Transcript SHH - Faculty Bennington College

MUTANTS
genetic variation in human development
Lecture 7
Fall 2006
Bennington College
Overall comments for Manar Maget/Bodyshock Essay
Really great work - amazing variety of public
health issues raised
great insights and discussion
of Manar’s situation and the
Bodyshock program in general
things to work on:
Attention to detail - make sure to address/answer all
of the questions the assignment asks for.
PLEASE read over your paper BEFORE you print it out.
Nothing says “I couldn’t be bothered to check my work
ahead of time” like hand-written corrections at the last
minute…
For the sake of my quickly declining eyesight, please
at least 1.5 space everything (I still have no vision plan…)
POLYDACTYLY
(aka “digital enhancement”)
The “normal” number of digits varies
greatly between vertebrate species:
humans,
mice, cats,
dogs, elephants
pigs
camels
horses
-5
-4
-2
-1
Relatively common for extra digits to occur:
1:3000 Europeans (usually thumbs)
1:300 Africans
(usually pinkies)
POLYDACTYLY
extra pinkie finger
extra pinkie toe
Same guy, by the way…
POLYDACTYLY
bonus thumbs
POLYDACTYLY
usually genetic
generally dominant
runs in families over many generations
What governs the formation of the
“correct” number of digits?
John Saunders and Mary Gasseling
addressed this question using
surgically manipulated chicken embryos
limb bud
For correct limb and digit development, three specialized
cell clusters are of primary importance:
the apical ectodermal ridge (AER),
the progress zone (PZ), and
the zone of polarizing activity (ZPA).
Saunders and Gasseling
The result was a palindromic duplication of the wing and digits
mirror image polydactyly
They named this area of mesodermal tissue the
Zone of Polarizing Activity
Postulated that the ZPA secreted a polarizing morphogen
morphogen intensity
numerous potential candidates for the morphogen
that dictates digit number and polarity
Numerous mouse models for polydactyly used
to investigate possible morphogen candidates
Doublefoot
Sasquatch
Extra Toes
Doublefoot
skeletal phenotype of right hindlimbs in WT and
Doublefoot mutant mice at embryonic day 17.5
Crick, A. et al., Developmental mechanisms underlying polydactyly
in the mouse mutant Doublefoot. J. Anat., 202: 21-26, 2003.
Doublefoot
In Doublefoot mutant mice, the expression pattern of SHH
itself is normal, but there is ectopic expression of SHH signaling
pathway components
ectopic expression - when a gene is expressed in cells that
it is not normally expressed in.
The sonic hedgehog signaling pathway
SHH
SHH receptor
(Patched - PTC)
Smoothened
is activated by
the binding of SHH
to PTC
activated smoothened
signals for the expression
of SHH target genes
Cytoplasm
Nucleus
CELL
Doublefoot
Patched expression
in embryonic day
10.5 embryos
SHH expression
in embryonic day
10.5 limb buds
Crick, A. et al., Developmental mechanisms underlying polydactyly
in the mouse mutant Doublefoot. J. Anat., 202: 21-26, 2003.
Extra toes
Mouse extra-toes mutant stained for
SHH expression at embryonic day 11.5
WT
forelimb
Hx +/forelimb
Hx +/hindlimb
Blanc, I. et al., Unusual pattern of sonic hedgehog expression
in the polydactylous mouse mutant Hemimelic extra-toes.
Int. J. Dev. Biol. 46: 969-974, 2002.
Extra toes
Appearance of newborn polydactylic limbs
in mouse extra-toes mutant
forelimb paw (7 digits)
hindlimb paw (6 digits)
hindlimb skeleton
Blanc, I. et al., Unusual pattern of sonic hedgehog expression
in the polydactylous mouse mutant Hemimelic extra-toes.
Int. J. Dev. Biol. 46: 969-974, 2002.
To initiate gene expression (or in some cases, to prevent gene expression),
regulatory proteins (homeobox proteins and other transcription factors)
bind to DNA upstream of the start of the target gene at regulatory elements.
The most recent evidence suggest that the Hx extra-toes
mutation is in a regulatory element upstream of the SHH gene.
Sasquatch
variable polydactyly phenotypes
associated with the Sasquatch mutation
(all pictures are of the hindlimb)
WT
Ssq/-
Ssq/strong
weak
adult
embryo
Sharpe et al., Identification of Sonic hedgehog as a candidate gene responsible for the
polydactylous mouse mutant Sasquatch. Current Biology. 9: 97-100.
Sasquatch
forelimb
hindlimb
Sharpe et al., Identification of Sonic hedgehog as a candidate gene responsible for the
polydactylous mouse mutant Sasquatch. Current Biology. 9: 97-100.
