第十章基因和发育

第十章基因和发育 The Genetic Basis of Development By Hongwei Guo, Peking University, 2008.12

概述 • 遗传信息的载体—DNA和基因 • 遗传信息的传递—中心法则 • 遗传信息的调控—基因表达 • 基因表达调控的事例—疾病和发育 • 拟南芥花发育的基因调控 • 果蝇胚胎发育的基因调控

Eye Antenna Leg Wild type Mutant • Use mutants to deduce developmental pathways Common Methodology Arabidopsis Drosophila

Model organisms DROSOPHILA MELANOGASTER (Fruit fly) CAENORHABDITIS ELEGANS (Nematode) ARABIDOPSIS THAMANA (Arabidopsis) The organism chosen for understanding broad biological principles is called a model organism. MUS MUSCULUS (Mouse) DANIO RERIO (Zebrafish) No human, why?

Arabidopsis thaliana (拟南芥): a genetic model plant • The advantages of using Arabidopsis as a model : • a small genome size: 125 Mb • a short generation time: 6-8 weeks • easy to grow, very small, and produces a lot of seeds • self-pollination, easy to cross • can be easily transformed

Flower Development • Transition from the vegetative to the reproductive phase.

Transition from Vegetative toReproductive Development • Controlled by developmental and environmental signals. • This transition is called flowering(开花) or bolting (抽苔) and results in the formation of the inflorescence meristem (花序分生组织), which produces floral meristem (花分生组织).

Shoot Apical Meristem (SAM)

Flower development in Arabidopsis Vegetative meristem Inflorescence meristem Transition to reproduction: Genes & other factors Floral meristem Flower: sepals, petals, stamens, and carpels Flower organ development: Organ identity genes

Flower Organs

The floral meristem produces 4 sets of floral organ primordia in concentric rings (called whorls) • Sepals - outer ring, 1st whorl • Petals- interior to the sepals, 2nd whorl • Stamens - interior to the petals, 3rd whorl • Carpels- inner ring, 4th whorl 拟南芥花的构成

Genetic Approach to Flower Development • Look for mutants • Analyze mutant phenotypes • Characterize genetic interaction • Clone genes – analyze gene expression • Sequence – biochemical properties • Transgenic plant – gain of function

Organ Identity Mutants Wild-type ap2 pi ap1 ap3 ag

class Genes mutants phenotype APETALA1 (AP1) APETALA2 (AP2) ap1 ap2 A APETALA3 (AP3) PISTILLATA (PI) ap3 pi B C AGAMOUS (AG) ag Eliott Meyerowitz group, Caltech, 1980s-90s

B B A C C A Sp P St C C St P Sp The ABC Modelfor Floral Organ Identity (Sp) (P) (St) (C)

The ABC Model • Three classes of gene products • Combinatorial interactions to give rise to the four types of floral organs • A = sepals identity • A+B = petals identity • B+C = stamens identity • C = carpels identity • A and C mutually repress each other

Floral homeotic genes in Arabidopsis Function Gene products APETALA1 (AP1) APETALA2 (AP2) A APETALA3 (AP3) PISTILLATA (PI) B C AGAMOUS (AG)

B B A A C C Sp P St C C St P Sp B B C C St C C C St St St C Loss of A function-ap1, ap2 mutants ap2 ap1

B B A A C C Sp P St C C St P Sp A A C C Sp Sp C C C C Sp Sp Loss of B function -apetala3/pistillata mutants pi ap3

B B A A C C Sp P St C C St P Sp B B A A Sp P P Sp Sp P P Sp Loss of C function–agamous mutant ag

B B A A C C Sp P St C C St P Sp A A Sp Sp Sp Sp Sp Sp Sp Sp Loss of B and C Functions-- Results in all Sepals ap3 ag

B B A A C C Sp P St C C St P Sp Thus, leaves are default structures ---花是由叶变态而成的最直接的证据--- Loss of A,B, C Functions results in a ‘flower’ with leaves in place of floral organs ap1 ap3 ag L L L L L L L L

