WT wg

A>P <A loss of P <A A> P P Mirror image duplication

WT en

WTen anterior posterior

CHRISTIANE NUSSLEIN-VOLHARD Max Planck Institute Tuebingen, Germany

Saturation mutagenesis: -identify mutations in all possible genes involved in embryo patterning -saturation reached at ~5 mutations per gene

Saturation mutagenesis: -saturation reached at ~5 mutations per gene tossing marbles into bins How many bins are there? -identify mutations in all possible genes involved in embryo patterning

Saturation mutagenesis: -saturation reached at ~5 mutations per gene 15 marbles tossed into bins Probability of missing fourth bin in 15 trys: -identify mutations in all possible genes involved in embryo patterning

Saturation mutagenesis: -saturation reached at ~5 mutations per gene 15 marbles tossed into bins = Probability of missing fourth bin in 15 trys: -identify mutations in all possible genes involved in embryo patterning

Saturation mutagenesis: -saturation reached at ~5 mutations per gene 15 marbles tossed into bins = Probability of missing fourth bin in 15 trys: ~99% probability there are only 3 bins -identify mutations in all possible genes involved in embryo patterning

Genes that control pattern and polarity in the embryo: Maternal genes: 1. anterior-posterior -bicoid -nanos 2. terminal -torso 3. dorsal ventral anterior posterior dorsal ventral Zygotic genes: 1. Gap genes 2. Pair-rule genes 3. Segment polarity genes 4. Homeotic genes early late

Triple mutant: -no information nanos, torso double: -only bicoid bicoid, torso double: -only nanos bicoid, nanos double: -only torso

Maternal genes: Bicoid Hunchback Gap genes: Hunchback Kruppel Knirps Giant Pair-rule genes: Even-skipped Ftz Runt Prd Odd-skipped Segment polarity genes: Engrailed Wingless Decapentaplegic Hedgehog Homeotic genes: Ubx abdA abdB Antp

WTknirps

Knirps phenotype expression

Kruppel Mirror image duplication phenotype expression

Maternal genes: Bicoid Hunchback Gap genes: Hunchback Kruppel Knirps Giant Pair-rule genes: Even-skipped Ftz Runt Prd Odd-skipped Segment polarity genes: Engrailed Wingless Decapentaplegic Hedgehog Homeotic genes: Ubx abdA abdB Antp

Pair rule: Even-skipped phenotype expression

Hairy Runt Eve Ftz

Segment polarity: patched

WT wg

Maternal genes: Bicoid Hunchback Gap genes: Hunchback Kruppel Knirps Giant Pair-rule genes: Even-skipped Ftz Runt Prd Odd-skipped Segment polarity genes: Engrailed Wingless Decapentaplegic Hedgehog Homeotic genes: Ubx abdA abdB Antp

Lecture 3…

WT en

WT wg

knirps kruppel giant Even-skipped Gap genes Pair-rule stripes

bicoid hunchback knirps

Knirps phenotype expression

Ma et al. (1996) Development 122 (4): BICOID bindings sites in the Hunchback and Knirps enhancers The EMBO Journal (1998) 17, 5998–6009 Hunchback Knirps

The EMBO Journal (1998) 17, 5998–6009

Cell Jul 13;130(1): Cell Jul 13;130(1):

Is cooperativity sufficient? Spatial Bistability Generates hunchback Expression Sharpness in the Drosophila Embryo Francisco J. P. Lopes1,2,3*, Fernando M. C. Vieira3,4, David M. Holloway5,6,7, Paulo M. Bisch3, Alexander V. Spirov1,2 PLOS Computational Biology (2008) 4:e

BCD Hb n=~5 Positive feedback loop + Cooperative binding

Bi-stable dynamics

BCD Hb n=~5 Positive feedback loop + Cooperative binding

BCD Hb n=~5 Cooperative binding No feedback

No feedback – no bi-stability

lowering Hill co-efficient shifts curve, but still bi-stable

Giant Kruppel Giant

Wild Type bicoid Giant expression

Kruppel Mirror image duplication phenotype expression

Knirps phenotype expression

Pair rule: Even-skipped phenotype expression

knirps kruppel giant Even-skipped Concentration dependent effects of Hunchback Bicoid Fishhook

WT eve expression Stripe 2- enhancer lacZ Stripe 3+7 enhancer lacZ

WT kni hb kni tor kni hb tor Gap gene regulation of stripe 3

Stage 14 Later cellularized Knirps defines boundaries of stripe 3 and 7

Bicoid binding site deleted Expression partially restored by compensating removal of Giant repressor site

Deletion of GIANT binding sites expands band Even-skipped expression

Lecture 2 -segment polarity -wing polarity -evolution of development

WT wg

WTen anterior posterior

Segment Parasegment A P A P A P

EvenOddEven Parasegments Segments denticals wg en runt prd eve ftz prd runt ftz prd runt

