Download Segmentation genes (contd)

BioSci 145A Lectures 19 - Gradients, cascades, and
signaling pathways regulate development II
•
Office hours
– tu/th 2-3
– [email protected]
– 824-8573
•
review session Thursday bring questions
– we will also go over answers to last year’s final
examination
•
evaluations will be done on Thursday as well so please
come and give us your candid assessment of the
course
•
today
– patterning by zygotic genes
– cross-regulatory interactions among segmentation
genes
– cell-cell communication
– homeotic genes
BioSci 145A lecture 19 (Blumberg) page 1
©copyright
Bruce Blumberg 2000. All rights reserved
Zygotic D/V patterning
•
D/V patterning is the same in all organisms!
– subject of a famous debate in 1830 between Georges
Cuvier and his mentor Etienne Geoffroy St.Hilaire
• Geoffroy - function follows form - one body plan
• Cuvier - form follows function - no evolutionary
connection between four distinct forms
• Cuvier considered to have won debate until 1995
– simple observation suggests that most invertebrates are
upside down relative to vertebrates
• vertebrates - dorsal nerve cord and ventral
mesenchyme
• invertebrates have a ventral nerve cord and dorsal
mesenchyme
– the same signaling pathway is initiated on the dorsal
side of flies and ventral side of vertebrates (Geoffroy)
– ventral (vertebrate) or dorsal (invertebrate) structures
are induced by members of the TGF/BMP family
• both are small, dimeric polypeptides that act
through cell-surface receptors by activating
transcription factors called SMADS
• TGFs - transforming growth factors were first
identified as proteins that caused cancers when
loss-of-function mutations occurred.
• BMBs - bone morphogenetic proteins identified as
factors from cartilage that could induce the growth
of bone when injected into animals
BioSci 145A lecture 19 (Blumberg) page 2
©copyright
Bruce Blumberg 2000. All rights reserved
Zygotic D/V patterning (contd)
BioSci 145A lecture 19 (Blumberg) page 3
©copyright
Bruce Blumberg 2000. All rights reserved
Zygotic D/V patterning (contd)
•
•
TGF/BMP family members act through specific receptors
– high-affinity receptors are heterodimers of type I and
type II receptors
– first receptor in the family to be identified was the
activin receptor
• identified by expression cloning using a radiolabeled
ligand (activin) that was purified biochemically
– Wylie Vale’s laboratory at the Salk Institute
• Xenopus versions followed soon thereafter and it
was thought for a time that these were the key
players in early development
– the author of your textbook found that the
model was sexy (but the data were weak)
Dorsal determination in Drosophila
– Drosophila dorsal morphogen is decapentaplegic (dpp)
– receptors are encoded by three genes in Drosophila
• two type I receptors
– saxophone
– thick veins (tkv)
• one type II receptor
– punt
– phenotype of tkv and pun is the same as dpp suggesting
that these two genes encode the dpp receptor
– receptors phosphorylate smads that activate transcription
• mad = mothers against decapentaplegic
BioSci 145A lecture 19 (Blumberg) page 4
©copyright
Bruce Blumberg 2000. All rights reserved
Zygotic D/V patterning (contd)
•
BioSci 145A lecture 19 (Blumberg) page 5
©copyright
mechanism and
components are
conserved in
evolution
– extracellular
components are
ligand,
antagonist and
protease
– tolloid/xolloid=
BMP1
– intracellular
components are
smads and
receptor kinase
domains
Bruce Blumberg 2000. All rights reserved
Zygotic D/V patterning (contd)
•
conservation of mechanism should give you pause
– remember in Drosophila that both dorsal and ventral
have active morphogens that antagonize each other
– Drosophila
• dorsal pathway has
– dpp that actively specifies dorsal
– gurken->torpedo pathway antagonizes ventral
• ventral pathway has
– spatzle->dorsal pathway that ACTIVELY
promotes ventral (twi, sna)
– same pathway represses dorsal by repressing
dpp and zen
– Xenopus
• ventral pathway has
– BMP4 that actively specifies ventral
• dorsal pathway has
– noggin,chordin, follistatin and cerberus that all
antagonize BMP4 and/or wnt activity
– Isn’t there something missing ???
