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        Abstracts of papers presented at the 21st Genetics Society's Mammalian Genetics and Development Workshop held at the Institute of Child Health, University College London on 11 November 2010
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        Abstracts of papers presented at the 21st Genetics Society's Mammalian Genetics and Development Workshop held at the Institute of Child Health, University College London on 11 November 2010
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        Abstracts of papers presented at the 21st Genetics Society's Mammalian Genetics and Development Workshop held at the Institute of Child Health, University College London on 11 November 2010
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Spina bifida aperta is a common and disabling neural tube defect in humans; it occurs when the caudal progression of neural tube closure from the hindbrain/cervical boundary is interrupted. Spinal neural tube closure requires the formation of ‘hinge points’, which are localized regions of bending in the neuroepithelium. Dorsolateral hinge points (DLHPs) are essential for lumbosacral neural tube closure between the 16- and 30- somite stages: mice homozygous for the Zic2 loss-of-function allele, Kumba (Zic2 Ku/Ku) lack DLHPs and have a fully penetrant spina bifida. Both Bmp and Shh signalling can directly inhibit DLHP formation (Ybot-Gonzalez et al., 2007). Although Zic2 Ku/Ku mutants have a notochordal defect and diminished Shh expression, downstream effectors such as Ptc and Gli2 are unaltered. A change in neural tube patterning might also affect neuroepithelial bending, but the expression domains of the dorsal marker, Pax3, and ventral marker, Nkx6.1, are normal. However, the effector of Bmp receptor-mediated signalling, phospho-Smad 1/5/8, is upregulated in the Zic2 Ku/Ku neuroepithelium. In parallel with our pathway analysis, we have also identified an orphan nuclear receptor, GCNF/Nr6a1, that is downregulated in the Zic2 Ku/Ku neuroepithelium, and is linked to Bmp pathway activation (Lan et al., 2003). Our data so far suggest that Bmp receptor-mediated signalling, but not Shh signalling, contributes to the Zic2 Ku/Ku spina bifida.

Hitchhiker (hhkr), a mutant exhibiting spina bifida, exencephaly and polydactyly, was identified through an ENU G3 recessive screen at Harwell. The mutation was mapped to Tubby like protein 3 (Tulp3), and is predicted to cause loss of all functional domains. A yeast 2-hybrid screen using Tulp3 as bait identified multiple potential protein interactions, most robustly an interaction with Tripartite motif protein 71 (Trim71), a stem cell specific E3 ubiquitin ligase involved in the control of cell fate in C. elegans. This is particularly interesting as examination of cellular processes in the hindbrain of hhkr mutant embryos revealed a significant increase in proliferation in this region.

We are confirming this potential interaction using an immunoprecipitation approach, the preliminary results of which suggest the interaction can be verified. Examination of mRNA distribution confirms that Trim71 is expressed throughout neurulation in tissues relevant to the phenotypes of hhkr, and this expression is not altered in hhkr mutant embryos. We are investigating the dorsal-ventral patterning of the neural tube of Trim71 mutant embryos, and are crossing hhkr to Trim71 in order to examine any resultant genetic interaction, and refine the role of Tulp3 in promoting proper neural tube closure.

Primary cilia are essential for mouse development, where they have been shown to be important for key signalling events. They are necessary for Hedgehog (Hh) signalling and mouse mutants for cilia genes show defects reminiscent of perturbed Hh responses. We are developing a cell-based RNAi screen to identify new genes involved in cilia formation and/or function. We are screening a subset of candidate genes, identified by cross-species analysis plus proteomic and transciptomic studies. The screen provides two readouts, one image-based readout identifies genes required for cilia formation, assayed by high-throughput immunofluorescence microscopy. The second functional readout measures Hh responsiveness using a luciferase reporter, which is altered when cilia are perturbed. Preliminary results will be presented. Identification of novel ciliogenic genes could aid the search for the diverse functions of primary cilia in development and perhaps help explain the varied phenotypes seen in human ciliary diseases, known as ciliopathies.

Sexual development begins with the process by which the bipotential gonads of the embryonic urogenital ridge develop into either testes or ovaries. Sex determination occurs at around 11·5 dpc in the mouse and depends on the presence or absence of the Y chromosome and the associated activity of the testis-determining gene, Sry, in supporting cell precursors.

