Professor Pete Scambler
Professor Pete Scambler
TBX1, Congenital Heart Defect and the 22q11 Deletion (DiGeorge/Velocardiofacial) Syndrome
The group has a long standing interest in the 22q11 deletion
syndrome (22q11DS), which is the
most common interstitial deletion found in man with a birth incidence of
approximately 1:4000. It is associated with a wide range of congenital
abnormalities including congenital heart defects, hypo/aplasia of the thymus.
Hypo/aplasia of the parathyroids, learning difficulties, facial dysmorphism and
genitourinary problems. The complications of the cardiac defects are the major
cause of morbidity and mortality in the condition, especially in the early
years. Feeding problems and speech delay also complicate infant life. Recently,
it has become apparent that the deletion is also associated with psychiatric
problems, and it has been postulated that a schizophrenia predisposition locus
maps to the deletion region.
Haploinsufficiency of the transcription factor TBX1 plays a major role in the syndrome. Current research is directed at understanding more about the function of TBX1 during development, in particular its transcriptional targets, and also its partners. These experiments make use of models created by targeted mutagenesis and chromosome engineering, and involve comparing the transcriptome of early embryos using microarray, real time quantitative PCR and in situ hybridization technologies. These strategies are continually being refined, but have already implicated retinoic acid, SMAD, and Slit/Robo signaling downstream of Tbx1. As well as the mouse, we also make use of zebrafish and chick models to analyse these pathways.
We have shown that the pharyngeal surface ectoderm is an important signaling centre in the embryo, and Tbx1 is required in this tissue to control the contribution of the cardiac neural crest during embryogenesis. A major strand of our work is to identify the molecular involved in this process. There is potential clinical relevance. The failure of Tbx1-driven signaling results in abnormal innervation of the heart (and upper gastrointestinal tract). We are investigating if this contributes to the excess cardiac sudden death seen in adults with 22q11DS. Similarly, we collaborate with other groups in attempts to identify what developmental processes might underlie the strong predisposition to schizophrenia and other psychiatric illness in 22q11DS patients.
Tbx1 also has a major role within a group of cardiac progenitors originating in the second heart field. These cells have the ability to differentiate into heart muscle, endothelium and vascular smooth muscle. Future work will explore this aspect of Tbx1 function, with a view to contributing to efforts to utilize stem/progenitor cells for regenerative strategies.
DGS/VCFS-like features in the absence of 22q11 deletions appears to be due to multiple causes. We have shown that deletions in chromosome 5, 8 and 10, and mutations of TBX1, can mimic the syndrome to an extent and are searching for additional genes involved in the birth defects of such patients. This strand of our work led directly to the identification of mutations of the chromodomain protein CHD7 in this patient group, and to our interest in CHARGE syndrome. In addition, we and others have described atypical deletions of 22q11 that do not encompass TBX1 and yet are associated with congenital heart defect. HIC2 is a candidate gene for this distal 22q11 deletion haploinsufficiency (see HIC2 section for more detail).
PSC1 early growth
CHD7 and CHARGE Syndrome
CHD7 is a chromodomain protein involved in transcriptional regulation via alteration of chromatin. Heterozygous loss of CHD7 causes CHARGE, a disorder with congenital heart and vascular defects. We have demonstrated close phenotypic similarity between haploinsufficiency for TBX1 (22q11 deletion syndrome) and CHARGE. Tbx1 and Chd7 are in epistasis, indicating a likelihood that the two genes’ functions overlap within a common developmental pathway(s). Our genetic rescue experiments demonstrated a dosage sensitive requirement of Chd7 in pharyngeal ectoderm for great vessel development in mouse, and others have shown a requirement in neural crest differentiation with morpholino knockdowns in Xenopus. We have a conditional mouse mutant line which will be used to further probe the tissue requirements and tissue-specific dosage sensitivity towards Chd7. In particular, we wish to know how Chd7 controls progenitor cell differentiation and proliferation in the second heart field and the cardiac neural crest population. Genomic methods will be used to identify genes whose expression is altered by loss of Chd7 in ectoderm and in heart field progenitor cell populations. Stem cell culture will be used to identify roles in the very earliest stages of differentiation to cardiac cell types, and we will investigate if Chd7 is involved in the reprogramming process.
As mentioned in the appropriate section, Chd7 interacts with Tbx1, and both have important roles in cardiac progenitors. The respective teams work closely to identify the mechanisms underlying the epistasis, again with the long term goal of understanding how control of stem/progenitor cells may contribute to cardiac repair/regeneration.
We collaborate with Dr. Albert Basson, Kings College London, whose major interest is the role of Chd7 in the central nervous system.
FRAS1/FREM2 and Fraser Syndrome
Fraser syndrome comprises cryptophthalmos and cutaneous syndactyly with urogenital abnormalities. Autozygosity mapping assisted in the identification of the first Fraser syndrome gene (FRAS1). This gene is also mutated in the naturally occurring mouse "blebbed" line (bl). FRAS1 is a member of a family of similar proteins called FREMs. Frem1 is mutated in the head blebs mutant, Frem2 in myelencephalic blebs and FREM2 in some Fraser syndrome patients. A PDZ-domain adaptor protein called Grip1 is mutated in eye blebs. Each of these "blebbed" mutants show skin blistering during embryogenesis, indicating that these genes are required for skin epithelial integrity in utero. No blebs are seen in the developing kidneys. Efforts are aimed at understanding how Fras/Frem proteins participate in epithelial and basement membrane development, exploring the signalling pathways modulated by these proteins, and the mechanisms of renal morphogenesis disrupted in the mutants. Recent work has shown that lack of the Fras/Frem complex compromises the process of ureteric bud invasion of metanephric mesenchyme, but that this can be bypassed by the ex vivo addition of certain growth factors. We are attempting to achieve an in vivo rescue in order provide proof of principle that therapeutic intervention is possible for this group of disorders. In addition we have begun to uncover “late” functions for Fras1 in maintaining the glomerular filtration apparatus, and are using conditional mutagenesis to explore this further.
