第十章 基因和发育

The Genetic Basis of Development

第十章 基因和发育

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1. 拟南芥花发育的基因调控2. 果蝇胚胎发育的基因调控

Common MethodologyUse mutants to deduce developmental pathways

Drosophila

Arabidopsis

Eye

Antenna Leg

Wild type Mutant

Model organismsThe organism chosen for understanding broad

biological principles is called a model organism.DROSOPHILA MELANOGASTER

(Fruit fly)CAENORHABDITIS ELEGANS

(Nematode)

MUS MUSCULUS(Mouse)DANIO RERIO

(Zebrafish)

ARABIDOPSIS THAMANA(Arabidopsis)

No human, why?

Arabidopsis thaliana ( 拟南芥 ): a genetic model plant

The advantages of using

Arabidopsis as a model :

1. a small genome size: 125 Mb

2. a short generation time: 6-8

weeks

3. easy to grow, very small, and

produces a lot of seeds

4. self-pollination, easy to cross

5. can be easily transformed

• Transition from the vegetative to the reproductive phase.

Flower 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 ).

Transition from Vegetative toReproductive Development

Shoot Apical Meristem (SAM)

9

Flower development in Arabidopsis

Vegetative meristem

Inflorescence meristem

Floral meristem

Flower: sepals, petals, stamens,

and carpels

Transition to reproduction: Genes & other factors

Flower organ development:Organ identity genes

Flower Organs

拟南芥花的构成

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

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

ap2

ap1

pi

ap3 ag

Wild-type

class Genes

A

B

C

APETALA1 (AP1)APETALA2 (AP2)

APETALA3 (AP3)PISTILLATA (PI)

AGAMOUS (AG)

mutants

ap1ap2

ap3 pi

ag

phenotype

Eliott Meyerowitz group, Caltech, 1980s-90s

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

The ABC Model for Floral Organ Identity

A A

B B

C

Sp St CP St P Sp

C

C

(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

A

B

C

APETALA1 (AP1)APETALA2 (AP2)

APETALA3 (AP3)PISTILLATA (PI)

AGAMOUS (AG)

Loss of A function- ap1, ap2 mutants

A A

B B

C C

Sp P St C C St P Sp

B B

C C

St C C St St CC St

ap1

ap2

Loss of B function - apetala3/pistillata mutants

ap3

pi

A A

B B

C C

Sp P St C C St P Sp

A AC C

Sp C C SpSp C C Sp

Loss of C function – agamous mutant

A A

B B

C C

Sp P St C C St P Sp

A A

B B

Sp P P SpSp PP Sp

ag

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

Loss of B and C Functions-- Results in all Sepals

A A

B B

C C

Sp P St C C St P Sp

A A

Sp SpSpSpSpSp SpSp

ap3 ag

Loss of A,B, C Functions results in a ‘flower’ with leaves in place of

floral organs

A A

B B

C C

Sp P St C C St P Sp

L L L L L L L L

Thus, leaves are default structures

--- 花是由叶变态而成的最直接的证据 ---

ap1 ap3 ag

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

A A

B B

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

Exception: AP2 is expressed in all four whorls

A B C

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

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

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

35S::PI35S::AP3 c mutant

35S::PI35S::AP3 a mutant

35S::PI35S::AP3

A A

B B

C C

Sp P St C C St P Sp

PI/AP3 ： B function

A A

B B

C C

P St St PP St St P

E class: SEP1, SEP2, and SEP3 are required for B and C functions

wild typesep1 sep2 sep3

triple mutant

pi ag (BC)double mutant

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)

“ Revisionist” ABC Model

AB

CE

petallstamencarpelsepal

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

lfy

lfy + ap1ap1 ap1 + cal

Wild-type

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

IM

FM

LFY Expression

IM IM

LFY and AP1 Expression

LFY and AG Expression

IM

LFY, AP3, and PI Expression

FM precursor Sepal St + Ca Pe + St

IM: Inflorescence MeristemFM: Floral Meristem

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.

How Does LFY Activate ABC Genes?

Is this activation direct regulation?

---test of direct regulation

LFY

AP1

Activates Transcription

LFY

Protein X

Gene X

AP1

Activates Transcription

• 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

Similarly, LFY also directly regulates AG

The AG cis-regulatory elements reside in the second intron ， ~200 bp fragment

H S XB HE3

GUS2.98kbE2

• 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

44

45

Transition to reproduction

Vegetative phaseReproductive phase

Inflorescence

Flower

Factors regulating the transitions

Vegetative meristem

Inflorescence meristem

Floral meristem

• Genes (flowering-time genes and floral identity genes)

• Day length (photoperiod)• Temperature (vernalization)• Hormones (GA, etc)

LFY

A, B, C, E genes

（光周期） （春化）

Take home questions:

1. What are the common criteria for those model organisms?

2. If you want to make a plant flower early, what gene(s) will you overexpress in that species?

3. Can you turn a leaf into a floral organ? how?

4. Can you get a plant producing flowers with stamens and carpels outside while sepals and petals inside?

5. If you think ABC model is not correct or complete, what could be your evidence?