参考教材： 现代分子生物学（第三版），朱玉贤等。参考文献：•Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC (1998). Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature. 391: 806-11.•Lee, R. C., Feinbaum, R. L. and Ambros, V. (1993). The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 75, 843-854.•Y. Kobayashi and D. Weigel. (2007). Move on up, it's time for change—mobile signals controlling photoperiod-dependent flowering. Genes Dev 21, 2371–2384.•Laurent Corbesier, Coral Vincent, Seonghoe Jang, Fabio Fornara, Qingzhi Fan, Iain Searle, Antonis Giakountis, Sara Farrona, Lionel Gissot, Colin Turnbull and George Coupland. (2007). FT Protein Movement Contributes to Long-Distance Signaling in Floral Induction of Arabidopsis. Science 316: 1030-1033.•Margulies, Egholm, et al. (2005) Genome sequencing in microfabricated high-density picolitre reactors. Nature 437:376-380.

第十章 基因和发育

By Hongwei Guo, Peking University, 2011.12.13

第三讲 动物发育的基因调控

Fruit fly embryo development

Development• In multicellular organisms, life begins as a single

cell (zygote).• With few exceptions, somatic cells contain the

same genetic information as the zygote.• In development, cells commit to specific fates

and differentially express subsets of genes.• Cells identify and respond to their positions in

developmental fields.• Most developmental decisions involve changes

in transcription.

Pattern formation （形态建成）– Is the development of a spatial organization of

tissues and organs– Pattern formation in animals and plants results

from similar genetic and cellular mechanisms– Occurs continually in plants ， but is mostly

limited to embryos and juveniles in animals

Positional information– Consists of molecular cues that control

pattern formation

– Tells a cell its location relative to the body’s

axes and to other cells

Location, location, location!!!

Temporal information: e.g. CO, FT, FLC

Timing, timing, timing!!!

Development = Real-estate + Stock

（发育 = 房地产 + 股票）

Positional information

• Localization of mRNAs/proteins within zygote establishes positional information

----where developmental fields begin as a single cell

• Formation of concentration gradients of extracellular diffusible molecules establishes positional information in multicellular developmental fields

– works by signal transduction– diffusible molecules are known as

morphogens

Cytoplasmic determinants Differential induction

cytoskeletalelements

Polarity determination （极性确定）

• Pattern formation was extensively studied in the model organism Drosophila melanogaster---Fruit fly

• The fruit fly is small and easily grown in the laboratory.– It has a generation time of only two weeks and

produces many offspring (up to 300).

– Embryos develop outside the mother’s body.

– Sequencing of Drosophila genome was completed in 2000.

Drosophila as a genetic model

Isolation and characterization of developmental mutants

Nusslein-Volhard and Wieschaus concentrated their efforts on understanding early embryogenesis, they looked for recessive embryonic lethal mutations, and classified them according to their phenotype before death. Lewis did pioneering research on late embryogenesis, who discovered homeotic mutants - mutant flies in which structures characteristic of one part of the embryo are found at some other location.

果蝇发育的时间周期取决于温度，在 25℃ 下约9-14 天为一个周期，其中第一天为胚胎发育期。幼虫经历三个阶段，到第四天蜕皮分化后蛹化，在蛹中经过 5 天的变态，再发育为成虫。另外，还应加上受精所需的 1-2天，共约两周左右。成年果蝇存活约 9 天。

卵母细胞 (oocyte) 自身的细胞核不具转录活性，而由母源抚育细胞 (nurse cells) 、滤泡细胞 (follicle cells) 利用自身的基因和细胞资源提供遗传信息和营养物质，然后输入到卵母细胞中。这些被输入到卵母细胞的基因被称为母源影响基因（ maternal effect

genes ），对卵子的发育有重大影响。

Egg cell in the ovary is positionally determined

Bicoid mRNA, Nanos mRNA 是 nurse cell 分泌进入卵母细胞，前者与 RNA binding protein swallow, exuperantia 结合后在microtubule 作用下被铆钉在卵细胞前端； Nanos mRNA 则是与 RNA binding protein tudor, oskar 等结合被运动到了后端。此过程发生在卵细胞受精之前。

母源影响基因（ maternal effect genes ）

• Early syncitial( 合胞体 )development– zygotic nucleus divides 9 times with no cell

division• some nuclei migrate to posterior pole to give rise

to germ line

– 4 more mitotic divisions without cell division

Drosophila embryo development (1)

Drosophila development (2)

At 10 hours, 14 segments

–3 head–3 thoracic–8 abdominal

At 12 hours, organogenesis begins

At 24 hours, larva hatches

• Maternal effect genes– Encode for cytoplasmic

determinants that initially establish the axes of the body of Drosophila

Axis Establishment

Maternal effect genes

bicoid, nanos, hunchback, caudal

Drosophila larvae with wild-type and bicoid mutant phenotypes. A mutation in the mother’s bicoid gene leads to tail structures at both ends (bottom larva). The numbers refer to the thoracic and abdominal segments that are present.

