Greig cephalopolysyndactyly Polydactyly - extra digits Syndactyly - webbed digits

1. Genes in Development - November 5, 2000 Karen B. Avraham, Instructor Developmental malformation syndrome Greig cephalopolysyndactyly Polydactyly - extra digits Syndactyly - webbed digits Mutation in GLI3 gene on chromosome 7 Zinc finger gene Cranial, hand abnormalities

2. Waardenburg’s syndrome Mutation in PAX3 gene on chromosome 2q35 Paired-box transcription factor gene Deafness, white forelock, iris heterochromia

3. Brief outline of human development Fertilization Pre-embryonic stage first cell division zygote reaches uterine cavity formation of bilaminar disc formation of trialaminar disc Embryonic stage cranio-caudal and dorso-vental axes established cellular aggregation and differentiation -> tissue and organ formation Fetal stage rapid growth and development

4. Developmental genes discovered through mutations Frog Fruitfly (Drosophila) Zebrafish Worm (C. elegans) Mouse spontaneous ENU-induced transgenics knock-outs

5. Ways to study genes in development In situ hybridization Whole mount Sections

6. Life begins with a single cell Reaches maturity with trillions of cells combined into complex organism with many organ systems General body plan Insect 6 legs mammals 4 legs All must differentiate the anterior from the posterior end and the dorsal from the ventral side dorsal late embryo adult anterior posterior ventral early embryo

7. During establishment of body plan, cells adopt specific cell fates Cell fates: the capacity to differentiate into particular kinds of cells Determination: process of commitment to a particular fate As cells proliferate, decisions are made to specify fate of cells Cells make developmental decisions in context of decisions made by their “neighbors” Inner ear Single fate Totipotent uncommitted Eye

8. Genetic dissection of cell fates • 15 years ago • Description of mutant phenotypes • Microsurgical manipulations of embryos • Today • Combination of genetics and recombinant DNA techniques • Can now identify protein products contributing to these developmental events • Can fish out related genes from different organisms • Same basic set of regulatory proteins govern major developmental events in all higher animals

9. Every stage of human (and other) development is controlled by genes The cell cycle Interphase G1, G2, S Cell division (mitosis) prophase, metaphase, anaphase, telophase Apoptosis (cell death) Sperm development Ovum development Germ cell formation (male and female) Fertilization Cleavage and implantation Etc…..

10. Genes involved in early development: Transcription factors • Control RNA transcription from DNA template by binding to specific regulatory DNA sequences • Switch genes on and off by activating or repressing gene expression • Control many genes involved in segmentation, induction, migration, differentiation, and apoptosis (programmed cell death) • Three gene families in vertebrates • homeotic genes • paired box genes • zinc finger genes

11. Homeotic mutation • Homeosis - replacement of one body part by another • In place of normal antennae, an Antennapedia mutation causes antennal precursor cells to develop into a leg

12. Homeobox gene clusters in humans cluster Hox 1 Hox 2 Hox 3 Hox 4 Chromosome 7p 17q 12q 2q number of genes 11 9 9 9 • Conserved 180 bp sequence - homeobox • In each Hox cluster, there is direct linear correlation between position of gene and its temporal and spatial expression • CHX10 (14q) micropthalmia (congenital blindness) in humans • Hand-foot-genital syndrome (HFGS), 7p, HOXA13 in humans • Transgenic mice have multiple severe abnormalities (face & skull)

13. Paired-box (PAX) genes • Highly conserved DNA sequence that encodes ~130 aa • First identified in Drosophila • Encode DNA binding proteins • 8 Pax genes identified in mice and humans • Mutations in Pax1 cause vertebral malformations in mice • Mutations in Pax3 causepigmentary abnormalities in mice • Mutations in Pax6 cause small eyes in mice • In humans, mutations in PAX6 cause aniridia (no iris) • In humans, mutations in PAX3 cause Waardenburg’s syndrome • (rearrangements cause rare childhood tumor,alveolar rhabdomyosarcoma)

14. Zinc finger genes • Finger-like projection formed by amino acids between 2 separated cysteine residues which form complex with zinc ion • Many DNA binding proteins contain zinc fingers • GLI3 - Greig cephalopolysyndactyly • WT1 (Wilm’s tumor gene) • Increased risk of renal malignancy/ Denyss- Drash syndrome (abnormal sexual differentiation and disordered renal development)

15. Apoptosis C. elegans Drosophila mammals Suicide of supernumary, misplaced or damaged cells Activation of evolutionarily conserved molecular program Dysfunctions implicated in developmental abnormalities and disease