• Homologous pathways predicts a ventrally specified
antagonist that acts against dorsal
AND
• a positively acting dorsal factor
– Is anyone looking?
BioSci 145A lecture 19 (Blumberg) page 6
©copyright
Bruce Blumberg 2000. All rights reserved
Zygotic D/V patterning (contd)
•
D/V patterning illustrates several important concepts in
developmental gene regulation
– morphogens may work both by being present and
being absent
• presence of BMP-4 -> ventral mesoderm
• absence of BMP-4 -> neural tissue
– antagonists are frequently combined with agonists to
tune response of tissues
• dpp and sog
• BMP-4, noggin, and chordin
– multiple gene products may be required to produce
the same result
• noggin, chordin, cerberus, and follistatin all act
by inhibiting BMP-4
• single KO (knockout) of chordin or noggin are
not informative
• double KO produces expected result (no head)
– extracellular proteases may be required both to
activate morphogens and degrade antagonists
• active cleavage of BMP-4 required for function
• degradative cleavage of antagonist complexes by
tolloid/xolloid/BMP-1 required for function
– multiple and varied responses can be obtained by
altering the location or concentration of players in
the pathway
BioSci 145A lecture 19 (Blumberg) page 7
©copyright
Bruce Blumberg 2000. All rights reserved
Segmentation genes
•
•
BioSci 145A lecture 19 (Blumberg) page 8
©copyright
a fate map of the embryo
may be drawn at the
cellular blastoderm stage
– segments are overt
physical separations
formed in the larva
– parasegments mirror
gene expression ->
contribute to
adjacent segments
– embryonic regions
directly correspond
to adult structures,
even though they do
not contribute to
adult structures at all
these compartments are
formed by the successive
and combinatorial
interaction of a group of
segmentation genes
– mutations cause
specific, predictable
changes in larval
structures
Bruce Blumberg 2000. All rights reserved
Segmentation genes (contd)
•
•
•
three broad groups of
segmentation genes
– defined by the type
of defect that
mutations in each
causes
– gap genes - lof
mutations cause loss
of large, adjacent
chunks of the pattern
– pair-rule genes - lof
mutations cause
pattern deletion in
alternate segments
– segment polarity
genes - lof mutations
cause mirror image
duplications
these genes are successively expressed in the early embryo
a fundamental principle is that the boundaries established
between areas of gene expression are very important for
establishing the expression of subsequent genes
– Hans Meinhardt is the major theoretician
– The Algorithmic Beauty of Seashells is a “tour de force” in
understanding patterning mechanisms (beautiful too)
BioSci 145A lecture 19 (Blumberg) page 9
©copyright
Bruce Blumberg 2000. All rights reserved
Hans Meinhardt
BioSci 145A lecture 19 (Blumberg) page 10
©copyright
Bruce Blumberg 2000. All rights reserved
Hans Meinhardt (contd)
BioSci 145A lecture 19 (Blumberg) page 11
©copyright
Bruce Blumberg 2000. All rights reserved
Hans Meinhardt (contd)
BioSci 145A lecture 19 (Blumberg) page 12
©copyright
Bruce Blumberg 2000. All rights reserved
Segmentation genes (contd)
•
•
BioSci 145A lecture 19 (Blumberg) page 13
©copyright
Temporal and spatial
hierarchy
– maternal genes establish
gradients that activate
or repress gap genes
– gap genes, and
boundaries between
them combine to set up
the pattern of 7 stripes
for the pair rule genes
– pair rule genes borders
set up segment polarity
genes
– gap and pair rule genes
are expressed before
cellularization
– segment polarity genes
at time of cellularization
most of maternal, gap and
pair-rule genes are
transcription factors
– most types represented
• zn finger (hb, kr)
• nuclear receptor
(tll, kni)
• homeodomain (eve,
ftz)
• bHLH (hairy
Bruce Blumberg 2000. All rights reserved
Segmentation genes (contd)
•
•
•
•
Principle: combinatorial interactions among a small number
of genes specifies each region of the embryo
gap genes are regulated in two ways
– respond directly to bicoid
– regulate each other
– four bands are created thusly
• bcd -> hb
• hb -> kr
• hb -| kni, gt
**figure 29-22 is mislabeled**
• nos -| hb allowing
kni and gt
posteriorly
transition to 7 stripes
– high hb -| kr
– but some hb is required
for kr hence the decline
when hb is lost
– kni requires absence of
hb
– gets complicated
general principle is that
combinatorial interactions
among proteins control
where they are expressed.
