hypothetical pathway regulated during development of the inner ear, currently under investigation in our group:

 

Examples of ongoing projects, grouped by gene names, has been entered (see below).

Sven Enerbäck, MD, PhD


FoxI1

FoxI1
is a member of the forkhead family of winged helix transcriptional regulators. Forkhead genes have previously been shown to be instrumental during embryogenesis in mammals, in particular during development of the nervous system. Mice with a targeted disruption of the FoxI1 locus exhibit circling behaviour, pathological swim test and abnormal reaching response - all common findings in mice with vestibular dysfunction. These animals also fail to elicit a Preyer reflex, in response to a suprathreshold auditory stimulation, as seen in mice with profound hearing impairment. Histological examination of the inner ear reveals a gross structural malformation of the vestibular part of the inner ear as well as the cochlea. These structures have been replaced by a single irregular cavity in which neither proper semicircular ducts nor cochlea can be identified. We also show that at day 9.5 post coitum (p.c.) FoxI1 is exclusively expressed in the otic vesicle. These findings, implicate FoxI1 as an early regulator necessary for development of both cochlea and vestibulum and identify its human homologue FoxI1 (also known as FREAC-6, Fkh10 or HFH-3) as a previously unknown candidate deafness gene at 5q34.

Cochlea derived from wild type mouse.
In FoxI1-/- mice the entire cochlea is missing.

Wild type mouse E9.5 hybridized with a FoxI1 cRNA sense probe in a whole-mount hybridization experiment. No hybridization signal can be detected. The otic vesicle is marked with an arrow. Wild type mouse E9.5 hybridized with a Fkh10 cRNA antisense probe in a whole-mount hybridization experiment. At this developmental stage FoxI1 is exclusively expressed in the otic vesicle (arrow).
Sandra Rodrigo-Blomqvist , PhD student
Hilmar Vidarsson , PhD student

FOXC2


FOXC2 is a winged helix gene that protects against obesity hypertriglyceridemia, and insulin resistance Obesity, hyperlipidemia and insulin resistance are common forerunners of non-insulin dependent diabetes mellitus (NIDDM) a serious and increasingly prevalent disease in the industrialised part of the world. Thrifty genes are thought to conserve energy during periods of famine whereas they constitute a risk for developing obesity related conditions, e.g. NIDDM, when energy is abundant. The precise nature of such hypothetical thrifty genes is not known. We have showed that the transcription factor gene FOXC2 is exclusively expressed in white (WAT) and brown (BAT) adipose tissue during postnatal life. In mice over-expressing this gene, solely in WAT and BAT, intra-abdominal WAT has acquired a "brown fat-like" histology. The intra-abdominal WAT depot is reduced (see below) and the interscapular BAT is hypertrophic. Increased FOXC2 expression has a pleiotropic effect on gene expression in BAT and WAT. For instance, the BAT specific gene uncoupling protein-1 (ucp-1) is induced in intra-abdominal WAT and there are also increased steady state levels of mRNAs encoding: beta 1,2 and 3-adrenergic receptors, adipsin, CEBPalpha, PPARgamma, PPARgamma coactivator-1 (PGC-1), insulin receptor, insulin receptor substrate-1 (IRS-1), IRS-2, insulin responsive glucose transporter-4 (GLUT-4) and hormone sensitive lipase (HSL). As a consequence hereof, total body lipid content has decreased from 30% to 10% (p<0.0004) while no significant change in total body weight can be observed, serum triglycerides are down by more than 50% (p<0.004), serum glucose showed a significant 16% reduction (p<0.05), serum insulin levels are decreased by approximately 50% (p<0.03) and levels of free fatty acids (FFA) in serum are down by 27% (p<0.001). In an intravenous glucose tolerance test, mice over-expressing FOXC2, display significantly increased glucose elimination - a sign of increased insulin sensitivity. Even though it is likely that other pathways/genes are involved, the induction of ucp-1 in WAT, the hypertrophy of BAT, the decrease of intra-abdominal WAT depots and the upregulation of mRNAs encoding beta-adrenergic receptors suggest that an increased sensitivity in a beta-adrenergic/cAMP/protein kinase A (PKA) pathway, at least in part, can explain the findings in mice over-expressing FOXC2. We have demonstrated a PKA isozyme switch with increased levels of the regulative subunit RI-alpha. The PKA type I holoenzyme (RIalpha2C2) binds cAMP with higher affinity and activates more easily than the PKA type II enzyme, we have showed that such a shift in cAMP sensitivity exists in adipocytes from animals with an increased expression of FOXC2. Thus, the adipocytes will have a lower threshold for PKA activation by adrenergic stimuli as compared with wild type littermates. Since FOXC2, directly or indirectly, regulates triglyceride metabolism, adrenergic sensitivity and insulin action in adipocytes, in the way described here, it fulfils the criteria for an anti-thrifty gene. To our knowledge FOXC2 is the only known gene that, in a concerted action, can counteract most, if not all, of the symptoms associated with obesity including hypertriglyceridemia and insulin resistance. A likely consequence of such action would be prevention of NIDDM.


The intra-abdominal WAT depot is reduced in mice over-expressing
FOXC2, moreover the depot has acquired a "brown fat-like" histology.


We would like to propose a model in which the degree of FOXC2 induction, in response to a caloric load, perhaps sensed by cues like insulin and TNFalpha, determines the efficiency by which calories are converted to triglycerides (TGs). A strong induction would lead to a less efficient conversion, with calories ending up as heat rather than stored as TGs

Anna Cederberg, PostDoc
Michal Bell, Post Doc
Mats Grände, Post Doc

Rickard Westergren, PhD student
Daniel Nilsson, PhD student

Zahra Arani, PhD student