Head

Wild-type mother

Tail Tail

Mutant mother (bicoid)

T1 T2T3

A1 A2 A3 A4 A5 A6A7A8

A8A7 A6 A7

A8

Tail

Phenotype of bicoid

Embryos derived from bicoid(bcd) females lack head and thorax. Cytoplasmic transplantation experiment reveal that BCD activity is localized at anterior pole of wide-type embryos.BCD activity can induce anterior development in mutant embryo at any position along antero-posterior axis and suppress posterior development.

WT bcd bcd injected with cytoplasm

Hans Georg Frohnhöfer & Christiane Nüsslein-Volhard. (1986) Organization of anterior pattern in the Drosophila embryo by the maternal gene bicoid . Nature 324, 120 - 125

x

bcd/bcd

♂

♀

+/+

+/bcd

offspring

x

bcd/bcd

♂

♀

+/+

+/bcd

offspring

? +

Gradients of bicoid mRNA and Bicoid protein in normal egg and early embryo

Translation of bicoid mRNAFertilization

Nurse cells Egg cell

bicoid mRNA

Developing egg cell

Bicoid mRNA in mature unfertilized egg

100 µm

Bicoid protein in early embryo

Anterior end

1

2

3

Properties of Bicoid• Bicoid encodes a transcription factor that includes a

homeodomain;• Bicoid transcripts were concentrated in the cortical

cytoplasm in the form of an anterior cap;• Bicoid mRNA is transported to the apical nuclear

periplasm during syncytial blastoderm; and bcd transcripts are degraded rapidly during the first third of nuclear cycle 14;

• Bcd protein gradient is dictated by the bcd mRNA gradient and its subsequent translation;

• Bicoid mRNA co-localizes with Staufen (Stau), suggesting that the Bicoid mRNA gradient forms by transport of a Stau-bcd mRNA complex.

Formation of the bicoid morphogen gradient: an mRNA gradient dictates the protein gradient

(A-I) Similarity of bcd mRNA and protein gradients. Patterns of bcd mRNA and Bcd protein are visualized by double-staining of bcd mRNA with Alexa 647 (A,D,G, green) and Bcd protein with Alexa 555 (B,E,H, red) in Drosophila embryos during interphase of nuclear cycle 5 (A-C) and 13 (D-F), and 10 minutes after onset of nuclear cycle 14 (G-I; enlarged views of the anterior of the embryo), with the merge shown to the right (C,F,I). (J-L) Colocalization of bcd mRNA and Stau protein. Drosophila embryos were stained with an anti-Stau antiserum during interphase of nuclear cycle 5 (J) and 13 (K), and 10 minutes after onset of nuclear cycle 14 (L). Inset in L is a DIC image (see Fig. 5). All embryos are oriented with anterior to the left and dorsal side up. Confocal images were taken with a Zeiss LSM Pascal (A-I) or a Leica TCS SP (J-L) microscope.

Similarity of Bcd protein, bcd mRNA and Stau gradients

High concentrations of Bicoid activate the transcription of orthodenticle in the anterior of the embryo. (D) hunchback transcription is activated at a lower threshold concentration of Bicoid, and it is therefore expressed throughout the anterior half of the embryo. (Note that the posterior stripe of hunchback is under separate regulation from the terminal system.) (E) Bicoid represses the translation of caudal mRNA to produce a posterior to anterior gradient of Caudal protein.

How the Bicoid Gradient Works?

RNA binding and translational repression.

Transcriptional activation and translational repression.

mRNA protein protein in bcd

Bicoid dependent formation of caudal protein gradient

Bicoid protein (indicated by p83/p71) can bind to Caudal mRNA (two regions:1350-

1470; 1919-2028)

32P-labelled full size Caudal mRNA + fly embryo nuclear extract

Bicoid protein suppresses translation of Caudal 3’UTR containing mRNA.

Caudal 3’UTR

GAL4

Bicoid associates with the 5′-cap-bound complex of caudal mRNA and represses translation

Autoradiogram showing that in vitro translated 35S-labeled BCD (input) is capable of interacting with m7GTP-sepharose-bound recombinant eIF4E in the presence of in vitro transcribed cad 3′-UTR.

Upon binding to the caudal mRNA motif (BRE), BCD also binds to eIF4E, disrupting the interaction between eIF4E and eIF4G, which is required for loading the ribosome onto the caudal mRNA and initiation of translation.