Sasquatch
Sasquatch mutation induces ectopic expression of Shh,
elevated expression of FGF8, and ectopic expression of Hoxd13
-------------------hindlimb-----------------WT
+/-
+/-
+/-
-/-
-/-
forelimb
+/-
Shh
WT
Shh
Fgf8
-/-
-/-
Hoxd13
WT
+/-
WT
+/-
-/-
Sharpe et al., Identification of Sonic hedgehog as a candidate gene responsible for the
polydactylous mouse mutant Sasquatch. Current Biology. 9: 97-100.
Sonic hedgehog seems to be a good candidate for the
ZPA morphogen…
-altered regulation (too much or too little Shh produced)
- abnormal pattern of expression (ectopic expression)
- expression of other components in the Shh signaling pathway altered
Sonic hedgehog seems to be a good candidate for the
ZPA morphogen…
- Altering Shh levels can result in mirror image polydactyly in chicken wings
- Shh mutant mice lack paws (similar to acheriopody)
- expression of other components in the Shh signaling pathway altered
mutations in either a transcription factor itself or in its
the DNA of its regulatory elements can alter function
There are at least 10 genes that, when mutated,
affect the activity of the Shh signaling pathway
- forebrain separation
- facial geometry
- digital determination
crosstalk and feedback between signaling pathways
make it complicated to determine exactly what
controls what
for example: If the AER and FGFs secreted from it fail,
the result is failure of limb formation
one role of Shh is to maintain and shape the activity
of the AER
one function of the AER is to maintain and shape
Shh production if the ZPA
Signaling pathways are not independent, but rather
are interconnected and often reciprocal in nature
The role of Programmed Cell Death in fine tuning digit formation
~ day 32 first signs of bone formation are visible - “condensation”
first to develop are those bones closest to the body:
humerus --- radius, ulna ---wrist and palm bones --- digits
by day 38 the ends of the limb buds look like paddles
the ectoderm between the paddles forms involutions and the extra
cells “die off” to clearly define the 5 digits
Programmed Cell Death - occurs not only during development
but also in some adult tissues
used to balance cell proliferation to maintain constant cell numbers
in tissues that undergo cell turnover:
liver
blood cells (~5 x 1011 killed daily)
also used as a defense mechanism to protect against viruses
and DNA damage that can lead to cancerous cells
During development, Programmed Cell Death is used extensively:
gets rid of larval tissues during insect and amphibian metamorphosis
gets rid of excess neurons (up to 50% of all neurons initially made
end up being destroyed - the one that make their proper connections
with their target cells cause those cells to secrete growth factors which
block initiation of the programmed cell death pathway
eliminates the extra tissue between digits during finger and toe formation
(so we don’t end up with webbed hands and feet like ducks…)
Apoptosis - the distinct series of cellular changes that
occurs during Programmed Cell Death
Apoptotic cells and fragments are effectively removed
by macrophagesand neighboring cells
partly due to “eat me” signals normally not displayed
on the cell surface (certain lipid moieties, for example,
phophatidylserine)
Apoptosis is different than accidental cell death
from injury - those cells will swell and burst,
releasing their contents into the extracellular
space and causing inflammation.
Digit formation also depends upon Hox genes (homeotic genes)
Hox gene mutations result in:
short big toes and bent pinkies (single mutant allele of Hoxd13)
synpolydactyly (extra and often fused digits)
missing forearm bones, fingers, and toes (deletion of 9 Hox genes)
Hox gene mutations also affect other appendages that grow
outward from the body (i.e. genitalia)
What can Hox genes tell us about our origins?
are our limbs ≈ fins?
are our fingers ≈ fin rays?
very different type of bone…
what about “closer: relatives, the lobe-finned fishes?
lungfish
coelacanth
Pros:
the lobe-finned fishes seem to have cognates
of our humerus, radius and ulna.
even have some small bones that could be
cognates of our digits
made of the right kind of bone…
Cons:
the geometry is all wrong
But still the fin buds have AER, ZPA, FGFs, Shh, and Hox genes
fin -vs- limb
Hoxd13 (or fish orthologue) is expressed in the ZPA of both
in fin buds, Hoxd13 only expressed for a short time
and over a short range
in limb buds, Hoxd13 stays on much longer and reaches
all the way across the outermost part of the ZPA
fin -vs- limb
Does Hoxd13 have the power to specify our digits?
Do ∆hoxd13 mice have fins instead of arms and paws with digits?
NO! but they do have small deformed digits and 6 instead of 5
Suggests that perhaps the common ancestor of modern land-dwelling
vertebrates had more digits than what we now have and that Hox genes
have evolved to trim down and define this number
Some contenders for this common ancestor
(from the Devonian era ~360 million years ago)
Acanthostega
8 digits
Some contenders for this common ancestor
(from the Devonian era ~360 million years ago)
Icthyostega
6 or 7 digits