BioSci 145A lecture 19 (Blumberg) page 14
©copyright
Bruce Blumberg 2000. All rights reserved
Segmentation genes (contd)
•
•
gap genes directly regulate
transcription of pair rule
genes
– each pair rule gene is
expressed in a pattern
of 7 stripes that can be
directly traced to
combinatorial
interactions among gap
genes (example
coming)
– always expressed in
pairs
• one in even
• other in odd
parasegments
primary pair rule genes are eve and hairy – first ones expressed
– they affect expression of other pair rule genes
– expressed in stripes that are 3-4 nuclei wide
– stripes in which a gene are expressed correspond to the
regions that are missing in loss-of-function mutations
– compartments are fundamental units of morphological
development in Drosophila
• boundaries are respected
• these are set by segmentation gene expression
BioSci 145A lecture 19 (Blumberg) page 15
©copyright
Bruce Blumberg 2000. All rights reserved
Segmentation genes (contd)
•
example of how
combinatorial
regulation of pair-rule
gene expression occurs
– eve promoter has
various binding
sites for gap genes
in 480 bp
• bicoid
• hunchback
• giant
• Kruppel
– these were
identified by the
usual types of
experiments
• footprinting
• transfection
• biochemistry
– note competition
on four sites
– general rule is that broadly distributed proteins are needed
for activation (e.g. bicoid, hunchback)
– localized proteins set borders by repression (kr, gt)
BioSci 145A lecture 19 (Blumberg) page 16
©copyright
Bruce Blumberg 2000. All rights reserved
Segmentation genes (contd)
•
Generalized theoretical model for pattern formation uses a combination
of long acting and local factors
– Meinhardt shows that all complex patterns can be created by
combining the activity of a long acting repressor and local
activator (or vice versa)
– http://www.eb.tuebingen.mpg.de/abt.4/meinhardt/primary.html
– provides a computer program that you can use to alter the stability
and diffusibility of these morphogens and observe results on
patterning
•
pair rule genes are first
expressed in broad domains
that touch anteriorly and
have a gap posteriorly
– pattern is later refined
by repression
– the repression is
mediated by engrailed
protein that is
expressed as a single
cell wide stripe
BioSci 145A lecture 19 (Blumberg) page 17
©copyright
Bruce Blumberg 2000. All rights reserved
Segmentation genes (contd)
•
Segment polarity genes
– segment polarity genes are expressed at or after the
cellular blastoderm stage
– engrailed is expressed as a single cell wide stripe at
the anterior border of both eve and ftz expression
– how can it be that en is exclusively transcribed in a
single cell width?
