Genetic Regulation of Development Lecture Notes PDF

Genetic Regulation of Development Farhad K. Shahian, DDS, PhD  Embryonic Body Axis Formation 1) The dorsal-ventral (D-V) axis: BMP gradient. 2) The antero-posterior (A-P) axis: Hox genes. - Colinearity - Activation by retinoic acid. Retinoid receptors. Growth factor si...

Genetic Regulation of Development Farhad K. Shahian, DDS, PhD  Embryonic Body Axis Formation 1) The dorsal-ventral (D-V) axis: BMP gradient. 2) The antero-posterior (A-P) axis: Hox genes. - Colinearity - Activation by retinoic acid. Retinoid receptors. Growth factor signals a morphogen gradient can be generated by a source of growth factor (such as BMP) or by a localized source of inhibitor (such as Chordin). Both mechanisms are used.  The BMP gradient induces different tissues in mesoderm and ectoderm (because the DNA-binding partners are different) Mesoderm Ectoderm BMP signaling differentiation BMP signaling differentiation Lateral plate Somite Notochord Epidermis Neural CNS crest The best example of a morphogen is the gradient of BMP signaling that controls D-V tissue differentiation. Chordin binds BMP growth factors in the extracellular space. Chordin establishes a BMP4 activity gradient at gastrula. Noggin, has similar activity. Chordin inhibits  Epidermis CNS Ventral Dorsal Mesoderm Spemann’s Organizer Endoderm At gastrula a gradient of BMP4 is established by a ventral source of BMP4 and a dorsal source of Chordin and Noggin, two BMP antagonists secreted by the dorsal organizing center.  In situ hybridization Chordin mRNA is expressed in Spemann’s organizer. Chordin protein is secreted and diffuses in the embryo.  Cell-cell communication is controlled by surprisingly few signal transduction pathways: 1) TGFβ/BMP Serine/Threonine kinase receptors 2) Receptor Tyrosine kinases such as FGF, EGF, IGF, Insulin 3) Wnts 4) Sonic Hedgehog 5) Notch 6) G protein-coupled receptors (7-transmembrane receptors) 7) Nuclear hormone receptors  Anterior-Posterior Axis Formation Hox genes  Homeobox genes Controlpattern formation and share a consensus sequence of 180 nucleotides. Homeodomain (60 amino acid domain) three alpha helices (a3) align in the major groove of DNA TALE proteins (Exd): - Bind to DNA first - Alter the DNA conformation slightly - Assists in binding of other TFs - Stabilize bound homeodomain proteins.  Homeobox genes exhibit two patterns of localization 1. Some are scattered throughout the genome. 2. Hox genes: other homeobox genes are clustered within a small region and in a very specific sequence. These Hox genes are highly conserved in organisms ranging from flies to humans.  Hox genes and the development of body plans Homeotic transformations in humans. A cervical vertebra transformed into a thoracic one with ribs.  Antennapedia Mutations Wild-type Mutants  Hox complexes: arose by repeated duplication and mutation of an ancestral homeobox gene. This formed an ancestral HOX complex. In some organisms, including most vertebrates, the HOX complex has been duplicated four times.  Hox genes are organized into paralogy and orthology groups Paralogy group (within a species): - Paralogues have often evolved different functions - Diverged after a duplication event Orthology group (different species) - Orthologs maintain basically the same function in the different species. - Diverged after a speciation event - Orthologous Hox genes from different species can replace one another in function (Hox d4 substitutes for Dfd in Drosophila). Retinoic acid (RA) influences Hox gene expression. Overexposure in the human fetus: - absent or defective ears, - absent or small jaws -cleft palate - aortic arch abnormalities - thymic deficiencies - abnormalities of the central nervous system. Overexposure in mice: Axial truncation Reduction in the sizes of the first and second pharyngeal arches, which normally form the jaw, ear, and other facial bones (Gilbert, 2003; Ligas, 2000). The truncated embryo exhibits a posterior region having the characteristics of the anterior region of an embryo At very high concentrations, the cells do not differentiate to form the posterior of the embryo at all (Ligas, 2000). Retinoic acid disrupts development by: - altering the expression of Hox genes/the anterior–posterior axis - Inhibition of neural crest cell migration from the cranial region of the neural tube (Gilbert, 2003). The retinoic acid-bound RARs: - bind to DNA enhancer sequences and activate particular genes (homeotic genes). - Excess RA results in more anterior Hox genes are expressed in typically posterior regions (Ligas, 2000). Pax2/5 and Pax6 transcription factors subdivide the early neural tube into three divisions The expression patterns of Pax2 and 5 and Pax6 genes demarcate the midbrain and forebrain primordia at the neural plate stage. Vertebrates have four Hox complexes, with about 10 genes each. They display colinearity: a) Temporal colinearity: genes on one end of the complex are expressed first, those on the other (posterior) end are turned on last. b) Spatial