1. 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!

2. Methods in Developmental Biology Research : In Situ Hybridization and Immunohistochemistry

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

4. 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

6. 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

8. 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

9. How do you “see” your probe? Radioactivity Digoxigenin + Labeled Antibody Fluorescence

10. Whole Mount in Situ

11. 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

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

13. FISH: Detecting Genomic Sequence on Chromosomes

14. FISH: X and Y Chromosome Markers in the Prostate

15. FISH allows for detection of subtle chromosome abnormalities Metaphase FISH Chromosome 4q (green) Loss of material from terminal end of one chromosome

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

17. 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

18. Conservation of patterning between flies and mammals

19. Hox Gene Expression Determines Leg Segments

20. Spectral karyotyping (SKY) -visualization of all an organisms chromosomes together each labeled with a different color -technique is useful for identifying chromosome abnormalities

21. 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

23. 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

24. 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.

25. Immunohistochemistry : Metastatic Rhabdoid Carcinoma Look for a brown precipitate!

26. 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

27. In situ hybridization of whole embryo can reveal patterns of gene expression during development RNA or DNA probes and labeled antibodies are used.

28. 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.