Transcript PPT - Bruce Blumberg

Bio 108 - 3/13/2000
Molecular Genetics of Pattern Formation I
•
Contact information
office hours W/F 3-4
phone 824-8573
[email protected] (preferred contact mode)
•
Lectures posted at
http://blumberg-serv.bio.uci.edu/bio108-w2000/index.htm
http://blumberg.bio.uci.edu/labtemp/bio108-w2000/index.htm
•
Many students are concerned with who will write the test
and what will be covered
– both Dr. Cho and I will contribute to the exam
• very likely that the questions will be similar to
those given in previous years
– everything in the assigned chapters is fair game
– anything lectured about is fair game
– You need to learn everything.
• ultimately, you will need to know this stuff for
MCAT and GRE
• may as well learn it now while we are here to
explain the parts that may not be completely clear
BioSci 108 lecture 26 (Blumberg) page 1
©copyright
Bruce Blumberg 2000. All rights reserved
Genesis of the body plan
•
•
•
As we have been discussing, the structure of an organism
is controlled by the action of its genes.
– mechanisms by which genes controlled development
remained a mystery until the early 1980s
Drosophila is the key system that allowed progress
– genes that regulate development were identified by
mutations that cause body parts to be absent,
duplicated or formed in inappropriate places
• famous Drosophila screen by Christianne
Nusslein-Volhardt and Eric Wieschaus (Nature,
1980 287, 795-801)
• led to the identification of the major classes of
patterning genes and categorization into a
coherent framework
– opened up the field -> Nobel prize
Why Drosophila?
– T.H. Morgan abandoned amphibians in the 1930s
because he presciently realized genetics were
required to answer major embryological questions
– small, easy to keep, short generation time
– genetics allowed identification of genetic loci long
before it was possible to clone genes
• field was ripe for picking when recombinant
DNA tools became available in late 1970s
• understanding of tools since 1980s have made it
possible for anyone to do these experiments
BioSci 108 lecture 26 (Blumberg) page 2
©copyright
Bruce Blumberg 2000. All rights reserved
Genesis of the body plan (contd)
•
•
•
Why Drosophila? (contd)
– fancy genetic techniques allow difficult questions to
be answered
• mitotic recombination and compartment
boundaries
– in situ hybridization was first perfected and widely
applied in Drosophila
• no easier organism to do in situs in than
Drosophila
– polytene chromosomes allowed relatively precise
mapping of mutations
• later advances allowed chromosomes to be
microdissected and genes in a region to be
cloned
Reward: genes identified in Drosophila turn out to
have close counterparts in virtually all animals,
including humans
Anatomy of the fly (Fig 21-47)
– adult fly has characteristic structures
– important to note that each part has characteristic
patterns of hairs and pigmentation
• allows identification of subtle differences in
mutants (much like cockroach leg bristles)
BioSci 108 lecture 26 (Blumberg) page 3
©copyright
Bruce Blumberg 2000. All rights reserved
Segmentation
•
•
•
Stages of development (Fig 21-48)
– egg -> adult takes 9 days
• very large differences between larval stages and
adult -> metamorphosis is important
• other insects have more direct development primary differences are in size, e.g.. grasshopper
– larval stages are called instars
– in the pupa, larval structures are recycled and the
adult form (imago) appears
Regions of the early embryo map to adult structures (Fig
21-49)
– but they do not actually give rise to them except
indirectly via imaginal disks
– fly larva does not show overt segmentation until
gastrulation
– at the completion of major morphogenetic
movements (~10 hrs) all of the future segments are
visible
ancestral insects had numerous identical segments (like a
millipede)
– evolution has led to the modification of certain
segments to produce specialized structures
– the segments are different from each other but built
according to a similar plan
BioSci 108 lecture 26 (Blumberg) page 4
©copyright
Bruce Blumberg 2000. All rights reserved
Segmentation (contd)
• adult fly has
– head with various appendages
• head appendages are homologous to legs
– three thoracic segments (T1-T3)
• all 3 have legs
• T2 has wing
• T3 has haltere (wing in dragonfly)
– 8-9 abdominal segments (A1-A9)
• no legs or wings
– terminal structures nonsegmented (acron and telson)
• these segments can be mapped to the larva (Fig 21-50)
– fly can be discussed in terms of segments or
parasegments
• segments correspond to physical units
• parasegments correspond to patterns of gene
expression.