• expression is important because en sets
boundaries of actual compartments
• engrailed is a homeodomain protein with a
strong repression domain -> dominant repressor
– two models proposed
• combinatorial model - as with gap gene control
of pair-rule genes, the overlapping expression of
pair-rule genes may regulate segment polarity
genes
• boundary model - interactions involving cell-cell
communications at boundaries are important for
causing subdivisions to arise within a
compartment (like positional confrontations)
– boundary model seems more probable
• en is initially expressed in response to ftz and eve
expression at boundaries
• establishment is followed by a completely
different maintenance phase
BioSci 145A lecture 19 (Blumberg) page 18
©copyright
Bruce Blumberg 2000. All rights reserved
Segmentation genes (contd)
•
maintenance of expression at boundaries
– en and wingless are expressed in adjacent cells
– after en expression starts an autoregulatory loop is
established that makes expression of en and wg
required for each other’s expression
– wg product is a secreted protein that induces cells to
express en
• en expression allows cells to secrete hedgehog
• hh induces wg expression in adjacent cells
– mechanism keeps the boundary sharp
• reciprocal short range interactions that stabilize
cell fate are very important in development
– e.g. in fly eyes, legs and wings
– developing or regenerating vertebrate limb
BioSci 145A lecture 19 (Blumberg) page 19
©copyright
Bruce Blumberg 2000. All rights reserved
Cell-cell communication
•
•
BioSci 145A lecture 19 (Blumberg) page 20
©copyright
many segment polarity
genes are secreted,
transmembrane, kinases,
cytoskeletal components,
suggesting that cell-cell
communication is now
important
wingless pathway is very
interesting for a variety of
reasons (Larry Marsh, Ken
Cho)
– like many others, it
begins with the
expression of an
extracellular ligand
that leads to the
expression of a
transcription factor
– unique feature:
• transcriptional
mediator armadillo is a
component of the
cytoskeleton
• interacts with
cadherins to
promote adhesion
Bruce Blumberg 2000. All rights reserved
Cell-cell communication (contd)
•
•
wingless (contd)
– arm is inactive when phosphorylated by zeste-white3
– activation of wg signaling pathway blocks
phosphorylation of arm and it now translocates to the
nucleus and heterodimerizes with pangolin to
activate transcription
a nearly exact copy of this pathway is operational in
vertebrate cells
– only the names have been changed
– remember the colon cancer example from a few
lectures ago?
– APC tumor suppressor gene normally binds to catenin (arm)
– loss of APC results in inappropriate activity of target
genes and increases cell growth
BioSci 145A lecture 19 (Blumberg) page 21
©copyright
Bruce Blumberg 2000. All rights reserved
Homeotic genes
•
Homeotic genes control the identity of the particular
segments in which they are expressed
– homeotic mutations “transform” one compartment
into another
– practically speaking, this means that they transform
one body part into another
– virtually all are transcription factors that regulate
each others expression as well as other target genes
• most are homeodomain proteins
– homeotic genes interpret the information laid down
by the segmentation genes.
• expression coincides with the peak expression of
the segment polarity genes
– phenotype of mutation in one homeotic locus
depends not only on the gain or loss of function in
that gene but also how other homeotic genes alter
their expression to in response to the loss.
• genetic loci are large and complex with multiple
promoters and enhancers operative in particular
temporal and spatial patterns.
– two major loci in Drosophila (HOM-C)
• bithorax - BX-C
• Antennaedia - Ant-C
– these are together in one large complex in most
insects and other animals including vertebrates
(HOX-C)
BioSci 145A lecture 19 (Blumberg) page 22
©copyright
Bruce Blumberg 2000. All rights reserved
Homeotic genes (contd)
BioSci 145A lecture 19 (Blumberg) page 23
©copyright
Bruce Blumberg 2000. All rights reserved
Homeotic genes (contd)
•
•
Homeotic genes lead to a sequential elaboration of pattern
– complex is transcribed in one direction
– each gene in the complex acts on a successively more
posterior region of the animal
– formation of a particular compartment (structure)
depends on the expression of all preceding genes and a
new activity encoded by the next gene in the cluster
• loss-of-function mutations transform the segment
toward the next more anterior
• gain-of-function transform segments toward the next
more posterior structure
Ant-C patterns head through T2
– many weird mutations all put legs where other
structures belong
• antennapedia
• proboscipedia
• nasobemia
– antp is an example of a gene that is required to suppress
the formation of head structures
• lof alleles transform T2-> T1
• gof alleles transform antenna->leg
BioSci 145A lecture 19 (Blumberg) page 24
©copyright
Bruce Blumberg 2000. All rights reserved
Homeotic genes (contd)
•
BX-C patterns T2 though the abdomen
– only 3 proteins are encoded by BX-C
• how can three proteins specify the identify of 10
segments?