16. Regulatory cascades: complex network of genes coordinate developmental pathways • Cells achieve different roles through series of “on-off” decisions • Conditions within cell allow a master switch to be regulated • Once master switch is activated, it sets in motion a cascade of “downstream” regulatory events • In absence of activation of master switch, set of default signals remain in place MASTER SWITCH or ON OFF Downstream regulatory factors induced Default regulatory factors operate New development pathway induced Default developmental pathway maintained

17. Example: Sex Determination Early Drosophila embryo • Relies on regulation of one transcription factor by another • Ratio of X chromosome to sets of autosomes (X:A ratio) in early embryo establishes whether fly becomes male or female • Sexual differentiation carried out by master regulatory switch and several downstream sex-specific genes X:A = 1 X:A = 0.5 Sx/ON Sx/OFF maintenance tra/ON tra/OFF dsx RNA dsx RNA splice splice dsx-F dsx-M protein protein Repression of -specific structural genes Repression of -specific structural genes

18. Example: Development of male germ cells Germ cells highly specialized cells for transmitting genetic information to the next generation Separated from somatic lineages at early stage of embryogenesis Germ cell specification takes place during early gastrulation Germ-line precursors give rise to primordial germ cells (PGC) Germ-line precursors located in rim of epiblast adjacent to extra-embryonic ectoderm before gastrulation PGC identified in the gastrulating mouse embryo at 7.25 days postcoitum (dpc) Proliferating PGCs migrate into genital ridges around 10.5-11.5 dpc PGCs colonizing genital ridge differentiate into precursor cells of either male or female gametes under control of cell interactions in developing gonad

19. Genes involved in formation of germ cell precursors • Germ cell precursors - pole cells • Genetic studies in Drosophila has led to discovery of genes involved • Oskar, Nanos, Tudor • Vasa • member of DEAD-box family of genes encoding ATP- dependent RNA helicase • required for assembly and function of pole plasm • identified in many animal species, where it is expressed specifically in germ-cell lineages • C. elegans - P-granules of eggs • Xenopus - germinal granules of eggs • zebrafish • mouse - Mvh • Knock-out

20. Example: Vertebrate eye development • E8.5: the optic vesicle forms as out-pouching of forebrain • E9.0: optic vesicle contacts endoderm of head • E9.5: signals from optic vesicle induce lens placode • E10.0: lens placode invaginates to lens pit; optic vesible inaginates to create optic cup • E10.5: invagination of lens pit to form lens vesicle complete. Lens vesicle detaches from overlying ectoderm • E12.5: differentiation of optic cup into neuroretina and epithelium The mouse Whole mount in situ hybridization Pax6 expression in developing mouse eye

21. Ectodermally derived eye imaginal disc • Morphogenetic furrow moves from posterior to anterior • Progress of furrow driven by wave of ommatidial differentiation Drosophila

22. Genetic pathway controlling eye development BMP4/BMP7 Pax6 lens placode Dach toy ey dac dpp Eya Six3/Optx2 so eya Drosophila Mouse/Human

23. Vertebrate genes Drosophila homolog loss of function Aniridia, small eye no lens placode no lens placode no eye phenotype in BOR Eya1-/- Holopresencephaly microphthalmia Anophthalmia Pax6 Bmp4 Bmp7 Eya1 Six3 Optx2 Dach1 eyeless, twin of eyeless Dpp 60A eyes absent sine oculis Optix dachshund

24. The Human Genome Project • As of June 26, 2000 • Finished sequence • 24% of genome • Draft sequence • 85% of genome • 38,000 predicted genes

25. Comparative Mapping and Sequencing • Saccharomyces cerevisiae (Baker’s yeast) • 1996 • 15 Mb • 6000 genes • Caenorhabditis elegans (nematode) • 1998 • 99 Mb • 19,000 genes • Drosophila melanogaster (fruitfly) • 1999 • 120 Mb euchromatic genome • 13,000 genes Sequencing of the Mouse Genome • Finished sequence 20.3 Mb • 0.65 % of genome • Draft sequence • 180 Mb • 5.8 % of genome

26. Genes in Disease and Development • Cystic fibrosis (CFTR) • Huntington’s disease (Huntingtin) • Ataxia talengiesta (ATM) • Retinoblastoma (RB1) • Wilson’s disease (ATP7B) • Gaucher’s disease (2 genes) • Deafness (> 100 genes)