Jim Smith: Projects

The regulation of TGF-B Signalling

The TGF-b superfamily of growth factors, which include Nodals/Activin and Bone Morphogenetic Proteins (BMPs), are essential for early vertebrate development. They play a pivotal role in the formation of the body axis and generation and patterning of the three germ layers: endoderm, mesoderm and ectoderm.

Signalling is transmitted through type I and type II transmembrane serine/threonine kinase receptors by phosphorylating receptor regulated R-Smads: Smad 2 and 3 in the case of TGF-b (Nodals/Activin) and Smad 1, 5 and 8 for BMPs. In the canonical pathway, activated rSmads, bound in homo- or heteromeric complexes with a common partner Smad4, translocate into the nucleus where they regulate transcription of target genes. Although poorly understood, BMPs can also signal independently of Smad4.

Not surprisingly, TGF-b superfamily signalling is kept under tight control either through the control of ligand accessibility or by attenuating activated receptors and Smads via a variety of mechanisms.

Figure

Figure. Canonical TGF-β superfamily signalling activates gene transcription via phosphorylated r-Smad/Smad4 complexes, while in non-canonical signalling phosphorylated r-Smads form active complexes with PAWS. (B, C) USP15 enhances BMP signalling (B) knockdown of xUSP15 reduces BMP-dependent Smad1 phosphorylation. (C) xUSP15 stabilises levels of Alk3 by targeting it for deubiquitylation. (Click to view larger image)

We are interested in identifying mechanisms regulating TGF-b superfamily signalling and asking how these mechanisms influence the induction and patterning of the primary germ layers. One approach is to unearth new molecules that either modulate Smad activity or regulate their intracellular trafficking and turnover.

Selected publications

Al-Salihi, MA, Herhaus L, Dingwell KS, Wasmus L, Vogt J, Ewan R, Bruce D, Macartney T, Weidlich S, Smith JC and Sapkota GP (2014). USP15 targets ALK3/BMPR1A for deubiquitylation to enhance BMP signalling. Open Biology 4, 140065.

Vogt J, Dingwell KS, Herhaus L, Gourlay R, Macartney T, Campbell C, Smith JC and Sapkota G (2014). Protein Associated With Smad1 (PAWS1/FAM83G) is a substrate for type I bone morphogenetic protein receptors and modulates bone morphogenetic protein signalling. Open Biology 4, 130210.

Callery EM, Park CY, Xu X, Zhu H, Smith JC and Thomsen GH (2012). Eps15R is required for bone morphogenetic protein signalling and differentially compartmentalizes with Smad proteins. Open Biology 2, 120060.

The roles of T-box transcription factors in mesoderm formation and early development

The induction of mesoderm is among the earliest cell fate decisions occurring in bilaterian embryos as a consequence of zygotic genome activation and TGF-b signalling. This event leads into gastrulation, a period of complex tissue rearrangements (morphogenetic movements) segregating mesoderm from neural and endodermal tissue.

On a genetic level nascent mesoderm correlates with the activation of several T-box transcription factors such as Eomesodermin and Brachyury. In fact, these T-box transcription factors are essential for the maintenance of mesodermal cells and their derivatives. Without these transcriptional inputs cells in anatomical positions of mesoderm switch to a neural fate at least in part due to their common cell lineage.

Consequently, such embryos develop an excess of neural tissue at the expense of mesoderm. We are making use of classic embryological and state of the art technologies to dissect in vivo how naïve cells adopt these different cell fates to complete a well-formed vertebrate embryo.

Figure

Figure. Xenopus Brachyury (Xbra) marks precursor cells giving rise to mesoderm and neural tissues. Xbra is a sequence-specific transcription factor recognising a 9-bp motif. This binding motif was determined de novo from Xbra binding profiles at gastrula stage (ChIP-Seq) and confirmed in vitro by Surface Plasmon Resonance (SPR). Morpholino-mediated knockdown experiments revealed functional redundancy between Brachyury paralogues Xbra and Xbra3 in regulating axis elongation. (Click to view larger image)

Selected publications

Gentsch GE, Patrushev I and Smith JC (2014). Genome-Wide Snapshot of Chromatin Regulators and States in Xenopus Embryos by ChIP-Seq. Journal of Visualized Experiments, in press.

Gentsch G and Smith JC (2014). Mapping physical chromatin associations across the Xenopus genome by chromatin immunoprecipitation. CSH Protocols: doi: 10.1101/pdb.prot080614.