The boygirl (byg) mutant was identified at Harwell during an ENU-based screen. On the C57BL/6J background, XY byg/byg homozygotes exhibit embryonic gonadal sex reversal, associated with reduced and delayed expression of Sry. The defective gene in byg, Map3k4, is a component of the mitogen-activated protein kinase (MAPK) signalling pathway, however the other components of this MAPK pathway in sex determination have not yet been determined. I am currently focusing on the relationship between p38 MAPK, one of the MAPKs known to be activated downstream of MAP3K4, with the transcriptional regulation of Sry and sexual development more broadly. To do this I have utilized a conditional knockout approach to delete the p38β isoform using Sf1-Cre and Amhr2-Cre. To address the possibility that there could be functional redundancy amongst the four different p38 isoforms, I am working on siRNA-mediated knockdown approaches in organ culture models.

The Human Developmental Biology Resource (HDBR) is a unique resource funded by the MRC and Wellcome Trust. It provides human embryonic and fetal tissue for gene expression studies related to congenital disease, including both birth defects and inherited metabolic disorders. Use of the material should particularly illuminate developmental gene expression underlying aspects of functioning that characterize humans as opposed to lower animals (e.g. higher brain function, language). This research is essential if we are to introduce new methods for prevention of congenital defects and develop an improved understanding of “what makes us human”.

The HDBR has ethics approval for the collection, storage and distribution of material between 4 and 12 weeks of gestation. The material can be used to generate cell lines, stem cells, protein, RNA and DNA. In addition, paraffin wax and frozen sections of embryos and early fetuses are available for in situ hybridization and immunohistochemistry. For users who do not have experience in gene expression analysis the HDBR offers an in-house gene expression service using in situ hybridization and/or immunohistochemistry.

Further details can be found at

XLαs (eXtra Large αs) is an NH2-extended variant of the ‘α-stimulatory’ subunit of the trimeric G-protein, Gαs. Gnasxl, the transcript encoding XLαs, is derived through the use of an alternate promoter and first exon and shows strict maternal imprinting. At birth, knock-out mice display poor feeding, increased neonatal mortality and very limited adipose development, while adult survivors go on to develop a healthy exceptionally lean, hypermetabolic phenotype, showing increased sympathetic tone and weighing ~45% lighter with less than half the body fat of wild-type controls. To investigate the possible tissue-specific functions of XLαs a conditional knock-out is required. Since the complexity of the imprinted Gnas locus does not allow a standard approach to be used we designed a conditional gene trap strategy for the Gnasxl transcript. Tissue-specific Cre expression causes an inversion/activation of a silent gene-trap cassette, resulting in truncation of XLαs and the formation of a lacZ fusion marker protein. A neural-specific knock-out has been undertaken using the well established Nestin-Cre strain. Results from this cross have revealed both novel and unexpected insight into the tissue distribution and expression of XLαs.

Intrauterine growth restriction (IUGR) is the second leading cause of perinatal mortality and morbidity, after prematurity. Imprinted genes play an important role in placental and fetal growth in eutherian mammals. Our previous study showed a significant correlation between lower birth weight and increased PHLDA2 expression in term placentae without loss of imprinting. To investigate the increased expression of PHLDA2 in smaller birth weight babies, sequence analysis of the promoter region was performed in the Moore cohort (n=285). A tandem 15 bp duplication was found in most individuals (86%), while a rarer, single copy number variant (CNV) in the remainder was found to reduce PHLDA2 promoter efficiency in vitro. Meta-analysis of the polymorphism frequency and birth weight data from the Moore (n=263), Rayne (n=339) and ALSPAC (n=9,327) cohorts showed that a maternally inherited CNV in the baby results in increased birth weight. Moreover, when the mother is homozygous for this polymorphism, the influence on the baby's birth weight is twice as high. This suggests a cumulative effect of the CNV through the mother's mother. To our knowledge, this is the first evidence of genetically inherited regulation of imprinted gene expression, which has a direct and significant effect on fetal birth weight.

Dopa Decarboxylase (Ddc) is an enzyme that plays a fundamental role in the biosynthesis of catecholamine neurotransmitters and serotonin. A short form transcriptional variant of Ddc called Ddc_exon1a, which originates from an alternative promoter at exon 1a, is highly expressed in the trabecular cardiomyocytes during development of pre-natal heart and is progressively silenced during postnatal development. Ddc_exon1a has been shown to be epigenetically regulated via genomic imprinting in mouse heart in a tissue-specific and transcriptional variant-specific manner.

