Transcript PowerPoint 프레젠테이션

Chapter 23
Homeotic mutation
Developmental
Genetics
Central themes of developmental genetics
- pattern formation, construction of complex form,
operation through developmental program
Logic of building the body plan
- Cell fate : differentiate into particular kinds of cells
: positional information  developmental field
- Fate refinement : asymmetric division  different regulatory instruction
: decision by neighboring or paracrine signals
- Specify fate option of cells of a given cell lineage in step by step manner
: totipotent  fate refinement  cell lineage
Major decisions in building the embryo
- simple binary decisions
: separation of the germ line from the soma
: establishment of the sex of the organism
- specification decisions
: establishment of the positional information
: anterior-posterior axis segment
dorsal-ventral axis germ layer
: production of the various organs, tissues, systems
Applying regulatory mechanisms to developmental decisions
- Simple developmental pathway
: the concentration of some
key molecules determine
the “on” or “off” binary choice
- making a pathway decision and
subsequently remembering that
decision are both key to cell fate
commitment
Decision making in developmental pathway
- Simple developmental pathway
: During development,
regulatory mechanisms
at any of level can control
the production of active
protein products
Regulation of protein activity
Gene regulation at levels other than transcription
Initiation: examples
Tissue-specific regulation at the
level of DNA structure
Tissue-specific amplification
of the number of copies
of a gene leads to high
levels of gene expression
in that tissue
: Eggshell gene copy number is
increased by somatic DNA rearrangement
only in the follicle cell
: each ACE region contains an amplification
control element  act as a special site for
the initiation of tissue-specific DNA
replication in follicle cells of the ovary
Structure of the two eggshell-gene clusters
in Drosophila melanigaster
Transcript processing and tissue-specific regulation
The production of an active protein can be regulated by controlling
the pattern of splicing of an initial transcript into an mRNA
Germ line specific expression of Drosophila
P elements: examples of tissue-specific
regulation by RNA splicing
a) Somatic and germ line mRNA structure
for the wild type P element
b) Modified P element transgene, P {2, 3};
in all tissues, transposase is active
Transcript processing and tissue-specific regulation
P transposase derived from a wild type P element:
P element is inactive in soma (red eye)
P transposase derives from the P { 2,3} transgene:
P transposase is active in soma (red and white)
Somatic expression of P transposase in Drosophila
containing P(w+) transposon
Posttranscriptional regulation
* A certain sequences of 3’UTR of mRNA
- regulate the degradation or the translation efficiency of mRNA
- target sites for proteins that digest mRNA molecule or that block
their translation
* A 3’UTR sequences interact with a regulatory RNA molecule
ex) in C. elegans
- premature adult development, or reiterated, producing delayed adulthood
- RNA product of the lin-4 repress translation of lin-14 mRNA
(complementarity between lin-4 RNA and 3’UTR of lin-14)
* A 3’UTR act as sites for anchoring an mRNA to particular structures
within a cell
Regulatory instructions are also contained within noncoding regions of mRNAs
Posttranslational regulation
* Enzymatic modifications of proteins
- phosphorylation
- acetylation…..
* Multiprotein complexes
- protein-protein interactions
Binary fate decisions: pathways of sex determination
* In many species, sex determination is associated with the inheritance of
a heteromorphic chromosome pair in one sex (XX-XY system)
* The XX-XY system of flies and mammals arose independently
Drosophila sex determination: every cell for itself
Every cell in Drosophila independently determines its sex
Phenotypic consequences of different x-chromosome-to-autosome ratios
Drosophila n = 4
one sex chromosome
three different autosomes
X : A > 1 or = 1 -> female
X : A < 0.5 or = 0.5 -> male
(A : number of autosome set)
Basics of the regulatory pathway
Sexual phenotype is carried out by
a master regulatory switch and several
downstream sex-specific genes
The pathway of sex determination and differentiation in drosophila
Regulatory switch; the activity of Sxl (Sex lethal) protein
1. Setting the switch in the “on” or “off” position
<The initiation & maintenance
of the Sxl switch>
- NUM (numerator gene in X ch.)
