An Introduction to Model

Organisms of Development

Gas: 2000 liters of methane gas released/day!

What makes a good model organism?

Gas: 2000 liters of methane gas released/day!

Size : 6 tons 250kg food eaten every 100kg of elephant dung/day

Gestation : 23 months Females give birth to single offspring every five years

Sexual maturity at age 12

What makes a good model organism?

Size : 1 mm in length Live on a diet of bacteria

Gestation : 500,000 offspring in 1 week from single organism

Sexual maturity in 3 days

Genome : Sequenced!

Drosophila melanogaster

Caenorhabditis elegans

Arthropods

Nematodes

Xenopus laevisAmphibians

Mammals

Mus musculus

Homo sapiens

Caenorhabditis elegans

Nematode Worm

Nematodes account for an estimated four of every five animals in the world !

Smooth-skinned, unsegmented worms first used as a model organism by Sydney Brenner in 1965

C. elegans is diploid and has five pairs of autosomal chromosomes (named I, II, III, IV and V) and a pair of sex chromosomes (X).

Most adults are hermaphrodite (XX) but .05% of lab populations are male (XO)

Lifespan is 2 to 3 weeks

Worms are usually kept on petri plates and fed E.coli

About 10,000 worms fit on a single plate

959 Somatic cells

- all visible with microscope

- 300 neurons

- 81 muscle cells

- 131 cells undergo programmed cell death

Developmental fate of every cell is known in C. elegans

8 to 17 rounds of division are required for cell differentiation depending on tissue type and cell function

• C. elegans genome contains 19,000 genes and is fully sequenced

• 70% of human genes have worm homologues

• C. elegans will be the first and possibly only animal that we know

everything about at a cellular and molecular level

• Gene function studies have become relatively simple with the

recent discovery of RNAi

Using RNA interference for local and systemic gene silencing in C. elegans

A. C. elegans hermaphrodite expressing GFP transgenes in the pharynx and the nuclei of body-wall muscle cells

B. C. elegans hermaphrodite expressing GFP transgenes + GFP double stranded RNA in the pharynx

A. B.

Small RNAs as regulate gene expression during development

• Look for heterochronic defects in mutagenesis screen

-- cells behave as if in an earlier or later developmental stage

• Regulatory cascades unveiled which involve small RNAs (21-22 nts)

• Lin-4 and Let-7 encode short untranslated RNAs and function by binding to complementary sequences in mRNAs of specific genes controlling development

• Lin-4 expression allows cells to progress from larval stage 1 to 3

• Let-7 expression allows cells to progress from late larval to adult stages

Lin-4

Developmental mRNA

No translation of mRNA

Molecular genetic of life-span in C. elegans

C. elegans can live 2X longer under some experimental conditions: Mutations in DAF-16, AGE-1, DAF-2, or CLK-1; heat shock proteins; feeding behavior; free radical exposure and oxidative stress

Pathways involved appear to be connected

General features of longer life span include less reproduction, less growth and more DNA/cellular repair

Could be related to caloric restriction observations in mammals

Drosophila melanogaster

Common Fruit Fly

Most studied animal model

Life Cycle of 2 weeks (fertilization to sexual maturity)

Four pairs of chromosomes: the X/Y sex chromosomes and the autosomes 2,3, and 4

14,000 Genes, sequenced genome and 2/3 of human disease genes have fly homologues

Large repositories of mutant flies available

Conservation of patterning between flies and mammals

In situ hybridization of whole embryo can reveal patterns of gene expression during development

RNA or DNA probes and labeled antibodies are used.

Polytene Chromosomes

• Present in salivary glands of flies

• Originate from chromosomal duplication with no cell division

• Have patterns of dark and light bands unique for each chromosomal section visible with a light microscope

• Can be labeled with nucleic acid probes

• Can be used to determine binding site of labeled proteins

• Chromosomal rearrangments and deletions can be visualized

Antennapedia mutant: Antenna are transformed into metathoracic (second second thoracic segment) legs

Wildtype fly

Studying Organogenesis in Drosophila

Imaginal discs are groups of undifferentiated cells in larva that give rise to adult organs and structures

Transplantation and gene mis-expression studies allow characterization of organ formation at cell and molecular level

Organ-specific genes have mammalian homologues

Xenopus LaevisAfrican clawed toad

Advantages of using Xenopus as a model:

• Vertebrate model with fundamental features of land-dwelling vertebrates

• Oocytes are large and undergo external development

• Females can be stimulated to ovulate with hormones

•Development is rapid; fertilization to fully formed tadpole in a few days

Large size allows study of movement of cells within Xenopus embryos.

• Cleavage every 30 minutes

• Gastrulation at 10 hours

• 1 day to neurulation

• Germ layers and structural characteristics are easily observed

• Manipulation of embryo may involve surgery or mRNA injection

Study of Xenopus Development by Embryo Injections

• mRNAs and cDNAs can be injected to study role of genes and proteins

• Antisense to knockout expression

• Over- and mis-expression of protein of interest

• Alternate protein forms: dominant negative, constitutively active, etc.

• Reporter forms (GFP, etc.)

• Study maternal vs zygotic contributions

• Signaling molecules and chemical agents can be applied to determine affects on development

The Embryonic Signaling Center: Spemann’s Organizer

• Classic experiment first performed by Spemann and Mangold in 1924

• Grafted dorsal lip of an embryo onto a second embryo

• Gastrulation initiated at both sites

• Second whole set of body structures formed

Cell fate studies in Xenopus: Noggin

• Noggin expression permits cells to become brain and nervous system tissue

• No Noggin expression results in tissue becoming skin, bone

• Noggin is an inhibitor of BMPs which promote bone growth

• Use nucleic acid microinjection to knockout or over-express noggin

Mus Musculus

• Best model for mammalian development

• Life cycle approximately 9 weeks; 21 day gestation

• Litters up to 20 pups

• Genome sequenced

• Many inbred strains characterized (450 available)

• Genetic manipulations well developed

House Mouse

Embryos can be perturbed in various ways but give rise to normal mouse

• Allows genetic manipulation of embryo possible

• Early embryo can be split to yield two “twins”

• Two morulas can be combined to form a chimera

• Cells from an embryo can be injected into another blastocyst to form a chimera

Mouse embryos as a source of embryonic stem cells

• Culture inner cell mass (gives rise to whole embryo)

• ES cells will divide indefinitely without differentiating is cultured appropriately

• ES cells are totipotent; adult stem cells tend to be pluripotent

•Studying ES cells could lead to human therapies for various diseases

• ES cells good for genetic manipulation since whole mouse can be obtained after injection into blastocyst

Mouse embryo with Hox gene marker (created using methods described)

Motivations for understanding development: The cancer connection

• Many human disease gene homologues are required for development

• Cancer results in dedifferentiation of cells: development in “reverse”

• Embryonic lethality of knock-out mice has led to concentration on understanding mammalian development

• There are ways around lethality for studying gene function Egfr knockout: contribution of

genetic background