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February 06 Developmental biology: Drosophila segmentation and repeated units 1 * egg: generate the system * larva: eat and grow * pupa: structures in.

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Presentation on theme: "February 06 Developmental biology: Drosophila segmentation and repeated units 1 * egg: generate the system * larva: eat and grow * pupa: structures in."— Presentation transcript:

1 February 06 Developmental biology: Drosophila segmentation and repeated units 1 * egg: generate the system * larva: eat and grow * pupa: structures in larvae grow out to form adult fly: metamorphosis (Drosophila is a holometabolous insect)

2 February 06 Developmental biology: Drosophila segmentation and repeated units * Drosophila embryo: larva 3

3 February 06 Developmental biology: Drosophila segmentation and repeated units 4 Embryo stage: developmental patterning: most obvious characteristics in resulting larvae: segments Segments are partially coated with hairs: denticles Fate map How do you get from “empty bag” to organized (segmented) larva? What is required? What gene Activities? Where and When?

4 February 06 Developmental biology: Drosophila segmentation and repeated units 5 Loss of gene activity will tell us what it is required for: Mutagenize flies and screen for segmental phenotypes (Nüsslein-Volhard and Wieschaus): Idea is to find all possible genes that when ko’d could lead to a developmental anomaly… Approach: randomly mutagenize whole genome; Isolate single gene mutants; Study their phenotypes (in embryo) individual mutations created: 150 very clear segmentation phenotypes….

5 February 06 Developmental biology: Drosophila segmentation and repeated units 6 Segmental phenotypes (Nüsslein-Volhard and Wieschaus): * found 150 mutants that disrupt patterning in embryo on basis of cuticle defects. * amongst these, some have remarkable phenotypes: segments disappear in blocks gap mutants

6 February 06 Developmental biology: Drosophila segmentation and repeated units 6

7 February 06 Developmental biology: Drosophila segmentation and repeated units 7 mutant phenotype caused by loss of gene activity gap gene B In gap mutants blocks of consecutive segments are deleted. Clone gap genes: determine expression domain Area of gene expression corresponds to affected body area in mutant

8 February 06 Developmental biology: Drosophila segmentation and repeated units * From gradients to large blocks of expression 8

9 February 06 Developmental biology: Drosophila segmentation and repeated units 9 gap genes expressed in broad regions. Answer comes from group of mutants found in same mutagenesis screen: targets? all encode transcription factors:

10 February 06 Developmental biology: Drosophila segmentation and repeated units 10 Knirps protein Fushi tarazu pair rule protein knirps knirps action induces expression of one ftz stripe Only three blocks of gap gene expression: how to get seven stripes?

11 February 06 Developmental biology: Drosophila segmentation and repeated units 11 And we have functional test: in knirps mutant, stripe should disappear…. in fact kruppel mutant

12 February 06 Developmental biology: Drosophila segmentation and repeated units 12 Gap transcription factor proteins, each induce one stripe Combined action of two gap proteins (or gap + head/tail proteins) define stripe each again. Hunchback protein Kruppel protein 3To7Stripes!

13 February 06 Regulation of eve stripes 3 and 7 hb-Z kni eve 1 to 7 eve 1 and 7

14 February 06 The even-skipped (eve) stripes: a model to study how transcriptional regulation establishes positional information Regulatory elements are fused to a lacZ reporter gene and their expression is followed in transgenic flies. Eve has a long promoter element, which can be divided into regions that drive expression of various lacZ-transgenes (and indeed expression of eve) in different stripes (stripe-elements).

15 February 06 Developmental biology: Drosophila segmentation and repeated units 13 Trancript is made in nucleus: translated into cytoplasm. Transcription factor proteins are not transported out of cell: influence transcription in own nucleus In fly early embryo: transcription factor proteins can influence next door nuclei because there are no cell membranes to stop the proteins from diffusing. In fact, the diffusion is important, creates a gradient of activity: fine patterning.

16 February 06 Developmental biology: Drosophila segmentation and repeated units 14 coding region of pair rule gene Transcription regulation of typical pair rule gene: low affinity high affinity

17 February 06 Developmental biology: Drosophila segmentation and repeated units 16 pair rule genes = pair rule mutants! * in mutant embryo, every other segment is deleted. * pair rule gene expressed in seven stripes. In mutant: expression domains are deleted. * pair rule proteins are transcription factors (again)

18 February 06 Developmental biology: Drosophila segmentation and repeated units 17 Targets of pair rule transcription factor proteins? Pair rule protein segment polarity gene wingless expression wildtype paired mutant

19 February 06 Developmental biology: Drosophila segmentation and repeated units 18 How: pair rule stripes segment polarity stripes Each stripe of pair rule protein defines a stripe of segment polarity gene expression. Each segment polarity gene is thus induced by two different sets of pair rule proteins (7 + 7 makes 14). Fine tuning of stripe limits (anterior) by overlap of pair rule proteins 14 stripes of segment polarity gene expression: 14 segments!

20 February 06 Developmental biology: Drosophila segmentation and repeated units 19 So far, transcription factors were able to define areas surrounding the nuclei were gene is expressed: no cell membranes. Expression domains of genes Expression domains of proteins

21 February 06 Developmental biology: Drosophila segmentation and repeated units 20

22 February 06 Developmental biology: Drosophila segmentation and repeated units 21 Summary of early zygotic development in Drosophila embryos: Bicoid protein hunchback RNA Simple axis derived maternal system, divides egg into discrete units (mutants display loss of exactly the units). Transcription factor gradients create sets of “seven” identical units.

23 February 06 Developmental biology: Drosophila segmentation and repeated units 22 The action of the pair rule genes generates segmental stripes of expression of each segment polarity gene. In fact, pair rule protein action defines segments! Up until and including the pair rule proteins: all act within non-cellular environment, all are transcription factors. Segment polarity proteins act in cellular environment (not all transcription factors, become expressed at time of cellularization).

24 February 06 Developmental biology: Drosophila segmentation and repeated units 23 Further patterning within segments: next lecture


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