- DEM (denominator gene in
autosome)
- NUM and DEM (both bHLH)
forms dimers at random
- Only NUM-NUM dimers forms
active transcription factor
Regulatory switch
2. Maintaining the switch in a stable position
- The Sxl gene has two promoters
:- PE is active only early in embryogenesis
:- PL is active in every cell for the remainder of the life cycle
- SXL protein is an RNA-binding protein that alters the splicing
of the nascent Sxl transcript coming from late promoter
- Feedback or autoregulatory loop controlled at the level of RNA splicing
:- maintains SXL activity throughout development in flies with an X : A
ration of 1.0
Autoregulatory loop exemplifies how an early developmental decision
can be “remembered” for the rest of development, even after the
initial signals that established the decision have long disappeared
Regulatory switch
3. Propagating the decision
Two forms of tra mRNA
Different dsx mRNA are produced in both sexes
Alternative splicing of tra and dsx transcripts
Mutational analysis of Drosophila sex determination
“Treasure your exceptions”
* The effect of null mutation
- transform female into phenotypic male
- Sis-b (sisterless-b); a numerator gene
- Sxl (sex-lethal) and tra (transformer); the RNA splicing regulators
- Must be active for female development
* Dsx (doublesex)
- Dsx-/- leads to the production of flies that simultaneously
have male and female attributes
Sex determination in mammals:
coordinated control by the endocrine system
;- the presence or absence of a Y chromosome
Mammalian reproductive development and endocrine
organ control
The embryonic genital ridge consists
of a medulla surrounded by a cortex
- Female germ cells migrate into the cortex
& become organized into a ovary
- Male germ cells migrate into the medulla
& become organized into a testis
In the initial urogenital organization at the
indifferent gonad stage, precursors of
both male (Wolffian) and female (Mullerian)
ducts are present
Mammalian reproductive development and endocrine
organ control
* It is the presence or absence of a testis that determines the
sexual phenotype, through the endocrine release of testosterone
(in XY embryos lacking the androgen receptor, development
proceeds along a completely female pathway even though the
embryos have testes)
Setting the switch in the “on” or “off” position
Testis-determining factor on Y chromosome (TDF in human,
Tdy in mice), as same as SRY (human) –Sry (mice) gene
The wild type XY individual has SRY gene, which activates male
shunt pathway
Normal XX individual lacking SRY remains in the female default
pathway
Mutational analysis of mammalian sex determination
Sex reversal
- Sex reversed XX individuals are phenotypic males and
carry a fragment of the Y chromosome in their genomes
 sex reversal on Y gene (SRY)
A molecular map of the distal
part of the short arm of the
human Y chromosome
A transgenic mouse that proves Sry can cause the sex-reversal syndrome
PCR of genomic DNA shows that
Mouse 33.13 lacks a DNA marker
(Zfy-1) for Y chromosome
but, contains the Sry transgene
The external genitalia of sex-reversed
XX transgene mice 33.13 and
normal XY male sib (33.17)
Binary fate decision: the germ line versus the soma
In making the decision of germ line versus soma,
the embryo exploits its machinery for creating
asymmetries-the cytoskeleton, to localize a germ-line
determinant to a subset of early embryonic cells
Cytoskeleton of the cell
Intermediate filament (vimentin)
Microtubules (tubulin)
Microfilament (actin)
Roles: control of the location of the mitotic cleavage plane
control of the cell shape
directed transport of molecules and organelles
Cytoskeletal rods are polar structures
Different cytoskeletal system in the same cell
Intrinsic asymmetry of cytoskeletal filaments
The cytoskeleton serves as a highway system for the directed
movement of subcellular particles and organelles
;- polarity is essential
Polarity of subunits in
an actin microfilament
The distribution of tubulin in an
interphase animal fibroblast
Electron micrograph of two small
vesicles attached to a microtubule
Kinesin attaches and moves the
cargoes along the microtubules
A diagram of the kinesin protein
Movement of vesicles along microtubules
Localizing determinants through cytoskeletal
asymmetries: the germ line
The early development of C. elegans,
showing the early divisions of the zygote
The asymmetric distribution of P granules is microfilament dependent
In drosophila, microtubles provide the essential asymmetry of
germ-line determinants
Pole-cell formation at the syncitial stage of the early Drosophila embryo
Eggshell removed Drosophila embryo and longitudinal section
Forming complex pattern:
establishing positional information
Mutational analysis of early Drosophila development
Genetic assays for recessive zygotic and maternal effect mutations
The phenotypes of offspring are purely
a manifestation of their own genotype
The phenotypes of offspring are purely
a manifestation of their mother’s genotype
Cytoskeletal asymmetries and the Drosophila
anterior-posterior axis
Positional information along the A-P axis of Drosophila embryo;
creation of concentration gradients of two transcription factors
(the BCD and HB-M proteins)
BCD protein (bicoid gene product)
steeper gradient in the early embryos
HB-M protein (hunchback gene product)
shallower, longer gradient
Localization of mRNA within a cell is accomplished by
anchoring the mRNAs to one end of polarized cytoskeleton chains
* Bicoid mRNA is tethered to the – ends of microtubules (anterior pole)
* hb-m mRNA is uniformly distributed in the embryo
- nos mRNA is localized at the posterior pole
- NOS protein specifically block translation of hb-m mRNA
- produce the shallow
A-P gradient of HB-M protein
bcd mRNA
BCD protein
nos mRNA
NOS protein
The expression of the localized A-P determinant
* There are specific microtubule-association sequences located within
the 3’UTRs
- bcd mRNA 3’UTR localization sequences are bound a protein that
can also bind the – ends of the microtubules
The effect of replacement of the 3’UTR
of the nanos mRNA with the 3’UTR of the
bicoid mRNA on mRNA localization
and embryonic phenotype
Studying the BCD gradient
Genetic changes in the bcd gene alter anterior fate
Exoskeletons of larvae derived
from wild type and bcd maternal
effect lethal mutant mothers
Concentration of BCD protein
affects A-P cell fates
Left : wild type, normal phenotype
Right : bcd, anterior head and
thoracic structures missing
The position of the cephalic
furrow arises farther forward
the posterior according
to bcd+ gene dosage
Studying the BCD gradient
Bcd mRNA can completely substitute for the anterior determinant
activity of anterior cytoplasm
The bcd anteriorless mutant phenotype can be rescued by wild
type cytoplasm or purified bcd+ mRNA
Cell-cell signaling and the Drosophila dorsal-ventral axis
Positional information can be established through cell-cell signaling
by means of a concentration gradient of a secreted molecule
The D-V positional information : DL protein activity
The distribution of DL in response
to the SPZ signal (spaetzle gene product)
DL protein is in the nucleus ventrally, throughout
the cell laterally and in the cytoplasm dorsally
Dl; dorsal
Cact; cactus
Spz; spaetzle
A mature oocyte
Cell-cell signaling and the Drosophila dorsal-ventral axis
The signaling pathway that leads to the gradient of nuclear versus
cytoplasmic localization of DL proteins
The two classes of positional information
I. Localization of mRNAs within a cell
Unicellular field
II. Formation of a concentration gradient
of an extracellular diffusible molecule
Multicellular field
Morphogen; concentration-dependent
determinants of form
Forming complex pattern: utilizing positional
information to establish cell fates
Initial interpretation of positional information
Positional information ; a gradient of transcription factor activities
(AP axis: BCD & HB-M, DV axis: DL)
 Activation of cardinal genes
- the first genes to respond to the maternally supplied positional information
- the zygotically expressed genes
Review of Drosophila embryology
A syncitium-stage embryo, common cytoplasm toward the
periphery and the central yolk-field region
A cellular blastoderm embryo,
columnar cells
Changes during cellularization
Review of Drosophila embryology
3-hour Drosophila embryo
10-hour Drosophila embryo
Newly hatched larva
Segmentation identity of cells along the A-P
axis is already fated early in development
* A-P cardinal genes: gap genes
* The gap genes can be expressed in a series of distinct domains
by having promoters that are sensitive to the concentrations of
A-P transcription factors
Early blastoderm expression patterns of protein from three gap genes:
hb-z, kr, and kni
Early embryonic expression pattern of gap genes
Refining fate assignments through transcription-factor interactions
* The gap genes activate a set of secondary A-P patterning genes
; pair-rule genes in a repeating pattern of seven stripes
 the correct number of segments
Late blastoderm expression patterns
of protein from two pair-rule genes:
ftz (gray), and eve (brown)
* There is a hierarchy within the pair-rule genes
; combination of pair-rule genes
 transcriptional regulation of the segment-polarity genes
(the correct number of segments)
* Repeating expression pattern of primary pair-rule genes ?