Ddc imprinting is controlled via a differentially methylated region (DMR) located in a CpG Island (CGI2) within the adjacent imprinted gene Grb10. A bioinformatic screen using ChIP-seq data from an ES-cell line has identified a CTCF binding site at CGI2 which we have confirmed in vivo using ChIP-qPCR. Furthermore, we have preliminary data which show that binding of CTCF at CGI2 is allele-specific in somatic tissues. It has been proposed that a CG rich region (CGI3) adjacent to CGI2 may control the different imprinting patterns of Grb10 found in different tissues. We are using ChIP, bisulphite sequencing and bioinformatic analysis in order to assess the role of CGI2 and CGI3 in imprinting of Ddc.

The UniProt Knowledgebase (UniProtKB) aims to provide the scientific community with a comprehensive and authoritative resource for protein sequence and functional information. Given the importance of mammalian model data in biomedical research, a major focus is the high-quality curation of human proteins and their orthologs in other mammals. Manual curation involves (1) the extraction of experimental results from scientific literature to enrich protein records with a wide range of functional information, (2) the manual verification of each sequence and clarification of any discrepancies, and (3) the assessment of the output of a range of analysis programmes to ensure that sequence features are correctly reported. The process also facilitates the standardization of experimental data – a step necessary for development of methods that enable the semi-automated transfer of annotation to uncharacterised or related proteins. UniProtKB/Swiss-Prot currently contains the complete manually reviewed human proteome, comprising approximately 20,300 canonical sequences and 14,700 isoforms, and an additional 45,000 reviewed entries from other mammals such as mouse, rat, apes, cow, sheep and dog. It offers a number of features that are specifically useful in the context of developmental research and is a valuable resource for those working in the field. Ongoing efforts continue to improve the quality of mammalian sequences in collaboration with HAVANA, Ensembl, HGNC and RefSeq, and to extend the coverage of curated proteins in mammalian species. All data are freely available from

Genomic imprinting provides a useful paradigm to study epigenetic mechanisms. Imprinting refers to the expression of a gene from one chromosome homologue in a parental-origin-specific manner, which is dependent on epigenetic control by a cis-acting imprinting control region (ICR). Using high-throughput data extraction with subsequent analysis, we have found that particular histone modifications at imprinted genes are more likely to associate with either imprinting repression or more canonical ‘developmental’ repression (McEwen & Ferguson-Smith, 2010). Furthermore, we have identified a specific tri-mark signature comprising H3K4me3, H3K9me3 and H4K20me3 at all ICRs in mouse ESCs. We have further examined this epigenetic signature as a predictor of novel ICRs. Non-imprinted loci enriched with the tri-mark are identified, suggesting an intriguing function for this rare epigenetic profile. We have shown that publicly available whole genome data can be mined and analysed in order to generate novel findings for functional categories. Our results identify epigenetic profiles at imprinted loci that are likely to associate with distinct mechanisms of gene repression. This form of analysis is therefore a useful tool to elucidate the complex epigenetic code associated with genome function and determine underlying features conferring epigenetic states.

McEwen, K. & Ferguson-Smith, A. (2010). Distinguishing epigenetic marks of developmental and imprinting regulation. Epigenetics & Chromatin 3:2.

The role of epigenetics in human disease is not fully understood. Epialleles may act independently of DNA sequence to cause disease, known as pure epialleles. Alternatively, they may act to modify the effect of sequence variation such that disease manifests, known as facilitated epialleles. Or they may be obligatory epialleles, themselves determined by the DNA sequence. Delineating each type is vital for understanding disease pathogenesis.

We have collected a unique group of patients with diseases and syndromes of growth disturbance of unknown genetic cause, whose inheritance patterns and phenotypes point to the involvement of epialleles in their pathogenesis, i.e. asymmetric growth restriction or overgrowth, mosaicism and cases of discordant inheritance in monozygotic twins. Within our group we include patients with Silver Russell Syndrome, a potential genomic-imprinting related growth disorder, whose aetiology has been correlated with hypomethylated DNA epialleles on chromosome 11.

One of the most important and well studied epigenetic modifications is DNA methylation. To establish the role of DNA methylation in the disease phenotypes of our patients we carried out genome-wide methylome analyses, using methylated DNA immunoprecipitation followed by Next Generation Sequencing (MeDIP-seq). We present preliminary bioinformatic analyses of MeDIP-Seq data, showing candidate differentially methylated regions identified by comparing patients with healthy controls.

The imprinted Gnas cluster contains a complex group of paternally, maternally or biallelically expressed genes. Within this cluster are Gnasxl, which is exclusively paternally expressed and Gnas, which is maternally expressed in specific tissues. Both Gnasxl and Gnas determine the alpha subunit of the Gs protein. Deficiency of either Gnasxl or Gnas is associated with severe phenotypes shortly after birth.