– 1/2 segment out of register with physical
segments
» implies that each segment is derived from
two parasegments
– invented to explain why gene expression did
not correspond with segments
– convenient to think of embryo in terms of
parasegments when discussing patterns of gene
expression that elaborate the embryo
BioSci 108 lecture 26 (Blumberg) page 5
©copyright
Bruce Blumberg 2000. All rights reserved
Drosophila has no cells early in development
•
Fundamental weirdness of Drosophila (and other insects)
is that first 13 mitotic divisions occur without cell
division (Fig 21-51)
– end up with ~6000 nuclei in a single cell, the egg
– this means that regulatory molecules are free to
diffuse around the embryo
• fundamentally different from other animals
which always have cells
• patterning mechanisms are therefore a simplified
version of what goes on in other animals
– first nine nuclear divisions occur very rapidly and
synchronously (as with Xenopus)
• at this point the nuclei are randomly distributed
throughout the egg cytoplasm
• migrate to the surface - syncytial blastoderm
– four more divisions occur -> cell membranes form
• cellular blastoderm - after cellularization, DNA
replication slows and transcription begins
• pole cells (at the posterior end) separate a few
divisions earlier -> primordial germ cells
– as in the amphibian embryo, the rapid rate of DNA
replication and nuclear division prevents
transcription from occurring
• early development runs on mRNAs and proteins
deposited during oogenesis
• patterning is determined by maternal information
BioSci 108 lecture 26 (Blumberg) page 6
©copyright
Bruce Blumberg 2000. All rights reserved
Drosophila has no cells early in development (contd)
•
Drosophila cellular blastoderm corresponds to the hollow
blastula of the amphibian embryo
– gastrulation movements are quite different from
amphibians and mice but end result is the same:
• germ layers are formed
• endodermal cells invaginate to form the gut
• mesoderm surrounds the gut rudiment and
comes to lie between endoderm and epidermis
– cells are determined at the cellular blastoderm
• fate map using marking techniques (Fig 21-52)
• future segmented areas are outlined
• positions of most tissues are maintained,
however gut forms by invaginating at both ends
BioSci 108 lecture 26 (Blumberg) page 7
©copyright
Bruce Blumberg 2000. All rights reserved
Developmental hierarchy
•
Developmental genes in Drosophila act according to a
strict developmental hierarchy
– early acting genes divide the embryo in broad strokes
– the later acting genes sequentially refine these
subdivisions as development proceeds
MATERNAL GENES (egg polarity genes)
anterior group (e.g. bicoid)
posterior group (e.g. nanos)
terminal group (e.g. torso)
dorsoventral group (e.g. dorsal)
ZYGOTIC GENES
gap genes (e.g. hunchback)
pair rule genes (e.g. even-skipped)
segment polarity genes (e.g. engrailed)
Homeotic selector genes (e.g. antennapedia)
BioSci 108 lecture 26 (Blumberg) page 8
©copyright
Bruce Blumberg 2000. All rights reserved
Maternal genes pattern the early embryo
• Two coordinate systems are required to specify the
position of any cell in the embryo (Fig 21-52)
– four groups of egg polarity genes were identified in
searches for mutations that affect polarity
• 12 genes were identified that affect dorsoventral
(D/V) polarity
– loss-of-function in any one of 11 genes
gave dorsalized embryos (no ventral
structures)
– other one gave ventralized embryos (no
dorsal structures)
– these 12 genes are part of a pathway that is
required to set up a D/V morphogen
gradient in the early embryo
• anteroposterior genes (A/P) can be divided into
three subsystems (Fig 21-53)
– anterior group (4 genes) - head and thoracic
segments lost
– posterior group (11 genes) - abdominal
segments lost
– terminal group (6 genes) - non-segmented
structures at both ends of the embryo lost
– each of these three subsystems sets up its
own morphogen gradient
– these four primary spatial signals organize the early
embryo by setting up broad patterns interpreted by
later acting genes that refine the pattern sequentially
BioSci 108 lecture 26 (Blumberg) page 9
©copyright
Bruce Blumberg 2000. All rights reserved
Patterning genes are laid down in the egg
•
•
•
egg polarity genes are transcribed in the cells surrounding
the developing oocyte and transported there as maturation
proceeds
– transcribed from the maternal genome during
oogenesis
– products act before, or soon after fertilization
– phenotype of embryo is determined by genotype of
mother, not the embryo
– almost all recessive
mutations in these genes lead to effects two generations
downstream (Fig 21-54)
– maternal effect mutations identified by screening for
mutant embryos from apparently normal mothers
• usual mutations are dependent 50% on genotype
of father
• maternal effect mutations depend solely on
genotype of mother
– phenotype of offspring is dependent on
genotype of mother, not embryo
• homozygous offspring appear normal but are
sterile when mated with wt fathers
• alternative figure shows comparison of std and
maternal effect mutations
bottom line - components placed into the developing
oocyte by follicle cells are responsible for setting up the
initial pattern (Fig 21-55)
BioSci 108 lecture 26 (Blumberg) page 10
©copyright
Bruce Blumberg 2000. All rights reserved
Dorsovental patterning
•
•
Dorsoventral patterning involves an extracellular
signaling cascade not dissimilar to vulva induction in C.
elegans.
