1 Genetics of biological processes EPh - 2016 Miretti Plos 2008

2 Possible responses Apoptosis Diffenerentiation Self-renewal Who am I?Mutation Transform-ation Who am I? Where am I? Polarization, cell migration Cell proliferation

3 Cell fates Differentiation REGENERATIVE THERAPY POTENCY

4 Early phase of embryonic development In 16 day develops to multipotentFertilization Implantation Multipotent cells

5 Merrel-Stanger, Nat. Rev. Mol. Cell. Biol. - 2016

6 Canonical vs. stochastic models of differentiationSignificance of regenerative medicine !?!

7 Decreasing potencies during differentiationTotipotens Pluripotens Multipotens The totipotent zygote formed by the fusion of egg and sperm divides to form the inner cell mass (ICM) and the extra-embryonic (EE) tissue of the blastocyst. When isolated from the blastocyst in vitro, the cells of the ICM can be maintained in culture as pluripotent embryonic stem cell (ESC) lines. During the development of the embryo, the pluripotent stem cells in the ICM become increasingly restricted in their lineage potential and generate tissue-specific, multipotent stem cells. These include epidermal stem cells (bulge cells) that form skin and hair, haematopoietic stem cells in the bone marrow that give rise to all haematopoietic cells, neural stem cells in the subventricular zone of the brain, gastrointestinal stem cells that are located in the crypt of the small intestine, oval cells that give rise to liver (not shown), and mesenchymal stem cells that reside in the bone marrow and can form bone, stromal cells and adipocytes (not shown)88, 115. Unipotens

8 Stem cells - Regenerative medicineJohn B Gurdon és Shinya Yamanaka 2012

9 Developmental potencies – Epigenetic conditionsLevel of differentiation Totipotent Zygote Pluripotent ICM, embryo, iPS Multipotent Stem cells of adult Unipotent Differenated cells Epigenetical conition Total methylation of DNA Active X chrs; Differentiation - genes repr. Promoter hypometihylation X inactivation; Germ-line specific genes - repr. Promoter hypomethylation

10 Transcription factor mediated „reprogramming”

11 Steps of „reprogramming” to pluripotent level

12

13 Who am I? Origin = Position= lineage positional identityWhere am I? Endogen factors Asymmetric cleavage Receptors, transcription factors – uneven distribution Exocrine factors Morphogenes = Signal molecules Cell-cell interactions Cell-matrix interactions

14 Preferred directions in embryonic developmentAnterior- posterior (head-tail axis) (Wnt, HOX) Dorso-ventral ( SHH) Proximal- distal (SHH) Right-left symmetry

15 Governing molecules I. Morphogenes:Direction of development is influenced by concentration gradients Oocyte or embryo origin Transcription factors Molekules: Hedgehog protein family TGFβ family (BMP) WNT family (Drosophila Wingless mutation) FGF

16 Signal pathways Other members: FGF – Ras – MAPK pathwayRA (retinoic acid)

17 Hedgehog protein familySonic hedgehog Expressed in the notochord and then in the ventral part of the neural tube Influences on: CNS, skeletal muscl. and limbs Edward B. Lewis TGFβ family BMP SHH BMP: Bone morphogenetic protein

18 Concentration of morphogen itself serves as informationExample: Differentiation of mesoderm Morphogen: Activin (TGF family)

19 Activation of signal pathways

20 Governing molecules II.Homeobox / homeotic genes: Hox genes (homeobox sequence) 60 AA, helix-turn-helix structure proteins Positional information – longitudinal axis Max. 13 box Spatial colinearity

