Download Introduction to development

March 2, 2011
Introduction to development
Gene expression
What cellular functions are needed to
carry out development?
What cellular functions are needed to
carry out development?
Divide
Grow
Differentiate
Die
Move
Adhere
Secrete
Signal
Cell biological
properties need to
be coordinated in
space and time
Where is the “program” for development
encoded?
Where is the “program” for development
encoded?
In the genes
Other influences on development:
Maternal components
Environmental signals
Why do different cells behave differently?
Why do different cells behave differently?
Have different genes?
Have different histories?
Experience different environments?
Chance?
Have different gene expression states?
Why do different cells behave differently?
Have different genes
Have different histories
Experience different environments
Chance
Have different gene expression states
05_02_DNA.jpg
Genes
05_10_Genes_info.jpg
Carry out functions
Steps in gene
expression
07_37_Protein.produc.jpg
Regulation of gene expression
08_03_control.steps.jpg
mRNA localization control
(tether mRNA to localized proteins)
mRNA turnover control
(plant microRNAs)
Protein turnover control
(ubiquitylation and proteasome digestion)
Protein localization control
Regulation of gene expression
08_03_control.steps.jpg
mRNA localization control
(tether mRNA to localized proteins)
mRNA turnover control
(plant microRNAs)
Protein turnover control
(ubiquitylation and proteasome digestion)
Protein localization control
Regulation of transcription:
Parts of a gene
coding strand
Regulation of transcription
08_13_gene.activation.jpg
Transcription factors
Dimer
(Two
proteins)
05_24_Chromatin pack.jpg
Nucleosomes
(histones)
package DNA
Histones
affect gene
expression
Some transcription factors affect histones
08_14_chromatin.struc.jpg
Histone modifications affect gene expression
05_30_histone tails.jpg
H3K27m
4
Can affect
recruitment
of
transcription
factors to
promoter
Multiple transcription factors regulate most genes
08_15_Reg. proteins.jpg
Modularity of the Drosophila even-skipped promoter
08_18_reporter.gene.jpg
Coordinated regulation of multiple genes
Developmental functions
Signals can regulate activity of transcription factors
Maintaining gene expression states
08_23_cell.memory.jpg
(a positive feedback loop)
Maintaining gene expression states
08_24_chromatin.state.jpg
The histone code also needs to be
maintained in daughter cells
Maintaining gene expression states – DNA methylation
How does one detect which genes a
particular cell expresses?
Southern blot
10_14_1_Southrn.blotting.jpg
Southern blot – DNA on blot
Northern blot – RNA on blot
10_14_2_Southrn.blotting.jpg
Northern blot hybridization
(tests one gene at a time)
RT-PCR: 1) Reverse transcription
(tests one gene at a time)
RT-PCR: 2) Polymerase Chain Reaction
(tests one gene at a time)
RT-PCR summary
Quantitate on gel or by measuring
rate of DNA product accumulation
(tests one gene at a time)
In situ hybridization of developing flowers with ARF6 probe
Earlier stages,
longitudinal
sections
Later stages,
transverse
sections
Reveals which cells in a complex tissue express the gene
Rb-Related expression in Arabidopsis embryos
by in situ hybridization
Why is this pattern punctate (spotty)?
From Wildwater et al., Cell 123: 1337-1349 (2005)
Making reporter gene fusions
coding strand
Promoter
Reporter gene
Examples: lacZ, GUS, GFP
Insert construct into transgenic organism
PARF6::ARF6::GUS fusion expression in ovules
Promoter:protein:reporter
fusion gene
In situ hybridizations
Promoter:reporter fusion gene in a transgenic fly embryo
08_18_reporter.gene.jpg
Immunolocalization of Snail and Twist proteins in Drosophila embryos
Reveals protein location
anti-Snail
anti-Twist
Kosman et al., Science 254: 118-122 (1991)
Utility of looking at expression of single
genes at a time:
Markers of cell type, differentiation
Visualization of regulatory events
Utility of looking at expression of many
genes at once:
Global view of tissue identity
Comparing different tissues or states
Global view of regulatory events