Biology 3330 Molecular biology of development Winter 2008

1. Biology 3330 Molecular biology of development Winter 2008 Instructor: D. Law, CB 4018, 343 8277, dlaw@lakeheadu.ca Course website (rarely updated) http://flash.lakeheadu.ca/~dlaw/3330.html All course info regularly updated on WebCT Office hour: Tuesday 9:30-10:30 AM, or by email appointment

2. Course intent • Not to rehash information about life cycles that you learned in first year (there will only be a brief review) • We will concentrate instead on the molecular techniques used to probe the changes that occur during development • Biochemistry (proteomics), molecular biology (genomics, transcriptomics, metabolomics) • How can we visualize these changes? • Currently, these changes are defined as mostly gene expression changes: have to see differences in the expression of which macromolecule? • More crucial is the question of what is happening at the protein level since proteins do all of the work in the cell

3. Course outline Course objectives include exploration of the following concepts: • terms and concepts used in developmental biology • experimental model organisms amenable to study of developmental biology • common cross-species themes in • DNA repair and recombination • the regulation of gene expression • biochemical changes during development • adaptive responses to abiotic and biotic stresses • specific examples of the above with respect to several plant models: • potato shoot development • maize seed development • recovery and response of photosynthesis to stress • fruit ripening

4. Course outline (cont’d) • experimental laboratory methods used to examine the above questions • cell culture • epigenetics • protein:protein interactions • DNA and protein detection techniques • hot topics in molecular biology and biochemistry: genomics, protein structure, array technology, stem cells and genetic diseases • student development of real-world scientific skills • oral and presentation skills via student classroom and poster presentations • job-searching techniques and what you can do with a BSc

5. Textbook • Alberts et al. Molecular Biology of the Cell, 4th edition. 2002, Garland Science, NY. • Same text as for Cell Biology (2230) • There will also be supplemental readings that will include • Journal articles • Other material provided on reserve at the library

6. Evaluation and marking scheme Weight (% of final mark) • 5 assignments @ 4% each 20 • Oral presentation (in class) 15 • Poster presentation (early April) 15 • Course participation 7.5 • Final exam (in April) 42.5 TOTAL 100%

7. Assignment information These are geared towards • finding scientific resources relevant to molecular biology • increasing student job-hunting skills through effective CV writing • researching the scientific background of potential future supervisors (for those continuing to graduate school) or companies There will also be two developmental biology oriented assignments. Due dates for these assignments will be throughout the term. Assignments and due dates will be posted shortly on WebCT.

8. Presentation info • There will be two presentations by each student during the course • Each is worth 15% of your final mark • Both will be part of a group effort (groups of 2, different groups for each presentation) • One will be oral and take place during the lecture period, starting January 30 • These oral presentations will take up much of the course • The other is a poster presentation in the Agora in early April • Info on both of these is available on the website • Topics lists will follow shortly

9. Lecture schedule • I will lecture for the first 4 weeks, then intermittently thereafter • Much of the course will be taken up with oral presentations by students (starting Jan. 30) • Lectures will be posted online at least the evening before the date they are given

10. Why study developmental biology? • The progression of organisms through their life cycle has always been fascinating research • Many scientific questions revolve around understanding and decoding the biochemical changes that power physiological transitions between developmental stages Uncredited pictures from this lecture are from “Analysis of Biological Development,” 2nd ed., Klaus Kalthoff • e.g., the development of a mouse embryo from unfertilized egg to multicellular blastocyst

11. Embryology is a key subdiscipline of developmental biology • Embryonic development towards adult form not “linear”: the initial form taken seems alien • e.g., a 6-week human embryo is barely recognizable and has many features out of proportion in the newborn • A single fertilized cell develops into all of the cells in the individual • Cells in embryos are able to direct their fate based on positional cues and the set of instructions in their genes • Embryos build themselves one step at a time: progressively stepping through development

12. Epigenesis • = The successive building of new structures from preexisting ones • First proposed by Aristotle in the 4th century BC • Until the 17th century most biologists preferred to think of development as preformation: development = growth • Each individual is fully formed in the germ cell and then simply grows • Divided into spermists versus ovists • Anatomists pointed out that sperm, eggs or embryos do not resemble small adults! Homunculus: the spermist view

13. The animal life cycle occurs in distinct stages • Stage-specific adaptations favor survival until adulthood • Spatial and temporal cues actually govern cell differentiation and developmental progression • 3 major periods define the animal life cycle • Embryogenesis: fertilization  completion of histogenesis • Postembryonic development: period of growth between completion of organ development and adulthood • Direct versus indirect development • Adulthood: sexual maturity until death

14. Vigorous cell movement and rearrangement Life cycles for many organisms are “standardized” • e.g., for frogs Decreasing blastomere size Single blastula cell = blastomere Adulthood Fertilized egg = zygote Postembryonic development Embryogenesis