We describe here a new mutation in the Gnas cluster designated Caspa. Upon maternal transmission of the mutated allele we observe a reduction in expression of Gnas in imprinted tissues, and global up-regulation of Gnasxl transcript levels. These mice present with a marked hyperactive phenotype that is observed from the day after birth. This phenotype is generally lethal; most Caspa/+ mice die before weaning. This lethality appears to be associated with a failure to gain weight. The Caspa/+ phenotype appears similar to that observed in mice with paternal uniparental partial disomy for distal chromosome 2 (PatDp.dist2). We hypothesize that the phenotype observed in the Caspa/+ mice is due to over-expression of Gnasxl. Rescue experiments in which the Caspa/+ mutation is crossed with either a mutation that corrects for Gnas expression or a mutation that corrects for Gnasxl expression supports this hypothesis.

Significant advances in the understanding of pituitary gland development in mouse have lead to the identification of a number of genes implicated in the pathology of hypopituitarism, with or without syndromic features such as Septo-Optic Dysplasia (SOD). To date, a total of 15 mutations in the paired-liked homeodomain transcriptional repressor Hesx1 have been documented. Here, we characterize three novel HESX1 mutations identified in patients with hypopituitarism. We demonstrate that mutations occurring within the homeodomain impede DNA binding of mutant protein however repressor activity is maintained.

Hypopituitarism has also been associated with mutations in human Sox2, a SOXB1-HMG box transcription factor. The molecular mechanisms responsible for this remain poorly understood. To study the role of Sox2 in the pituitary, we utilized the Hesx1-cre knock-in mouse to genetically ablate Sox2 from the developing pituitary. Our preliminary data demonstrate that these mutants display profound anterior pituitary hypoplasia in addition to a defect in the specification of the Pit1+ lineage. Taken together, we highlight that it is essential to investigate factors required for normal development of the pituitary. This will ultimately lead to a better understanding of hypopituitarism and SOD in humans. This work is supported by a CHRAT studentship.

The majority of in-vitro derived human preimplantation embryos are chromosomally abnormal/mosaic but whether the same pattern exists in-vivo is unknown. This would be impossible to demonstrate in humans. Hence we chose murine embryos to study this difference owing to their ease of manipulation and compared the incidence of mosaicism between in-vivo and in-vitro cultured embryos. Fluorescent In situ hybridization (FISH) was developed using probe for chromosomes 2 and 11 on two groups of mouse embryos. In vitro group were obtained 48 hours following superovulation and mating of the female mice and cultured in vitro until the blastocyst stage. In vivo group were obtained by sacrificing female mice on day 5. FISH was performed on all blastocysts obtained. The same experiment was repeated in a different lab using exactly the same mouse species and the same protocol to validate the obtained results. The comparison between the incidence of mosaicism of the in vivo group and in vitro group indicated a significant difference with a p value <0·05 (8% vs. 31%). These results were repeated in the second lab with a p value <0·05 (12% vs. 46%). Our data show that murine embryos are an efficient model to study the effect of culture conditions on aneuploidy. Moreover, in vitro cultured embryos showed a significant increase in mosaicism in comparison to the in-vivo group.

Prion diseases are transmissible spongiform encephalopathies caused by the aberrant conformational conversion of PrPc to PrPSc. These disorders are characterized by prolonged, clinically silent incubation periods that are influenced by a number of factors including host genetic background. The major genetic determinant of incubation time is variation within the prion gene, Prnp, however, quantitative trait analyses have identified other modifiers, including HECTD2, that contribute to this effect. HECTD2 is thought to be an E3 ubiquitin ligase with no known substrates. To investigate the role of HECTD2 in prion disease it is important to confirm its effect on incubation time using mouse models and to define its function by identifying its substrates.

To assess the effect of HECTD2 on incubation time, a knockout mouse model is being characterized and challenged with a range of prion strains. To identify the substrates, the yeast-two-hybrid system was employed. Preliminary studies indicate that the screen identified a range of different interactors including those expressed in the central nervous system. This includes Stmn2, a gene previously implicated in vCJD susceptibility.

The characterization of HECTD2 will allow us to understand the molecular basis of incubation time with the ultimate goal of providing new therapeutic targets.