colinearity: the more anteriorly expressed genes are in one end, the more posterior ones at the other end of the gene complex. c) Anterior Hox genes are activated sequentially by retinoic acid. Hox genes can be aligned in 13 groups of paralogues. Temporal and spatial colinearity: order of Hox genes in DNA follows the antero-posterior body axis. Retinoic acid receptor is a DNA-binding protein that works as a ligand- activated transcription factor. Many hydrophobic hormone receptors work in this way. Nuclear receptors work very differently from cell surface receptors. (RA) RA Hox complexes have a retinoic acid receptor response element (RARE) in the DNA before paralogue 1. This DNA enhancer element controls expression of many genes in the complex. RARE  Pharyngeal arch 1 does not express any Hox gene. It gives rise to maxillary and mandibular structures. Retinoic acid can cause cleft palate and micrognathia The Influence of Genetics in Normal Dentition, Dental Anomalies, and Malocclusion Farhad K. Shahian, DDS, PhD  Aetiology of malocclusion The relative contribution of genes and the environment to the aetiology of malocclusion has been a matter of controversy. Genetic mechanisms are predominant during embryonic craniofacial morphogenesis. Environment is thought to influence dentofacial morphology postnatally, particularly during facial growth.  Inconsistency in MZ Twins (Townsend et al. 2009) MZ co-twins showing mirror-imaging for missing lower second premolars and different expressions of third molar development, emphasizing the role of epigenetic influences on dental development.  Epigenetics Differences in gene expression that do not involve changes to the underlying DNA sequence A change in phenotype without a change in genotype Factors influence epigenetic changes: DNA methylation Histone modification (acetylation) Non-coding RNA (ncRNA)-associated gene silencing  Factors Influences Human Dental Anomaly Multifactorial Model Genetic Epigenetic Environmental influences  Molecular genetics in dental development The first sign of tooth development is a local thickening of oral epithelium, which subsequently invaginates into neural crest-derived mesenchyme and forms a tooth bud. Subsequent epithelial folding and rapid cell proliferation result in first the cap, and then the bell stage of tooth morphogenesis. During the bell stage, the dentin producing odontoblasts and enamel secreting ameloblasts differentiate.  Molecular Mechanism of Dentogenesis Strict genetic control of odontogenesis, which determines the position, number, size and shape of the teeth. Bell stage - Cyto-differentiation of dental epithelial cells near the mesenchyme differentiate into ameloblastes - The adjacent mesenchymal cells differentiate into odontoblasts - Mesenchyme surrounding the tooth bud will develop forming the supporting structure of the tooth (PDL,…)  Morphogenesis and Differentiation More than 200 genes involved A crucial role was attributed to those transcription factors that have a homeodomain. During tooth morphogenesis, expression of the homeobox genes is under the control of signaling molecules (e.g. growth factors)  Important factors in tooth morphogenesis The family of fibroblast growth factors (FGF) Transforming growth factors (TGF, including BMP4 - bone morphogenetic protein 4), The family of Wnt (Wingless) Sonic hedgehog (Shh)  Odontogenesis The general scheme of dentition is determined even before the development of visible teeth. The proximal area of the molars to be developed is characterized by the expression of growth factors FGF8 and FGF9 BMP4 is expressed in the distal region of the presumed incisors Mentioned transcription factors define spatially the domains of expression of the homeobox genes in the developing jaw. Basically every combination of homeobox genes expressed is a “code” that specifies the type of the tooth  Dental agenesis Congenital lack of one or more teeth is the most frequent anomaly in humans. Hypodontia: 1–6 teeth are missing (excluding 3rd molar) Oligodontia: more than 6 missing (excluding 3rd molar) Anodontia: the complete absence of teeth (rare) - Large family in China (Autosomal recessive) Genetic defect in hypodontia/oligodontia MSX1 - hypodontia PAX9 - oligodontia AXIN2 - oligodontia associated with colono- rectal cancer EDA1 - oligodontia  Msx1 Homeobox Gene Early expression Migrating neural crest cells First branchial arch. Initiation of odontogenesis Mesenchyme adjacent the future primary epithelial thickening During odontogenesis MSX1 is also expressed at high levels in the dental mesenchyme at the cap and bell stages Mutations in human MSX1 also cause cleft lip/palate and tooth agenesis  PAX9 Homeobox Gene Plays a critical role in dental formation at all stages of odontogenesis Is strongly expressed in the oral mesenchyme during the early stages of tooth development PAX9 is responsible for BMP4 expression which further regulates expression of MSX1. Animal model studies have proven that PAX9- deficient knockout mice exhibit missing molars due to an arrest of tooth development at the bud stage  AXIN2 Gene Axin2 regulates the stability of β-catenin When cells receive Wnt signals, β-catenin binds to stabilized transcription factors (e.g.TCF family) Changes in the functioning of the Wnt signaling pathway leads to colorectal cancer predisposition. Oligodontia due to AXIN2 gene mutation is more severe than MSX1 and PAX9 genes More missing molars, premolars, upper lateral incisors and lower incisors, but upper central incisors are present.  