Hairy Runt Eve Ftz

EvenOddEven Parasegments runt eve hairy hunchback giant kruppel bicoid

EvenOddEven Parasegments Segments denticals wg en runt prd eve ftz prd runt ftz prd runt

Hedge hog

Segment polarity: patched

En mRNA Hh protein Hh is a short range signal

wg en pppp ptc Cells on the en side secrete hh peptide, but lack the hh receptor encoded by ptc In the absence of the hh peptide wg is repressed by the ptc signalling pathway hh wg hh wg en ci

Armadillo (beta-catenin) frizzled wg TCF dsh

gene phenotypevertebrate ortholog Wingless (Wnt1) segment polarity, many others Wnt1 DWnt2Pigment cells gonads; Kozopas 1998Wnt7 adult muscle Kozopas 2002 Trachea (with wg) Llimargas 2001 DWnt3/5 Axon Guidance (through Derailed)Wnt5 Yoshikawa 2003 DWnt4Cell Movement in ovary Cohen 2002Wnt9 Dorsoventral specificity of retinal projections (Sato 2006) DWnt6 Wnt6 DWnt8Antagonist Dorsal, no ortholog immunity phenotype (Gordon et al, 2005; Ganguly et al, 2005) DWnt10 Wnt10

Gene Phenotype of Knockouts or other functions 19 mammalian Wnts Wnt1midbrain, cerebellu; neural crest derivatives; hymocyte number Wnt2placental defects Monkley, 1996 Wnt2b/13retinal cell differentiation Kubo, 2003, Kubo, 2005 Wnt3early gastrulation defect; Axis formation; Hair growth; medial-lateral retinotectal topography; hippocampal neurogenesis Wnt3a vestigial tail; neural crest; hippocampus;Segmentation oscillation clock; left right asymmetry Wnt4kidney defects; sex determination; side-branching in mammary gland; number of thymocytes migration of steroidogenic adrenal precursors into the gonad Jeays-Ward 2003 Anterior-posterior guidance of commissural axons. Wnt5a truncated limbs, truncated AP axis, reduced number proliferating cells Yamaguchi 1999 Distal lung morphogenesi; chondrocyte differentiation, longitudinal skeletal outgrowth; Inhibits B cell proliferation and functions as a tumor suppressor Defects in posterior growth of the female reproductuve tract Wnt5b Wnt6 Wnt7alimb polarity; uterine patterning during the development of the mouse female reproductive tract Delayed maturation synapses in Cerebellum Wnt7bPlacental developmen; lung hypoplasia; macrophage-induced programmed cell death Wnt8a Wnt8b Wnt9aJoint integrity Wnt9bmesenchymal to epithelial transitions Wnt10a Wnt10b decreased trabecular bone; myogenic and Adipogenic program; overexpression inhibits adipogenesis Wnt11Ureteric branching defects; cardiogenesis Wnt16

WTen anterior posterior

ptc en

dpp en ptc Cells on the en side secrete hh peptide, but lack the hh receptor encoded by ptc In the absence of the hh peptide dpp is repressed by the ptc signalling pathway hh dpp hh dpp en

ptc en

MAD signaling factor is activated by the dpp receptor: phosphorylation state of MAD forms a signaling gradient

Conservation of Hedgehog

haltere

ANTp loss of function transforms leg to antenna-like appendage

Antennapedia Wild type Antp c.a. 1949

Ubx mutant ? ?

fly Dragon fly crustacean Ubx abd A Evolution of development

~550 MYA

Segment polarity: patched

wg en runt prd eve ftz ptc

En en-  En en-  En en-  het DNA replication Mitotic Cross-over Mutant daughter cell

Creating genetic mosaic flies by mitotic recombination

WTen anterior posterior

Hunchback is sensitive to ~10% changes in BICOID protein concentration

1. The gradient is approximately (but not necessarily exactly) an exponential decay in intranuclear (and cytoplasmic) concentration that is established rapidly (less than 90 min). 2. Bcd diffuses relatively slowly (D = 0.3 mm2/s) in the cortical cytoplasm containing the nuclei. 3. The Bcd gradient is stable over nuclear cycles 10– 14, when the number of nuclei is growing by a factor of two with each division and Bcd is concentrated and released from nuclei in a dynamic process. In particular, the initial postinterphase concentration in nuclei in successive cycles is constant to at least 10%. 4. Bcd is not simply trapped in nuclei; rather, it is in dynamic equilibrium between influx and efflux with the cytoplasm and, possibly, intranuclear degradation. 5. The spatial shape of the Bcd gradient (the length constant for an exponential decay) scales with embryo length over a factor of five range in lengths in different dipteran species (Gregor et al., 2005).