– actually two pathways
• a dorsally acting pathway acts to inhibit the
action of ventral gene products
• a ventrally acting pathway acts to specify ventral
and also inhibit the action of zygotic dorsalizing
factors that we will talk about next time
– all of so-called maternal dorsal group genes are
actually genes responsible for forming ventral
structures
• mutations in these dorsalize the embryo
ventralizing pathway (dorsal gene product) (supp figure)
– seven genes are required to localize the ventral
morphogen spatzle
– spatzle is the ligand for toll which is the key player
– toll is a transmembrane tyrosine kinase receptor
homologous to the IL-1 receptor
• IL-1 is required for antibody-mediated immunity
in vertebrates!
– after several steps, cactus protein is cleaved which
releases dorsal protein from the cytoplasm into the
nucleus
– dorsal is the ventral morphogen
BioSci 108 lecture 26 (Blumberg) page 11
©copyright
Bruce Blumberg 2000. All rights reserved
Dorsovental patterning (contd)
•
•
Dorsal protein acts on zygotic target genes in two ways
(Fig 21-56)
– activates expression of
• twist and snail which are required for mesoderm
and other ventral structures
– represses expression and action of
• decapentaplegic which is a dorsal morphogen
in regions where dorsal is high enough to repress dpp but
too low to activate twist, neurogenic ectoderm is formed
– this is very similar to how neural tissue is induced in
vertebrate embryos!
– the book is behind the times in its description of how
neural tissue is induced in vertebrates so wait for
Developmental Biology to worry about this
BioSci 108 lecture 26 (Blumberg) page 12
©copyright
Bruce Blumberg 2000. All rights reserved
Terminal patterning
•
terminal system is patterned through the action of a
transmembrane receptor
– torso is the key player and it is another receptor
tyrosine kinase
– torso ctivation ultimately leads to the production of
two transcription factors
• tailless - a nuclear receptor that probably
functions as a constitutive repressor
• huckebein - a zinc finger protein that can both
activate and repress transcription
– tailless is required for the formation of terminal
structures
– huckebein is required for mesoderm and endoderm
patterning
•
important point to get from the D/V and terminal systems
is that these use transmembrane signaling cascades to
specify pattern
– very much like the situation in vertebrate embryos
– very unlike the A/P patterning system
BioSci 108 lecture 26 (Blumberg) page 13
©copyright
Bruce Blumberg 2000. All rights reserved
Anteroposterior patterning
•
•
•
A/P patterning is performed by opposing morphogen
gradients that work on the same target gene
– active before the cellular blastoderm stage
– morphogens regulate gene expression directly by
diffusing along the embryonic A/P axis
Existence of an anterior and posterior morphogens was
suspected from experiments in which the embryo was
punctured and cytoplasm allowed to leak out (Fig 21-58)
– injection of anterior cytoplasm from wt embryos was
able to rescue anterior structures
– injection of anterior cytoplasm into the posterior of
wt embryos could cause duplicated anteriors
– injection of posterior cytoplasm into anterior led to
duplicated posteriors
taken together, these experiments demonstrated that the
factors which determined anterior and posterior were
located at the ends of the embryos and were diffusible
– elegant confirmation of the morphogen gradient
model
BioSci 108 lecture 26 (Blumberg) page 14
©copyright
Bruce Blumberg 2000. All rights reserved
Anteroposterior patterning (contd)
•
Posterior system involves many genes -most involved in
localizing the next most downstream gene
– mutations cause loss of:
• abdominal structures (all)
• germ cells (all but two most downstream)
– posterior and germ cell pathways branch at tudor
– apparently, each of these gene products is required
for localizing the next one in the pathway
– book mentions oskar - about in the middle of the
pathway
• one of the targets of oskar is vasa
• vasa protein found in germ cells of most species
– end result of the posterior gene cascade is the
localization of the nanos mRNA
– nanos protein diffuses toward the anterior and forms
a morphogen gradient
• specifies degree of posterior
• can replace posterior cytoplasm in injection
experiments or rescue experiments
BioSci 108 lecture 26 (Blumberg) page 15
©copyright
Bruce Blumberg 2000. All rights reserved
Anteroposterior patterning (contd)
•
•
anterior system involves only a small number of genes
– most of these are responsible for localizing bicoid
mRNA
– mutations in the bicoid gene cause the loss of
anterior structures
– increase in the number of copies of the bicoid gene
cause increases in the extent of the anterior
bicoid protein is the active anterior morphogen (fig 2159)
– concentration of bicoid protein, or the number of
bicoid genes directly influences patterning
– bicoid is a homeodomain protein that functions by
directly activating the expression of its target gene
hunchback
– wherever bicoid is injected into an embryo, head
structures form (supp figure)
• this proves that it is a true morphogen that acts
directly to pattern the anterior
– a vertebrate relative of bicoid exists, called
goosecoid
• goosecoid is involved in patterning the anterior
mesodermal tissues in the vertebrate embryo
• works by repressing transcription of target
genes, rather than activating
– can’t diffuse either since vertebrate
embryos always have cells
BioSci 108 lecture 26 (Blumberg) page 16
©copyright
Bruce Blumberg 2000. All rights reserved