• two RNAs that do not encode proteins are also
produced
– mutations in these RNAs cause phenotypes!
– some recent data from other systems
suggesting that RNAs can act as
transcriptional coactivators
– numerous cis-acting mutations throughout the
complex
• many genetic loci are really regulatory
sequences in one of the three coding units
– BX-C loss of function is lethal
• segments all look like T2 (not unlike ancestral
insect)
• like Ant-C, BX-C acts to refine structure in terms
of a combinatorial code
• mutations in cis acting sequences cause
inappropriate gain or loss of function in
particular compartments
BioSci 145A lecture 19 (Blumberg) page 25
©copyright
Bruce Blumberg 2000. All rights reserved
Homeotic genes (contd)
BioSci 145A lecture 19 (Blumberg) page 26
©copyright
Bruce Blumberg 2000. All rights reserved
Homeotic genes (contd)
•
Homeodomain is highly conserved
– vertebrates have Hox complexes (HOX-C)
– typical vertebrate has four
• four genome duplications in vertebrate lineage
• most fish have had an additional duplication and
may have 7-8 Hox complexes.
– genes at the same position in different clusters are
called paralogs (e.g. Hox-A1, B1, D1)
BioSci 145A lecture 19 (Blumberg) page 27
©copyright
Bruce Blumberg 2000. All rights reserved
Homeotic genes (contd)
•
Hox genes are clustered
– organized and transcribed in the same direction
along the chromosome
– like HOM-C genes, the downstream genes in the
cluster specify more posterior compartments
– all homeodomains in HOM-C and HOX-C have the
same in vitro DNA-binding specificity
• corresponding vertebrate gene can rescue
Drosophila mutations
• specificity must be conferred by flanking
sequence and context of proteins expressed in
the cell
• depending on set of proteins expressed, various
Hox genes can be transcriptional activators or
repressors
– these genes are considered to act together to specify
a Hox code that determines position along the A/P
axis
• gain- and loss-of-function experiments
completely support this model
• some functional redundancy exists between
paralogs
– knockouts do not completely ablate
structures expressing the gene
BioSci 145A lecture 19 (Blumberg) page 28
©copyright
Bruce Blumberg 2000. All rights reserved
Homeotic genes (contd)
BioSci 145A lecture 19 (Blumberg) page 29
•
Hox-C and HOM-C genes
are expressed at similar
positions in the
developing embryo
– implies that the Hox
code is an ancient and
successful mechanism
to specify position
•
still an open question why
the organization of the
clusters has not changed
– it was once thought
that there were a few
“master promoters”
that transcribed the
entire complex at one
time
– promoter bashing has
identified control
elements for each
gene but it remains
unclear why no
rearrangements are
tolerated
©copyright
Bruce Blumberg 2000. All rights reserved
Summary
•
genetic control of patterning
– combinatorial interactions among transcription
factors is the primary mechanism for determining
position and establishing pattern in the body
– genes and mechanisms are highly conserved among
all animals
– pattern is sequentially elaborated
• early acting genes divide the embryo in broad
strokes
• later genes refine this pattern
– repression is an important means of patterning
• it wasn’t so long ago that many researchers
believed that repression was not a viable
patterning mechanism
– reciprocal interactions at boundaries of gene
expression are a primary means of refining and
subdividing patterns
– patterning genes regulate the expression of other
patterning genes as well as structural genes
– many patterning pathways are aberrantly regulated in
various cancers
BioSci 145A lecture 19 (Blumberg) page 30
©copyright
Bruce Blumberg 2000. All rights reserved