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

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

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

Course outline

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

• Alberts et al. Molecular Biology of the Cell, 4th edition. 2002, Garland Science, NY.

Textbook

• 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

Evaluation and marking scheme

• 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%

Weight (% of final mark)

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.

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

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

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

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

Epigenesis

Homunculus: the spermist view

• = 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!

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

Life cycles for many

organisms are “standardized”

• e.g., for frogs

Decreasing blastomere size

Fertilized egg = zygote

Vigorous cell movement and rearrangement

EmbryogenesisPostembryonic development

Adu

lthoo

d

Single blastula cell = blastomere

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

• 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

Embryonic cells have predictable developmental fates

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

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

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

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

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!

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

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

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

Researchers are not limited to model species for research

• Developmental biologists can now drill down below the cell level to the networks of gene expression that control maturation

• 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)

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)

glycolysis

True vue ofmetabolism

Lipid metabolism

Nucleotide metabolism

Etc.

Pyruvate kinase

GAP dehydrogenase

Phosphoenolpyruvate carboxylase

Hexokinase

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