Curly tail (ct) is the best characterized mouse model of spinal NTDs, which closely resemble the corresponding birth defects in humans, with multifactorial etiology influenced by environmental and genetic factors. Spina bifida in ct/ct embryos results from reduced proliferation in the hindgut endoderm, which causes excessive curvature of the caudal region of the embryo, and inhibits closure of the posterior neuropore. The ct mutation is a hypomorphic allele of Grainyhead-like-3 (Grhl3) with reduced expression due to a putative regulatory mutation. This is confirmed by the Grhl3-transgenic curly tail strain which reinstatement of Grhl3 levels results in normal closure of the posterior neuropore. A two-dimensional protein electrophoresis analysis was used to compare ct/ct and matched wild-type embryos at the time of posterior neuropore closure in order to investigate the pathogenesis of NTDs in the ct strain. Migration differences have been found to result from a genomic polymorphism leading to a variation in lamin B1 protein sequence. In vitro analysis were suggestive lamin B1 variant could affect the dynamics of the nuclear lamina. Analysis of sub-strains of mice carrying different combinations of the Lmnb1 polymorphism and the Grhl3 mutation suggest that Lmnb1 could potentially act as a modifier of NTD risk in curly tail mice. This work is supported by the MRC.

Carrying a mutation in Tubby like protein 3 (Tulp3) gene, Hitchhiker (hhkr) displays multiple developmental defects including spina bifida, exencephaly and polydactyly. We and others have recently reported that Tulp3 functions as a negative regulator of the Sonic hedgehog signalling.

To investigate the molecular function of Tulp3 by identifying its interacting partners, we carried out a yeast 2-hybrid screen using Tulp3 as bait. This screen identified 11 potential protein interactions, all of which were expressed at the developmental stage that is important for neural tube closure. Rho-guanine nucleotide exchange factor (Rgnef, also called p190RhoGEF), is one of the potential interacting partners of Tulp3 identified in this screen.

Analysing the expression domain of Rgnef mRNA, revealed that it is an excellent candidate gene for regulating Shh signalling with Tulp3 during patterning of the neural tube. It is most highly expressed in cells where a high level of Shh is present i.e. the floor plate. This expression domain of Rgnef is not affected in hhkr. To confirm this potential interaction we performed co-immunoprecipitation study and our preliminary results suggest that Tulp3 and Rgnef may form a protein complex.

T-associated sex reversal (Tas) describes the formation of ovotestes or ovaries in XY mice harbouring the hairpin tail deletion (T hp) when on the C57BL/6J-YAKR genetic background. One explanation of this phenomenon invokes haploinsufficiency of an autosomal testis-determining gene in the T hp deletion that is postulated to reduce expression of the Y-linked testis-determining gene, Sry. Tas mimics certain human conditions, in which loss of one allele of a sex-determining gene is sufficient to cause XY sex reversal. We recently reported that mice heterozygous for a null allele of Map3k4 on C57BL/6J-YAKR exhibit a Tas-like phenotype: XY ovotestis development, testicular hypoplasia and occasional gonadal sex reversal. Haploinsufficiency of Map3k4 is therefore sufficient to phenocopy T hp/+ XY sex reversal. However, these experiments did not exclude the possibility that loss of other loci in the deletion or indeed that the deletion itself might also contribute to sex reversal, i.e. they did not demonstrate that Map3k4 haploinsufficiency is necessary for Tas. Here, we report the generation of a novel mouse line carrying a functional Map3k4 bacterial artificial chromosome (BAC) transgene. We are determining whether this transgene rescues abnormalities of C57BL/6J-YAKRT hp/+ gonad development which would suggest that only Map3k4 haploinsufficiency can cause T-associated sex reversal.

Effective methods for high-throughput screening and analysis are crucial for phenotyping the increasing number of mouse mutants that are being generated to investigate the role of genes in human diseases and development. Magnetic resonance imaging is an ideal phenotyping platform due to its inherent non-invasive and three-dimensional nature. We can image up to 40 embryos at E15.5 in a single overnight scan, generating an MRI dataset of all the embryos, which may be viewed on slice-by-slice basis. Furthermore, we have developed automated techniques for morphometric assessment of these large MRI datasets. Segmentation propagation enables rapid and non-invasive calculation of tissue volumes in a population using an average, composite atlas. Tensor-based morphometry is a fully automated technique that enables unsupervised and unbiased detection of local, volumetric changes in a population on a voxel-wise basis, which are not visible to the human eye. In addition, we are investigating active staining techniques for MR histology, which may allow specific cellular structures to be targeted using MRI, and developing diffusion tensor imaging techniques in the mouse embryo to investigate local microstructure, which may not be visible using histological and standard MRI approaches. This work is supported by the ESPRC and BHF.