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

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

4 Embryo stage: developmental patterning: Developmental biology: Drosophila segmentation and repeated units 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? February 06 4

5 Loss of gene activity will tell us what it is required for: Developmental biology: Drosophila segmentation and repeated units 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) 40.000 individual mutations created: 150 very clear segmentation phenotypes…. February 06 5

6 Segmental phenotypes (Nüsslein-Volhard and Wieschaus): gap mutants Developmental biology: Drosophila segmentation and repeated units 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 February 06 6

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

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

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

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

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

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

12 Gap transcription factor proteins, each induce one stripe Developmental biology: Drosophila segmentation and repeated units 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 3 To 7 Stripes! February 06 12

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

13 In fly early embryo: transcription factor proteins Developmental biology: Drosophila segmentation and repeated units 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. Trancript is made in nucleus: translated into cytoplasm. In fact, the diffusion is important, creates a gradient of activity: fine patterning. February 06 13

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

pair rule genes = pair rule mutants! 16 * in mutant embryo, Developmental biology: Drosophila segmentation and repeated units 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) February 06 16

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

18 pair rule stripes How: segment polarity stripes Developmental biology: Drosophila segmentation and repeated units pair rule stripes How: 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! February 06 18

19 So far, transcription factors were able to define areas surrounding Developmental biology: Drosophila segmentation and repeated units 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 February 06 19

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

21 Summary of early zygotic development in Drosophila embryos: Developmental biology: Drosophila segmentation and repeated units 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. February 06 21

22 The action of the pair rule genes generates segmental stripes of Developmental biology: Drosophila segmentation and repeated units 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). February 06 22

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