Regulatory element complexity of the primary pair-rule genes turns
an asymmetric (gap gene) expression pattern into a repeating one
* The establishment of segmental identity
; homeotic gene complexes
- ANT-C (segmental identity in head and anterior thorax)
- BX-C (segmental identity in posterior thorax and abdomen)
* Homeosis : the conversion of one body part into another
Wild type,
one copy T2
one copy T3
A bithorax triple mutant
homozygote transforms
T3 into a second copy of T2
Homeotic gene transformation of the third thoracic segment (T3) of Drosophila into an
extra second thoracic segment (T2)
Homeotic gene-encoded protein expression pattern in Drosophila
SCR
ANTP
UBX
ABD-B
Linear arrangement of the corresponding genes along chromosome 3
Segment identity is established through asymmetric gap-gene expression patterns
that deploy and asymmetric pattern of homeotic gene expression
A cascade of regulatory events
Hierarchical cascade that activates the elements forming the A-P
segmentation pattern in drosophila
Additional aspects of pattern formation
Memory systems for remembering cell fate
Two types of positive feedback loops in maintaining the level
of activity of transcription factors determining cell fate
The transcription factor binds to
an enhancer of its own gene,
maintaining its transcription
Each adjacent cell sends out a signal that activates receptors,
signal transduction pathways, and transcription factors (TF)
expression in the other cell (cell-cell interactions)
When the fate of a cell lineage has been established, it must be remembered
Ensuring that all fates are allocated: decisions by committee
Cell-cell interactions:
one type is the ability of one cell to induce a developmental commitment
in one neighbor of many, the other is the ability of one cell to inhibit its
neighbors from adopting its fate.
Adult Caenorhabditis elegans
The production of the C. elegans
vulva from the equivalence group
by cell-cell interactions
Fate allocation can be
made through
a combination
of inductive and
lateral inhibitory
interactions
between cells
Developmental pathways are composed of plug and play modules
Differences in the developmental context of different cell lineages -that is,
the transcription factors active in these cells- permit different inputs to,
and outputs from, a given developmental circuit
Different developmental decisions are made by using mixed
and matched combinations of pathway components
The many parallels in vertebrate and insect pattern formation
The similarity between the mammalian homeobox gene cluster
(Hox complex)
and insect ANT-C and BX-C homeotic gene cluster
(HOM-C)
The comparative anatomy of the HOM-C
and Hox gene clusters
paralogous
The expression domains and regions
of the Drosophila and mouse embryos
: the arrangements of the homeotic genes is
colinear with their spatial pattern of expression
Each Hox gene is expressed in a continuous block beginning at a
Specific anterior limit and running posteriorly to the end of the
developing vertebral column
The RNA expression pattern of three mouse Hox genes in the
vertebral column of a sectioned 12.5-day-old mouse embryo:
the anterior limit of each of the expression pattern is different
The phenotypes of the homozygous knockout mice are thematically
parallel to the phenotypes of homozygous null HOM-C flies
Enlargement of the thoracic and lumbar
vertebrae of a homozygous Hoxc-8- mouse:
L1 in WT mice has no ribs
homozygous Hoxc-8- mouse
right: clenched finger
Left: normal finger
The phenotype of a homeotic mutant mouse
Evolutionary conserved pathways
The signaling pathway for activation of the drosophila DL morphogen
parallels a mammalian signaling pathway for activation of NK-kB
Do the lessons of animal development apply to plants?
The general themes for establishing cell fates in animals are likely to be seen in plants as
well. But, the participating molecules in these developmental pathways are likely to be
considerably different from those encountered in animal development.
Flower development in Arabidopsis thaliana
A series of transcriptional regulator determine the fate of the four layers of the flower
Flower-identity gene expression and
the establishment of whorl fate