Transcript Pair-rule genes

参考教材： 现代分子生物学（第三版），朱玉贤等。
参考文献：
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第十章 基因和发育
第三讲 动物发育的基因调控
Fruit fly embryo development
By Hongwei Guo, Peking University, 2011.12.13
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
Temporal information: e.g. CO, FT, FLC
Development = Real-estate + Stock
（发育 = 房地产 + 股票）
Location, location, location!!!
Timing, timing, timing!!!
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
Polarity determination（极性确定）
cytoskeletal
elements
Cytoplasmic
determinants
Differential induction
Drosophila as a genetic model
• 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.
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。
Egg cell in the ovary is positionally determined
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。
母源影响基因（maternal effect genes）
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。
Drosophila
embryo
development
(1)
• 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 development (2)
At 10 hours,
14 segments
–3 head
–3 thoracic
–8 abdominal
At 12 hours,
organogenesis
begins
At 24 hours, larva
hatches
Axis Establishment
• Maternal effect genes
– Encode for cytoplasmic
determinants that initially
establish the axes of the
body of Drosophila
Maternal effect genes
bicoid, nanos, hunchback, caudal
Tail
Head
T1 T2
T3
A1 A2 A3 A4 A5
A6 A7
A8
Wild-type mother
Tail
Tail
A8
A7
Mutant mother (bicoid)
A8
A6
A7
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.
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
offspring
+/bcd
+/+
♂
x
♀
bcd/bcd
offspring
+/bcd
?
+/+
♀
x
♂
bcd/bcd
+
Egg cell
Nurse cells
1 Developing
egg cell
bicoid mRNA
2 Bicoid mRNA
in mature
unfertilized egg
Fertilization
Translation of bicoid mRNA
100 µm
3 Bicoid protein in
early embryo
Anterior end
Gradients of bicoid mRNA and Bicoid protein in
normal egg and early embryo
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
Similarity of Bcd protein, bcd mRNA and Stau gradients
(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.
How the Bicoid Gradient Works?
RNA binding and
translational repression.
Transcriptional activation and
translational repression.
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.
Bicoid dependent formation of caudal protein
gradient
mRNA
protein
protein in bcd
Bicoid protein (indicated by p83/p71) can
bind to Caudal mRNA (two regions:13501470; 1919-2028)
32P-labelled
full size Caudal mRNA + fly embryo nuclear extract
Bicoid protein suppresses translation of
Caudal 3’UTR containing mRNA.
GAL4
Caudal 3’UTR
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 m7GTPsepharose-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.
A
BICOID
NANOS
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
HUNCHBACK
CAUDAL
P
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)
?
?
Drosophila development (3)
--- Segmentation Pattern --• 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
• 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
Segmentation genes
of the embryo
Segment polarity genes
Homeotic genes of 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
KR
KNI
HB-Z
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 anteriorposterior 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" pairrule 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 pairrule 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.
2nd stripe
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 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 Engrailedexpressing 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
• homeotic transformation
---Structures characteristic of a particular part of the
animal arise in the wrong place.
• The identity of Drosophila segments is set by master
regulatory genes called homeotic genes
Eye
Antenna
Leg
Wild type
antennapedia
Drosophila homeotic
loci，whose genes
determine the identity of
body structures.
bithorax
All homeotic genes of Drosophila include a 180nucleotide 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
• Homeoboxcontaining 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
• Not all homeoboxcontaining genes are
homeotic genes that
are associated with
development.
 some don’t directly
control the identity of
body parts.
Adult
fruit fly
Fruit fly embryo
(10 hours)
Fly
chromosome
Mouse
chromosomes
Mouse embryo
(12 days)
Adult mouse
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
The spätzle gene encodes a component of the
extracellular signaling pathway establishing the
dorsal-ventral pattern of the Drosophila embryo.
spätzle is a maternal effect gene
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
Comparison of Animal and
Plant Development
Cell movement
Some animal cells may move to
other sites autonomously.
Plant cells are trapped in rigid cell
walls made of cellulose, which
prevents movement of cells and
tissues.
Symmetric / asymmetric development
Drosophila: Anterior-Posterior axis
---> Dorsal-Ventral axis
Flower: a radial pattern
Leave: 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?