Gentsch GE, Owens NDL, Piccinelli P, Faial T, Trotter MWB, Gilchrist MJ and Smith JC (2013). Cooperation of T-box transcription factors regulates neural and mesodermal fates in vertebrate embryos. Cell Reports 4, 1185-96.

Evans AL, Pedersen RA, Vallier L, Gilchrist M, Down T, Wardle FC and Smith JC (2012). Targets of Brachyury (T) in differentiating mouse embryonic stem cells. PLoS One 3, e33346.

Making hearts from human and mouse embryonic stem cells

Cardiovascular diseases are the leading cause of mortality in industrialised countries.

We are interested in unravelling human heart disease by studying heart development at the molecular and cellular level. Our ultimate goal is to apply developmental principles and lessons from model organisms in human embryonic stem cells to study how the heart develops and what makes this process susceptible to perturbation.

The human heart is very different from other mammalian hearts. By combining human stem cell biology and embryology we hope to: (i) uncover new links between early mesoderm patterning and congenital heart defects; (ii) provide insights into cardiac stem cell proliferation; and (iii) aid the generation of sufficient cardiac cells for use in drug trials and in stem cell-based therapies.

Figure

Figure. Upper panel: diagram depicting the development of the mouse heart. Cells, which give rise to the heart, are in green. Bottom panel: diagram depicting the differentiation steps hESCs undertake to become beating cardiomyocytes. (Click to view larger image)

 

Video of beating cardiomyocytes derived from human embryonic stem cells after 10 days of differentiation.

Selected publications

Bernardo AS, Faial T, Gardner L, Niakan KK, Ortmann D, Senner CE, Callery EM, Trotter MW, Hemberger M, Smith JC, Bardwell L, Moffett A and Pedersen RA (2011). BRACHYURY and CDX2 mediate BMP-induced differentiation of human and mouse pluripotent stem cells into embryonic and extraembryonic lineages. Cell Stem Cell 9, 144-155.

BMP signalling in vascular development

To generate a functioning vascular system, a wide array of blood and endothelial cell types must be generated, and those endothelial cells must assemble into intricate branching networks of tubes. We know of several major signalling pathways that are involved at different stages of this complex process. Among them, the importance of BMP signalling is clear from mouse knockout phenotypes, and from the propensity of heterozygous mutations in BMP pathway components to cause human vascular diseases. With these diseases in mind, our aim is to identify transcriptional targets of BMP signalling in the developing zebrafish vascular system, and to understand the biological functions of these target genes.

Using transgenic zebrafish in which endothelial cells express GFP, we can directly observe the effects of BMP inhibition on vascular development. We use a variety of approaches to manipulate BMP signalling, including inducible expression of modified receptors, application of chemical inhibitors such as LDN193189, and gene knockdown by antisense morpholino oligonucleotides (MOs).

We are using high-throughput sequencing to identify BMP-responsive genes in the zebrafish caudal vein plexus and cranial vessels. We have chosen to look at gene regulation in these structures because we know that BMP signalling is necessary for them to form normally (Figure 1). We can then generate mutants for any gene of interest using CRISPR and TALEN technology, as well overexpressing these factors at particular times and places in the embryo. We are also using similar techniques to discover new functions for genes that are strongly expressed in developing blood and endothelium.

Figure

Figure. Vascular effects of BMP inhibition. Top: The caudal vein plexus in the tail of the zebrafish embryo after treatment with DMSO or the BMP inhibitor LDN-193189. The plexus in the embryo treated with the inhibitor has fewer branch points at 32 hours hpf. Bottom: The cranial blood vessels are abnormally expanded in 48 hpf embryos injected with a MO that prevents normal splicing of transcripts for alk1, a type I BMP receptor. (Click to view larger image)

Selected publications

Cannon JE, Place ES, Eve AM, Bradshaw CR, Sesay A, Morrell NW and Smith JC (2013). Global analysis of the haematopoietic and endothelial transcriptome during zebrafish development. Mechanisms of Development 130, 122-131.

The regulation of the cell cycle and of transcription at the mid-blastula transition

Embryonic development is controlled initially by maternal transcripts and other factors that are laid down during oogenesis. In Xenopus, bulk zygotic transcription begins after twelve rapid and synchronous cell cycles, at the so-called the midblastula transition (MBT). At this time cell cycles become asynchronous and longer; several proteins translocate from cytoplasm to nucleus; maternal mRNA is degraded; the apoptosis programme is engaged; cells become motile; karyomeres (individual chromosomes surrounded by a nuclear envelope) are replaced by conventional nuclei; the distance between origins of DNA replication increases; and DNA replication slows down.