15. Modern embryology manipulated the embryo to gain clues about the function of organs • Developmental biologists research these stages by studying a new organism in the wild and the lab • Life cycle info is used to classify organisms evolutionarily and taxonomically • Experimental embryology (from the 19th century onwards) goes beyond observation and manipulates parts of the organisms • Early results indicated that cells eventually develop specific fates during embryogenesis

16. Embryonic cells have predictable developmental fates • Certain analytical strategies are used to detect these fates • One example: label a blastomere with a nontoxic dye to see where it ends up in the embryo when organ formation and histogenesis takes place • Developmental biologists favor model species that produce highly reproducible fate maps (e.g., the roundworm Caenorhabditis elegans) • Each worm contains exactly the same number of cells, allowing a very precise cell lineage (a/k/a cell fate) map to be drawn

17. When and how do cells acquire their developmental fates? The general strategy to examine development experimentally is controlled interference • Identify parameters that change the developmental process • Then change one at a time and observe any deviations from normality • Classic tools of developmental biology include hot needles, hair loops and microscopes

18. Physical strategies for determining cell fate • Destroy one of two blastomere cells with hot needle or separate by ligation with a hair-loop • non-destroyed cells each produce half-sized but viable embryos • Three strategies to confirm the developmental fate of cells: • Isolation: allow cells to develop without interacting with neighboring cells • Coculture with isolated companion cells to identify key interactions

19. The other 2 strategies of controlled interference are… • Removal: remove part as above but focus on development of remainder of embryo • Is the removed part providing a signal to the rest of the embryo guiding its developmental progression? • Transplantation: remove part of a donor and transplant elsewhere to a recipient of the • Same age • Different age • Same species • Different species

20. Many classic removal experiments focus on eye development • One is the removal of one optic vesicle to test whether it is necessary to form the eye lens • Yes, it is! Use hair-loop and glass needle to dissect and remove the optic vesicle • Genetic analysis of development was ignored by early classical (hands-on) embryologists

21. There are obvious links between genetics and developmental biology • Genetics = experimental research studying the transmission of genes between generations, specifically • The location of genes on chromosomes ( m________ ) • Description of phenotype • Raw material: mutants that possess an altered allele for a key gene • Types: • Null: loss of entire allele • Loss-of-function: reduced activity of protein • Gain-of-function: active gene when it is normally silent (usually due to alterations in regulatory region of gene) • Recall that most gene alleles are named after the mutant • for Drosophila red (wild-type) eye color, white+ = red!

22. Mutant alleles = classical embryology • These two approaches are functionally equivalent:Use genetics rather than dissecting tools to examine gene function • Embryo metabolism immediately post-fertilization largely controlled by long-lived maternal mRNAs (orange in diagram) • These include messages for proteins required for rapid cleavage: • DNA polymerase • Tubulin

23. Protein gradients determine how organisms develop • Additionally, concentration dependent messages are cytoplasmic determinants: establish differentiation of embryonic regions • Proteins encoded by these mRNAs in turn affect the expression of other mRNAs and proteins • Maternal mutations in key genes can affect survival of embryos: headless Drosophila! • Embryonic gene expression takes over during early cleavage (yellow in diagram on last slide) Wild-type Mutant

24. Homeotic mutations affect the location of body parts • These may be either loss- or gain-of-function mutations • e.g., replacement of antennae with legs (Antennapedia mutation) in Drosophila • These studies limited to species with large numbers of mutant stocks available: • Mus musculus • Danio rerio • Arabidopsis thaliana • Drosophila melanogaster • C. elegans • Not surprisingly, these represent many of the model systems of choice used in developmental biology studies

25. Researchers are not limited to model species for research • Many genes have conserved functions across phyla: can often make (tentative) conclusions on gene function in other species • Can also mimic mutations using advanced molecular techniques: these are hot! (e.g., DNA cloning; RNA interference) • Developmental biologists can now drill down below the cell level to the networks of gene expression that control maturation

26. Technological breakthroughs have powered this conceptual shift • Smaller: from frog embryos to isolated cells thanks to cell culturing, microscopy and imaging advances • Advances in molecular biology and biochemistry allow the examination of gene function (really, the function of the protein(s) encoded by the gene) and determination of the protein’s biological function in vivo • This has enabled the reductionist analysis of metabolism: putting together a picture of a complex system (metabolic pathways affecting developmental progression) from analysis of its individual parts (proteins) Hexokinase GAP dehydrogenase Pyruvate kinase Phosphoenolpyruvate carboxylase glycolysis Nucleotide metabolism Lipid metabolism Etc. True vue of metabolism

27. Reductionism must be combined with a wider view to understand metabolism • Another key reductionist view: ordered, differential gene expression is necessary for development • Genes have regulatory regions that control their expression • Important for reductionists to realize that interactions between isolated parts may also be crucial to examine • In vitro not necessarily = in vivo! (e.g.,) • A synthetic approach is necessary to keep developmental biologists’ heads away exclusively from the microscope and aware of the “big picture” • True not only in developmental biology but across disciplines