四种母源影响基因的mRNA 和蛋白沿果

蝇卵子和胚胎前 - 后轴分布的浓度变化图

A PBICOID

CAUDALHUNCHBACK

NANOS

Drosophila anterior-posterior axis

• Determined by gradients of BCD (product of bicoid) and HB-M (product of hunchback)

– BCD mRNA maternally deposited in anterior

– BCD mRNA tethered to “–” ends of microtubules via 3’ UTR

– HB-M protein gradient depends on NOS protein

• nos mRNA tethered to “+” end of microtubule via 3’ UTR

• NOS protein gradient blocks translation of hb-m mRNA, resulting in HB-M gradient

• opposite gradients of BCD and Nos determine A-P axis (Nos is a RNA binding protein that represses translation of HB)

?

?

• Developmental fate determined through transcription factor interactions

• Sequential activation of three sets of segmentation genes

1.Gap genes

2.Pair-rule genes

3.Segment polarity genes

Drosophila development (3)--- Segmentation Pattern ---

• Segmentation genes– Produce proteins that direct formation of

segments after the embryo’s major body axes are formed

Maternal effect genes (egg-polarity genes)

Gap genes

Pair-rule genes

Segment polarity genes

Homeotic genes of the embryo

Segmentation genesof the embryo

(segment identity genes)

Gap genes map out the basic subdivisions along the A-P axis.

• Mutations cause “gaps” in segmentation – Kruppel (Kr), knirps (Kni), Giant (Gt), Tailless (Tll),

hb-Z (zygotic Hunchback), and more– promoters have differential sensitivity to BCD

and/or HB-M

• Bifurcation of development: targets of gap gene encoded transcription factors– one branch to establish correct number of

segments– one branch to assign proper identity to each

segment

HB-Z HB-ZKR KNI

Gap gene expression--position-dependent sets of (I)transcriptional activator/repressor

and (II)post-translational protein turn over systems

1. Occurs in discrete domains and defines specific, overlapping sets of segment primordia.

2. Their protein products form broad and overlapping concentration gradients which are controlled by maternal factors and by mutual interactions between the gap genes themselves.

3. Once established, these overlapping gap protein gradients provide spatial cues which generate the repeated pattern of the subordinate pair-rule gene expression.

• Pair-rule genes define the modular pattern in terms of pairs of segments.

• Mutations result in embryos with half the normal segment number.

• Segment polarity genes set the anterior-posterior axis of each segment.

• Mutations produce embryos with the normal segment number, but with part of each segment replaced by a mirror-image repetition of some other part.

Region-specific combinations of different gap genes eventually generate the periodic pattern of pair-rule gene expression by the direct interaction with individual cis-acting "stripe elements" of particular pair-rule gene promoters. "primary" pair-rule genes (even-skipped, hairy, and runt) respond directly to gap information, they regulate the expression of the remaining so-called "secondary" pair-rule genes (fushi-tarazu, odd-skipped, paired, odd-paired, and sloppy-paired), which in turn play a more direct role in the establishment of segment-polarity gene expression. Pair-rule patterning, producing seven stripes, is a result of the action of gap genes, whose function is to define each of the seven stripes of primary pair-rule genes.pair-rule genes regulate engrailed (en) and wingless---the segment-polarity gene, resulting in the creation of the necessary fourteen evenly spaced segments on either side of the segmental border.A well-characterized target of pair-rule function is engrailed (en), which is expressed in fourteen stripes. Correct establishment of the fourteen engrailed stripes requires the activities of all the pair-rule genes. However, in general, mutations in individual pair-rule genes affect either odd- or even-numbered engrailed stripes.

The eve (even-skipped) promoter has binding sites for the proteins encoded by ：bicoid (bcd), hunchback (hb), giant (gt), Krüppel (Kr)•Binding of bicoid and hunchback proteins stimulates transcription of eve.•Binding of giant and Krüppel represses transcription.

Trapped in a valley between high levels of the giant and Krüppel proteins, expression of eve in the second stripe finally becomes limited to a band of cells only one cell thick. (A different set of promoter sites is used in the third eve stripe so expression is not repressed there.)

2nd stripe

2nd eve stripe

The pair-rule genes are expressed in striped patterns of seven bands(even-skipped) perpendicular to the anterior-posterior axis. They regulate the engrailed gene's transcription. Cells that make Engrailed can make the cell-to-cell signaling protein Hedgehog (green in lower picture). Hedgehog is not free to move very far and activates a thin stripe of cells adjacent to the Engrailed-expressing cells. Only cells to one side of the Engrailed-expressing cells are competent to respond to Hedgehog because they express the receptor protein Patched (blue in lower picture). Cells with activated Patch receptor make the Wingless protein (red in lower picture). Wingless protein acts on the adjacent rows of cells by activating its cell surface receptor, Frizzled. Wingless also acts on Engrailed-expressing cells to stabilize Engrailed expression after the cellular blastoderm forms. The reciprocal signaling by Hedgehog and Wingless stabilizes the boundary between each segment. The Wingless protein is called "wingless" because of the phenotype of some wingless mutants. Wingless also functioned during metamorphosis to coordinate wing formation.