B B A A C C Sp P St C C St P Sp

A, B, C gene mRNA expression pattern revealed by in situ hybridization AP1 AP3 AG A B C Exception: AP2 is expressed in all four whorls

flower homeotic genes • Floral homeotic genes or floral identity gene encode MADS-domain proteins • Proteins dimerize and bind to DNA to act as transcription factors • Are found in plants, fungi, animals

B B A A C C Sp P St C C St P Sp B B A A C C P P St St St St P P PI/AP3：B function 35S::PI 35S::AP3 c mutant 35S::PI 35S::AP3 a mutant 35S::PI 35S::AP3

E class: SEP1, SEP2, and SEP3 are required for B and C functions pi ag (BC) double mutant sep1 sep2 sep3 triple mutant wild type

SEP1, SEP2, SEP3 = E class • MADS box proteins (most similar to AP1) • Have redundant function • Single mutants show subtle phenotype • Triple mutant similar to bc double mutant • Interact with B and C proteins Q: How was this triple mutant obtained? (hint: reverse genetic approach)

B A C sepal petall stamen carpel E “Revisionist” ABC Model

Meristem Identity Genes • Floral transition activates genes important for reproductive meristems, called meristem identity genes • Promote the floral meristem identity • LEAFY (LFY) • APETALA1 (AP1) with CAULIFLOWER (CAL) • APETALA2 (AP2) • Maintenance of meristem identity • TERMINAL FLOWER -- inflorescence meristem • AGAMOUS– fully committed floral meristem

Floral Meristem Mutants Wild- type lfy ap1 ap1 + cal lfy + ap1

LFY: meristem identity gene • In strong lfy mutants, sepals, petals, and stamens are placed by leaf-like organs, bracts, carpels are formed by abnormal • LFY is expressed in the inflorescence meristem that will form the floral meristem, and in the floral meristem • The LFY protein is a plant-specific transcription factor

LFY is both necessary and sufficient for ABC gene expression • In lfy mutants, AP1 expression is delayed and reduced • In lfy mutants, AP3 and PI expressed is reduced • In lfy ap1 double mutant, AG expression is abnormal • Ectopic expression of LFY can cause ectopic AP1, AP3 and AG expression (what could be the phenotype of 35S::LFY?)

A Model for LFY Function in Activating ABC Genes FM precursor FM Sepal St + Ca Pe + St IM IM IM IM LFY Expression LFY and AP1 Expression LFY and AG Expression LFY, AP3, and PI Expression IM: Inflorescence Meristem FM: Floral Meristem

How Does LFY Activate ABC Genes? So, LFY can activate AP1 Expression • To study LFY activity, a fusion of LFY to a inducible protein, GR, was made. • In the absence of the glucocorticoid hormone, GR-LFY is inactive; • When the hormone is present, GR-LFY becomes active. • When LFY-GR was inactive, AP1 expression was not activated, when LFY-GR was active, AP1 was activated.

LFY Gene X Protein X LFY AP1 AP1 Activates Transcription Activates Transcription Is this activation direct regulation?---test of direct regulation

LFY binds to cis elements of the AP1 gene • EMSA (Electrophoresis Mobility Shift Assay) • ChIP (Chromatin Immunoprecipitation) • LFY activates AP1 expression when there is no new protein synthesis • Cycloheximide treatment (inhibit translation) • Therefore, the activation of AP1 by LFY is direct

H B X H S E3 E2 2.98kb GUS Similarly, LFY also directly regulates AG • This fragment contains two LFY-binding sites • If the sites are mutated, then LFY does not bind • These mutant fragments cannot support LFY induced expression The AG cis-regulatory elements reside in the second intron，~200 bp fragment

Transition to reproduction Flower ? Inflorescence Vegetative phase Reproductive phase

Factors regulating the transitions • Genes (flowering-time genes and floral identity genes) • Day length (photoperiod) • Temperature (vernalization) • Hormones (GA, etc) Vegetative meristem Inflorescence meristem Floral meristem

（春化） （光周期） LFY A, B, C, E genes

Take home questions: • What are the common criteria for those model organisms? • If you want to make a plant flower early, what gene(s) will you overexpress in that species? • Can you turn a leaf into a floral organ? how? • Can you get a plant producing flowers with stamens and carpels outside while sepals and petals inside? • If you think ABC model is not correct or complete, what could be your evidence?

第十章 基因和发育