In an effort to understand the MBT, we are asking whether these changes are all triggered by the same event, or whether one is primary and the others are initiated as a consequence. We focus on the changes in gene expression and in DNA replication that occur around the MBT.

High-resolution gene expression profiling of early Xenopus embryos reveals that there are distinct waves of gene expression: an early wave of polyadenylation of maternal transcripts followed by several waves of zygotic transcription. The experiments highlight the importance of maternal polyadenylation for the establishment of early zygotic transcription. Work in zebrafish embryos confirms that maternal transcripts provide the specificity required for proper initiation of zygotic transcription after genomic DNA acquires a competent state.

With respect to the cell cycle, we discovered that Y-RNAs become essential for DNA replication only after the MBT, and that four DNA replication factors-Cut5, RecQ4, Drf1 and Treslin-become limiting for the speed of DNA replication at the MBT. We are now exploring differences in the cell cycle before and after the MBT in more detail.

Selected publications

Langley AR, Smith JC, Stemple DL and Harvey SA (2014). New insights into the maternal to zygotic transition. Development 141, 3834-3841.

Collart C, Owens NDL, Bhaw-Rosun L, Cooper B, De Domenico E, Sesay AK, Smith JN, Patrushev I, Smith JC and Gilchrist MJ (2014). High-resolution analysis of gene activity during the Xenopus mid-blastula transition. Development 141, 1927-1939.

Collart C, Allen GE, Bradshaw CR, Smith JC and Zegerman P (2013). Titration of four replication factors is essential for the Xenopus laevis midblastula transition. Science 341, 893-896.

Harvey SA, Sealy I, Kettleborough R, Feynes F, White R, Stemple D and Smith JC (2013). Identification of the zebrafish maternal and paternal transcriptomes. Development 140, 2703-2710.

Collart C, Christov CP, Smith JC and Krude T (2011). An essential Y RNA-dependent pathway for the initiation of DNA replication is activated at the mid-blastula transition in Xenopus laevis. Molecular Cell Biology 31, 3857-70.

Deciphering the mechanisms of developmental disorders

This five-year study is funded by a Strategic Award from the Wellcome Trust, and involves researchers from the Crick, the Sanger Institute, King's College London, the Babraham Institute, the Institute of Child Health, and the Universities of Edinburgh and Oxford. It uses high-resolution 3D imaging and transcriptomics to characterise the developmental abnormalities that occur in mouse embryos after the deletion of individual genes. The resulting database will be available for the wider research community to catalyse new discoveries in developmental biology.

The work is important because one in 40 children in Europe are born with a genetic defect that can affect organs such as the heart, lungs or kidneys, or the formation of the spine and brain. Some defects are so severe they result in miscarriage or stillbirth. Studying these defective genes in mice, and uncovering the underlying molecular mechanisms, will lead to the development of better genetic screening strategies and diagnosis, and might result in new therapies.

This work will make extensive use of Wellcome Trust Sanger Institute's Mouse Genetics Programme, which is creating mouse strains with targeted disruptions in each of the 20 000 genes in the mouse genome as part of the International Knockout Mouse Consortium.

Figure

Figure. A mutation in the gene encoding Cysteine and Glycine-Rich Protein 1 causes the embryo to develop without a tongue (to within the red circle on the left). Wild-type embryo (+/+) to the left; homozygous mutant (Csrp-/-) to the right. (Click to view larger image)

Selected publications

Mohun T, Adams D, Baldock R, Bhattacharya S, Copp A, Hemberger M, Houart C, Hurles M, Robertson EJ, Smith JC, Weaver T and Weninger W (2013). Deciphering the Mechanisms of Developmental Disorders (DMDD). Disease Models and Mechanisms 6, 562-566.

Jim Smith

Jim Smith

jim.smith@crick.ac.uk
+44 (0)20 379 61103

  • Qualifications and history
  • 1979 PhD Middlesex Hospital Medical School, London, UK
  • 1979 Postdoctoral Fellow Harvard Medical School, Boston MA, USA
  • 1981 Postdoctoral Fellow, Imperial Cancer Research Fund, London, UK
  • 1984 Group Leader, then Head of Division and Head of Group, MRC-NIMR, London, UK
  • 2000 Director, Wellcome Trust/Cancer Research UK Gurdon Institute
  • 2009 Director, Medical Research Council National Institute for Medical Research, London, UK
  • 2014 Deputy CEO and Chief of Strategy, Medical Research Council
  • 2015 Director of Research, Francis Crick Institute