21

22 Human HOX genes

23 Expression of Hox in developmentShape of things to come. Changes to two Hox genes may have spurred evolution of the mammalian female reproductive tract. When the female ancestors of today's mammals evolved a uterus, placenta, and vagina, their egg-laying days were over. Now researchers have identified two genes that they suspect played a key role in creating the biological equipment needed for live birth. The two genes--HoxA-11 and HoxA-13--make transcription factors, proteins that bind to other genes and tell them when to turn on or off. Like other members of the expansive Hox family of transcription factors, HoxA-11 and HoxA-13 control development of the basic body plan; they're also expressed in the uterus and related tissues. The sequence of Hox genes is so consistent across species that biologists once thought that the genes had changed very little through the course of evolution. The new study, like others in recent years, suggests that some divergence among Hox genes has in fact contributed to the diversity of animal body plans. To investigate the history of HoxA-11 and HoxA-13, evolutionary biologist Vincent Lynch of Yale University and his colleagues examined the genes' sequences in a variety of animals, including frog, chicken, platypus, opossum, mouse, and human. They then reconstructed the sequences that likely existed in these animals' ancestors and used a standard technique to determine whether differences in the sequence likely arose by chance or by natural selection. Natural selection was responsible for changes in both genes in the common ancestor of today's marsupial and placental mammals, and for additional changes to HoxA-11 in the lineage that gave rise to placentals, the researchers report online this week in Proceedings of the Royal Society B. This timing of the changes to the Hox genes parallels stages in the evolution of the mammalian female reproductive tract, as a primitive uterus and placenta first appeared in the common ancestor of marsupial and placental mammals and then became more elaborate in placental mammals, Lynch says. Lynch hypothesizes that the changes to HoxA-11 and HoxA-13 could have altered development, perhaps by allowing them to bind to genes involved in creating different kinds of cells. The paper is the first to link the natural selection of developmental genes to a specific change in the way animals develop, the authors say. "It's a really interesting and suggestive correlation," says developmental biologist Matthew Ronshaugen of the University of California, Berkeley. These genes could have "played a major role in the evolution of the mammalian female reproductive tract," Ronshaugen says. Related sites Diseases: developmental abnormality (e.g. synpolydactylia, brachydactylia, Hand‑foot‑genital syndrome ) HOXA13 and HOXD13 MLL (therapeutic target)

24 Interaction of SHH and Hox expression Epithel-mesenchymal connectionsSadler, 9th ed. Fig Diagrams fo the mid- and hindgut regions. The morphogen shh is cecreted by gut endoerm and induces a nested expression of HOX genes in surrounding mesoderm. HOX expression then initieates a cascade of genes that “instruct” gut endoderm to differentiate into its regional identities. Signaling between the 2 tissues is an e.g. of an epithelial-mesenchymal interaction. Sadler, 9th ed.

25 Ultrasound 3D detection of face developmentWeek 4 Week 10 Teratogenic effects

26 Development of prominencies (frontal, maxillar, mandibular) and the influencing factorsFGF10 JAG NOTCH TBX22 PAX9 PITX1 OSR2 GABA Bensodiazepin FGF10 FGFR” SHH MSX1 LHX8 SHOX2 OSR2 Cholesterin TGBb3 LEF1 SMAD TWIST SNAIL Pi3Kináz RHO IRF6 Viruses MSX1 LHX8 TGFBR2 SHH HAND2 Smoking Dioxine 1 growing of upper frontal prominence 2 retraction of toung 3 -4 fusion of the two maxillar prominences

27 Development of teeth The developmental anatomy of early tooth morphogenesis and the formation of different tooth types: low-crowned molar, continuously growing molar with a complex cusp pattern, and continuously growing incisor lacking a complex cusp pattern. The primary dental lamina forms as a thickening of the oral epithelium at the site of the future tooth row. Tooth morphogenesis starts from the dental placodes where neural crest derived mesenchymal cells condense under the epithelial placode. The epithelium first buds into the mesenchyme and then grows to encompass the mesenchymal dental papilla during the cap stage.The epithelial cervical loop is formed during the cap stage on the lateral sides of the bud. Different developmental choices are made at this point leading to the formation of different tooth types. The epithelial stem cell niche in the cervical loop is maintained in continuously growing teeth, but disappears in teeth which develop roots such as all human teeth. Signaling centers are present at various stages of tooth development (placode, primary and secondary enamel knots). The enamel knots regulate tooth crown morphogenesis characterized by folding of the enamel epithelium. The locations of secondary enamel knots correspond to the future sites of tooth cusps. ERM, epithelial cell rests of Malassez; HERS, Hertwig's epithelial root sheath.