Drosophila Segmentation• Segment number

– gap gene products activate pair-rule genes• several different pair-rule genes• expression produces repeating pattern of seven stripes,

each offset– pair-rule products act combinatorially to regulate

transcription of segment-polarity genes• expressed in offset pattern of 14 stripes

• Segment identity– gap gene products target cluster of homeotic gene

complexes• encode homeodomain transcription factors• mutations alter developmental fate of segment

– e.g., Bithorax (posterior thorax and abdomen) and Antennapedia (head and anterior thorax)

Segment Identity

• The identity of Drosophila segments is set by master regulatory genes called homeotic genes

Eye

AntennaLeg

Wild type antennapedia

• homeotic transformation ---Structures characteristic of a particular part of the animal arise in the wrong place.

bithorax

Drosophila homeotic loci ， whose genes determine the identity of body structures.

All homeotic genes of Drosophila include a 180-nucleotide sequence called the homeobox, which specifies a 60-amino-acid homeodomain, part of a transcription factor.

interesting correlation

The homeodomain is responsible solely for binding to DNA. Proteins containing homeodomains may be either activators or repressors of transcription.

• The activator or repressor domains both act by influencing the basal apparatus.

• Activator domains may interact with coactivators that in turn bind to components of the basal apparatus.

• Repressor domains also interact with the transcription apparatus. The repressor Eve, for example, interacts directly with TFIID.

Gene products in Dm embryo development

Many are homeodomain-containing transcription factors

• Homeobox-containing genes often called Hox genes in mammals conserved in animals for hundreds of millions of years.

• Related sequences are present in yeast, animals (including human), but not in plants.

• MADS box genes in plants (in flower pattern formation)

• An identical or very similar nucleotide sequence in the homeotic genes of both vertebrates and invertebrates

Adultfruit fly

Fruit fly embryo(10 hours)

Flychromosome

Mouse chromosomes

Mouse embryo(12 days)

Adult mouse

• Not all homeobox-containing genes are homeotic genes that are associated with development.

some don’t directly control the identity of body parts.

Drosophila dorsal-ventral axis• Determined by gradient of transcription

factor DL (encoded by dorsal)– gradient established by interaction of spz

and Toll gene products deposited in oogenesis and released during embryogenesis

– SPZ-TOLL complex triggers signal transduction pathway in cells that phosphorylates inactive DL

• Phosphorylated DL migrates to nucleus, activating genes for ventral fates

spätzle is a maternal effect gene

The spätzle gene encodes a component of the extracellular signaling pathway establishing the dorsal-ventral pattern of the Drosophila embryo.

Generalizations• Asymmetry of maternal gene products

establishes positional information used for early development

• Successive rounds of expression of genes encoding transcription factors establish axes and body part identity

• Positive feedback loops maintain/stabilize differentiated state

• Differences in types and concentrations of transcription factors result in different outputs

Developmental parallels

• Early animal development follows fundamentally similar pattern

• Remarkable similarity among homeotic genes– one HOM-C cluster in insects– four HOX clusters in mammals

• paralogous to insect cluster• expressed in segmental fashion in early

development

Comparison of Animal and Plant Development

• In both plants and animals– Development relies on a cascade of transcriptional

regulators turning genes on or off in a finely tuned series

• But the genes that direct analogous developmental processes– Differ considerably in sequence in plants and animals– In plants, Homeobox-containing genes do not function

in pattern formation as they do in animals– MADS-box containing genes do this part

Some animal cells may move to other sites autonomously.

Comparison of Animal and Plant Development

Plant cells are trapped in rigid cell walls made of cellulose, which prevents movement of cells and tissues.

Symmetric / asymmetric development

Cell movement

Drosophila: Anterior-Posterior axis---> Dorsal-Ventral axis

Flower: a radial patternLeave: Adaxial-Abaxial axis

思考题：

1. How to obtain bcd/bcd female flies? How to obtain bcd/bcd male flies?

2. Can you imagine how mammalian segmentation is formed? What is the earliest signal to determine the cell polarity?

3. ABC model 中的 MADS 基因和 FT 开花素在进化上都很保守，但春花基因 FLC 只在十字花科中才有，你觉得是什么原因？这个现象暗示了什么？

Francis Crick

“Always ask questions, the bigger, the better.”

Crick at Salk Institute

How did flowers evolve? Darwin called this question an “abominable mystery”.

How is cell or organ polarity initiated?

How was development evolved?---evo-devo

How to know when to stop growing?

How is epigenetics caused and preserved?