Collaborators: Prof. Adrian Woolf, Manchester
University; Dr. Mieke van Haelst, University of Utrecht.
Frem2 expression in 14 day old mouse embryo
Hira, Stem Cells, Chromatin and Development
Hira is a chaperone for the replacement histone H3.3 and, in some species, a transcriptional co-regulator. Hira is implicated in a range of processes including fertilization, early development, chromatin dynamics, and cellular aging. Previous work has identified a general role for Hira in early development, with both chick neural crest knockdown experiments and constitutive knock out in the mouse indicating Hira is required for normal cardiovascular morphogenesis. Moreover, H3.3 itself appears to have a specific role in the rostral neural crest. At the molecular level, Hira is associated with H3.3 deposition within chromatin in specific regions of genes. It is not required for maintenance of a stem-cell like state, but is required during differentiation processes. The Hira project has two main aspects.
First, we explore how Hira affect the very earliest switches in gene expression observed during stem cell differentiation and how does this correlate with chromatin conformation. This work is undertaken using our dual targeted, Hira null embryonic stem cells which are cultured as embryonic “bods”. In collaboration with the group of David Allis, we plan to use these to investigate how the dynamics of transcription and H3.3 chromatin deposition are related. We are also in whether lack of Hira impacts specific signalling pathways within the early embryo.
Secondly, we investigate the role of Hira during embryonic development and subsequent maintenance of a differentiated phenotype. The main focus is on the cardiovascular system and neural crest, using conditional mutagenesis. However, our conditional mouse mutant will allow us to explore other aspects of Hira function, for example during fertilization and aging.
Collaborators: Prof. Richard Festenstein, Imperial College London; Dr. C. David Allis Rockefeller University, New York.
HIC2 and Cardiovascular Development
Rare patients have congenital heart defect and atypical deletions of 22q11 that do not involve TBX1. We investigated two families with balanced chromosome translocation breakpoints mapping within the distal atypical deletion region. HIC2 is the gene closest to the chromosome 22 breakpoint. Mice haploinsufficient for Hic2 have heart defects, and Hic2 is expressed in the cardiac crescent. We are currently investigating Hic2 further using conditional mutagenesis to explore its role in embryogenesis. We have conducted a yeast two hybrid experiment and identified protein interactors of Hic2. These point to Hic2 being involved in transcription regulation, as predicted from what is known about HIC1, but also in potentially novel mechanisms of post-transcriptional regulation, and post-transcriptional control of Wnt signaling.
High Resolution MRI Imaging
(with Francesca Norris (CoMPLEX student), Mark Lythgoe, CABI, UCL)
We analyse a large number of mouse embryos during the course of our work, and collaborate with UCL’s Centre for Advanced Biomedical Imaging www.ucl.ac.uk/cabi to develop magnetic resonance imaging (MRI) as a high throughput screening tool. The inherent non-invasive and three-dimensional nature of MRI makes it an ideal platform for phenotyping studies in the adult and embryo mouse. Mark Lythgoe and his team have developed an optimised preparation and scanning protocol for MR imaging of the mouse embryo at an isotropic resolution of ~18µm. This enabled the identification of cardiac and neurological phenotypic characteristics in Chd7 and Hesx1 mutant mouse embryos . Up to 40 embryos (≥ 15.5 days post coitum) can be imaged in a single scan, resulting in an MR data set that allows the entire cohort to be visualised on a slice-by-slice basis. Novel analytic tools are developed in parallel by the UCL Centre for Medical Image Computing (CMIC).
Atlas generation: MR atlases are the cornerstone of advanced computational techniques. These may be generated by combining individual MR images using registration techniques, to provide enhanced signal-to-noise ratio and visualisation of anatomical detail. We have developed an embryo atlas using MR images to enable automatic, high-throughput methods.
Tensor-based morphometry: Tensor-based morphometry is a fully automated technique that enables unbiased and unsupervised detection of local, volumetric differences in a population on a voxel-wise basis, which are not visible to the human eye.
Segmentation propagation: Segmentation propagation is a semi-automated technique that enables rapid acquisition of volumetric data in a population. This technique calculates a variety of volumes, including the heart, whole brain, olfactory bulb, pituitary gland and mesencephalic vesicle.
These segmented volumes may be propagated to any embryo MR dataset that has been registered to our embryo atlas, automatically generating volumetric data.
Cleary JO, Modat M, Norris FC, Price AN, Jayakody SA,
Martinez-Barbera JP, Greene ND, Hawkes DJ, Ordidge RJ, Scambler PJ, Ourselin S,
Lythgoe MF. 2011 Magnetic resonance virtual histology for embryos: 3D
atlases for automated high-throughput phenotyping. Neuroimage 54, 769-778.
Prof. Peter Scambler
Room 211 Molecular Medicine Unit
UCL Institute of Child Health
30 Guilford Street, London
WC1N 1EH UK
Tel: 0207 905 2635
Fax: 0207 905 2609 OR 7831 0488
Page last modified on 12 oct 12 12:35