Lecture 3 (1b) Segmentation in Drosophila melanogaster Flashcards
Aristotle - 6th century
Preformationism
• adult is pre-formed in egg or sperm
• all organisms were generated at the same time
(Homunculus - curled up in the head of each sperm)
Epigenesis
organisms are progressively built anew in each generation
in contrast to preformationism
DNA gives the instructions
The nucleus of the zygote and every cell that is produced during ontogenesis contains the complete genome
In principle, every cell…
somatic and germ line cells…
can be used to make a new animal
Differentiation - making cells different
The genotype contains a developmental program that unfolds and results in the expression of different sets of genes in different cell types
BY REGULATION OF GENE EXPRESSION
Factors that control gene expression regulate:
- chromatin structure
- initiation of transcription
- RNA processing
- initiation of transcription
In the context of axes formation and segmentation in Drosophila, we are discussing the regulation of
transcription by transcription factors
Chromatin-modifying enzymes provide initial control of gene expression by…
making a region of DNA either more or less able to bind the transcription machinery
• transcription factors then control levels of transcription
(Organization of eukaryotic genes)
Associated with most eukaryotic genes are control elements
segments of noncoding DNA that help regulate transcription by binding certain transcription factors
(Organization of eukaryotic genes)
Control elements and the transcription factors they bind are critical to the
precise regulation of gene expression in different cell types
Eukaryotic gene
a gene is a section of DNA required for the production of a biologically active RNA molecule
(Organization of eukaryotic genes)
Proximal control elements are located close to the
promoter
(Organization of eukaryotic genes)
Distal control elements, groups which are called … may be far away from a gene or even located in an intron
enhancers
Binding of … to … control elements regulates gene expression
Binding of TRANSCRIPTION FACTORS to ENHANCER control elements regulates gene expression
(The roles of transcription factors)
To initiate transcription, eukaryotic RNA polymerase requires the assistance of proteins called
transcription factors
(The roles of transcription factors)
General transcription factors are essential for the transcription of
all protein-coding genes
(The roles of transcription factors)
In eukaryotes, high levels of transcription of particular genes depend on control elements interacting with
specific transcription factors
(The roles of transcription factors)
A given gene may have different control elements (enhancers). Expression depends on the complement of
activator or inhibitor transcription factors present in the cell that can bind to theses control elements
General transcription factors are essential for the transcription of all
protein-coding genes
• TATA box binds TATA BINDING PROTEIN necessary for efficient RNA synthesis
• promoter elements bind transcription factors to help stabilize the RNA TRANSCRIPTION PROCESS
Enhancer elements are brought into contact with the
basal promoter
Basal transcription factors are required for
RNA polymerase II
to bind to promoter regions and initiate transcription
• cell specific transcription factors increase or decrease complex stability, thus increasing or decreasing gene expression
Liver cell nucleus
available activators for albumin =
albumin gene expressed
& crystalline gene not expressed
Lens cell nucleus
available activators for crystalline =
crystalline gene expressed
& albumin gene not expressed
Dorsal protein activates and represses different zygotic genes depending on
twist (?) nuclear concentration
Genes downstream of Dorsal activate other genes that further
subdivide cell fate
Dorsal structures are missing in
dpp mutant embryos/larvae
• we are analyzing the dpp H46 line
• • all structures above the H46 line are absent in the mutant embryos/larvae
Dpp mutant - weak phenotype
deletions of dorsal-anterior structures
Dpp mutant - strong phenotype
- dorsal expansion of denticle belts up to denticle belts encircling the whole embryo
- only patches of dorsal tissue present
In twist mutant embryos
ventral structures do not develop
• dorsalized embryos - all cells from dorsal cuticle
• embryos twisted
The anterior-posterior and dorso-ventral morphogens subdivide the embryo into
head, thorax, and abdomen
dorsal/ventral side
(respectively)
In Drosophila, a segmentation gene cascade results in the formation of segments
- maternal effect genes
- Gap genes
- pair-rule genes
- segment polarity genes
Activation of gap genes depends on the
concentrations of morphogens
Gap gene expression patterns
• established by MATERNAL MORPHOGENS
• maintained by MUTUAL REPRESSION
- high levels of Bicoid and Hunchback activate Giant
- Low levels of Hunchback activate Kruppel
- Caudal activates Knirps and Giant
Segmentation genes
gap genes
pair-rule genes
segment polarity genes
Pair-rule gene expression patterns divide the embryo into
precursors of the segmental units
Primary pair-rule genes are expressed before
secondary pair-rule genes
Primary pair-rule genes
- hairy
- even-skipped
- runt
Secondary pair-rule genes
- fushi tarazu
- odd-paired
- odd-skipped
- sloppy-paired
- paired
4 enhancers drive expression of
individual even-skipped stripes
Gap proteins regulate
pair-rule gene expression
Odd-skipped mutant phenotype
deletion of denticle belts in odd-numbered segments
Morphologically visible segments…
do NOT coincide with pair-rule gene expression
Segments contain
- posterior part of parasegment in front
* anterior part of parasegment behind
Segment polarity genes - members of the Wingless and Hedgehog signal transduction pathway
- engrailed
- wingless
- cubitus interruptus
- hedgehog
- fused
- armadillo
- patched
- gooseberry
- pangolin
Engrailed is activated in cells that show high concentrations of either
- Even-skipped
* Fushi tarazu
The expression of wingless is maintained by
interactions of the products of these genes
Engrailed expressed
posterior to wingless expression
Wingless also determines the cell fate in
epidermal cells anterior to engrailed-expresing genes
Wingless cuticle phenotypes
- fusion of denticle belts
* naked cuticle missing
In the practical, we use
- 4 promoter lacZ constructs
* in the embryo, the protein β-galactosidase is produced
β-galactosidase is only expressed in cells that
express the endogenous ftz gene
2-step indirect antibody staining
- primary antibody
- secondary antibody (clings to first)
- -> stainint
The anterior-posterior and dorso-ventral axes are established by
maternal morphogens
The segmentation gene cascade subdivides the embryo into smaller units -
parasegments and segments
