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.

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!

Methods in Developmental Biology Research : In Situ

Hybridization and Immunohistochemistry

Immunohistochemistry and in situ hybridization allow researchers to pinpoint the expression of their protein and nucleic acid targets, respectively.

Immunohistochemistry and in situ hybridization:

• Allow for specific detection of targets within tissues while maintaining the morphology of the tissue

• Identify specific cell types that are expressing the target, as well as, when the cell type is expressing that target

• Can be done in retrospect with paraffin-embedded specimens

Allows for the detection of nucleic acid sequences in cells and tissues– shows where, when, and in which

tissues a specific gene is being expressed

– can use both RNA and DNA probes– can utilize both tissue sections or whole

embryos

In Situ Hybridization

• Antisense probe– complementary strand to the mRNA– should hybridize to the target mRNA

• Sense probe– same sequence as the mRNA– should not hybridize to the target mRNA– can serve as a negative control

Choosing probes for in situ

How do you “see” your probe?

Radioactivity

Digoxigenin + Labeled Antibody

Fluorescence

Whole Mount in Situ

In Situ: Whole Mount vs. Sectioned Embryos

Whole mount in situ for Sonic Hedgehog in mouse embryos Heart section in situ for TBX5

in mouse

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

FISH: Detecting Genomic Sequence on Chromosomes

FISH: X and Y Chromosome Markers in the Prostate

FISH allows for detection of subtle chromosome abnormalities

• Metaphase FISH• Chromosome 4q (green)• Loss of material from terminal end of one

chromosome

• Interphase FISH sample• Trisomy 21 Down Syndrome• Chromosomal paint :21 (red); X (green)

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

• Puffing is where transcription is occurring.

•can be labeled with nucleic acid probes

• Can be used to determine binding site of labeled proteins

• Chromosomal rearrangments and deletions can be visualized

Conservation of patterning between flies and mammals

Hox Gene Expression Determines Leg Segments

• Spectral karyotyping (SKY)

-visualization of all an organisms chromosomes together each labeled with a different color

-technique is useful for identifying chromosome abnormalities

• SKY reveals translocations that can be missed by normal G-band staining

• Metaphase chromosome analysis using 24 chromosome paints• Unbalance translocation: material from chromosome 2 translocated to

chromosome 9

Allows for the detection of target antigens within tissue (usually proteins)

Utilizes antibodies to determine protein expression

- detects target within specific cells, gives a relative level of expression, and subcellular localization

- utilizes tissue sections and whole embryo mounts

Immunohistochemistry

Tracheal marker in Drosophilia embryo

Primary and secondary antibodies are used to detect protein of interest

Primary antibody recognizes epitope of interest (protein or protein with modification)

Secondary antibody recognizes first antibody and includes a marker visible under the microscope

Markers may be fluorescent or appear as a brown precipitate depending on method used.

Immunohistochemistry : Metastatic Rhabdoid CarcinomaLook for a brown precipitate!

immunohistochemistry

• Identifies cellular or tissue antigens by means of antigen-antibody interactions

• The site of antibody binding is identified by either direct labeling of an antibody or a secondary labeling method

• Top: JC virus• Bottom: endogenous

biotin

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

RNA or DNA probes and labeled antibodies are used.

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.