EDA1 gene Mutations cause X-linked hypohydrotic ectodermal dysplasia (HED) Hypoplasia or absence of sweat glands, dry skin, sparse hair and pronounced oligodontia. multiple missing teeth (oligodontia) and small, misshapen teeth The lack of central and lateral incisors as well as canine teeth of the maxilla and mandible (Chinese family) EDA is critical for ectoderm-mesoderm interactions which is essential for the formation of several structures that arise from the ectoderm, including the skin, hair, nails, teeth, and sweat glands.  Sonic Hedgehog Shh is expressed strongly in the epithelial cells (Placode) At a later stage Shh is expressed in the enamel knot (cuspal morphogenesis) The Gli zinc finger transcription factors (Gli-1, - 2, - 3) are known to act downstream of Shh. Double homozygous knockout mice for Gli-2/Gli-3: No teeth Double homozygous/heterozygous mutants (Gli-2 - /- , Gli-3 +/- ): Maxillary incisor development arrested, and all molars and the mandibular incisors were microdont.  Bone Morphogenetic Proteins The expression of Bmp's has been associated with epithelial- mesenchymal interactions involved in the development of a number of organs, including the teeth Bmp-2, 4, and 7 are all expressed in the presumptive dental epithelium during early tooth morphogenesis. Bmp-4 expression shifts from the epithelium to the condensing dental mesenchyme at the same time that the inductive potential for odontogenesis shifts from epithelium to mesenchyme. This suggests that Bmp-4 may be a principle component of the signal responsible for inducing odontogenic potential in the mesenchyme  Fibroblast Growth Factors Fgf-4, Fgf-8, and Fgf-9 are all expressed in epithelial cells during epithelial-mesenchymal (regulating odontogenic morphogenesis) Expression of Fgf-8 and Fgf-9 persists in the primitive oral epithelium until the beginning of the bud stage. Fgf-4 and Fgf-8 expressions are up-regulated at the cap stage in the enamel knot and later at the secondary enamel knots (the sites of future cuspal morphogenesis) Fgf-8 and Fgf-9 are the initiation of tooth development, and for Fgf-4 and Fgf-8 in determining coronal morphology.  The Role of the Enamel Knot Transient population of non-dividing epithelial cells Appear during the late bud stage of development at the site of the primary tooth cusps. Initially, the enamel knot expresses the Bmp-2, Bmp- 7, and Shh signalling molecules later, during the cap stage, it also expresses Bmp-4 and Fgf-4. It is thought that the enamel knot acts as a signalling centre, being responsible for directing cell proliferation and subsequent cuspal morphogenesis in the developing enamel organ (Vaahtokari et al., 1996a).  Both the primary and secondary knot cells express Fgf-4 and are non-dividing; Fgf-4 is known to stimulate proliferation of both dental epithelium and mesenchyme. It has been proposed that this induced cell proliferation of the enamel organ in conjunction with the lack of cell division in the enamel knot allows the growth and folding of the developing cusps (Jernvall et al., 1994). At the late cap stage, the cells of the enamel knot undergo apoptosis and disappear, presumably switching off its signalling function (Vaahtokari et al., 1996b). The enamel knot is seemingly necessary for morphogenesis of the tooth germ to progress from the bud to the cap stage.  Supernumerary teeth Caucasians: 0.2–0.8% of in the primary and 1.5–3.5% in the permanent dentition There is a male-to-female ratio of approximately 2:1 (most seen in pre-maxillary region) The most common type of supernumerary is a premaxillary conical midline tooth (mesiodens). Patients with supernumerary primary teeth have a 30–50% chance of these being followed by supernumerary permanent teeth. Supernumerary teeth are more common in the maxilla than in the mandible, with a ratio of about 5:1  Approximately 40% of patients with a cleft of the anterior palate having supernumerary teeth. There is a significant association between supernumerary teeth and invaginated teeth (dens in dente) In these patients, maxillary lateral incisor is the most affected tooth Mutants in RUNX2 cause supernumerary teeth RUNX2 is also involved in osteoblast differentiation Cleidocranial dysplasia (CCD) Clavicles: partly or completely missing The mandible is prognathic due to hypoplasia of maxilla the fontanelle failed to close and open skull sutures The permanent teeth include supernumerary teeth. Cementum formation may be deficient. Failure of eruption of permanent teeth. Bossing (bulging) of the forehead.