28 Sequence of events in expression of signal moleculesEpithelium cells Cap Early bell The sequential and reciprocal regulatory signaling between epithelium (red) and mesenchyme (blue) regulates the expression of specific transcription factors (boxes in side boxes). This picture is far from complete, but it illustrates the step-wise process and the reiterative use of same major signaling pathways. Missing in this schematic are modulators of signaling, in particular many signal inhibitors which add to the level of complexity. Mesenchymal cells Terminal differentiation of odontoblasts and ameloblasts Mineralization of teeth Defficiency of Pax9 and Msx results familiar tooth agenezis (hypodontia)

29 Developmental disorders of teethDisease Gene Locus Inheritance Other Emanel defects Amelogenesis imperfecta AMELX amelogenin Xp22.3-p22.1 XD 5% 14 mutations ENAM enamelin 4q13 AD MMP20 enamelysin 11.q22.3 AR Pigmented form KLK4 kallikrein 4 19q13.41 Taurodontizmus DLX3 Homeobox, BMP signal regulatory 17q21.3 Hypophosphatasia ALPL Alkalic phosphatase 1p36.1-p34

30 Therapeutic targets of cellular plasticityMerrel-Stanger, Nat. Rev. Mol. Cell. Biol

31 Characteristics of tumor cellsAngiogenezis induction Resistent to apoptosis Immortal Invasion and metastases Insensitive to antiproliferatíive signals Autonomous mitogen signal Characteristics of tumor cells (Hanahan és Weinberg 2000)

32 Sitting too much boosts cancer riskRead more:

33 Frequency of tumorous diseasesThe most important determinant of tumor formation is the age risk Risk groups: Childhood Young adult (< 30 yrs) Adult Oral tumors: 3-6%

34 Tumorigenesis – Genetical evolution (multiple hit theory)Adenomatosis polyposis coli (APC) – its inherited mutation increases dramatically the risk of adenocarcinomas 5q 12q 17p 18q A malignitás folyamata egymásra épülő progressziós lépcsöfokok 34

35 Tumor suppressor genesOncogenes dominant mutations somatic Tumor suppressor genes mutations are recessive on cellular level, but result frequently dominantly inherited predispositions Mutator genes DNA reparation germline and somatic mutations result dominant predisposition or AR tumorigenesis syndrome

36 Oncogenes turn to be active by:point mutation gene amplification chromosome translocation genome epimutation (hypomethylation, loss of imprinting – LOI)

37 Mutations characteristic to proto – oncogenes Insertion mutagenesis.Point mutation Amplification Chromosome ab. + Epigenetic effects: hypomethylation DNA rearrangement Insertion mutagenesis.

38 Point mutation - RAS H-RAS, K-RAS and N-RAS point mutation e.g. thyroid tumors

39 N-MYC gene amplification in neuroblastomaThe normal n-myc locus (2p2) and the HSR (homogeneously stained region)

40 Chromosome aberration – Reciprocal translocation Philadelphia chromosome (Ph1)ABL (Abelson cluster region) gene – coding thyrozine kinase enzyme BCR (breakpoint cluster region) New fusion protein – coded by bcr/abl Promoter of abl is lost High synthetic activity The non controlled proliferation of cells – TUMORS ( Leukemias: ALL, CML) Therapy: Gleevec (imitanib mesylate)

41 Burkitt’s Lymphoma specific translocationsTranslocation between chromosomes: 8 & 2 , 8 & , & 22 Chromosome 8 C-MYC oncogen Chr. 2, 14, 22 IG-H Immunglobulin enhancer region Oncogen activation

42 Tumor suppressors Loss- of- function mutations DeletionsSmaller – limited to one gene (p16 CDK inhibítor) Involves a full chromosome arm 3p (FHIT, RASSF1, LIMD1) – non papillary kidney cc. 1p – breast tumors Frequently AD Molecules: p53, Rb BRCA1 and 2 APC and DCC PTEN and PPA2 LKB1 p16 WT1 and WTX

43 Knudsons „two hit” hypothesis

44 Haploinsufficiency Mutation of one of the two tumorsuppressor allels -the remaining normal allel results a limited function only.

45 Inherited syndromes

46 Tumorigenesis The loss-of-function mutations are present in majorityColorectal carcinoma Prostata adenoma The loss-of-function mutations are present in majority © John Wiley & Sons, Inc. 46

47 Other factors influencing tumorigenesis I.Viruses Chemical substances (carcinogens) Azbest, vinyl-chloride and benzen Aryl carbohydrates of cigarette smoke Aflatoxin Irradiation: UV X-ray Radon, cosmic and gamma irradiations Immun-defficiencies Nutrition

48 Circular viral DNA recombination into host DNA

49 Nutrients (vegetables) can influence – decrease – the risk of tumorigenesis

50 Epigenetic factors, hypermethylated tumorsuppressor or mutator genes