MUTANTSgenetic variation in human development

Lecture 7

Fall 2006Bennington 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 allof 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 workahead of time” like hand-written corrections at the lastminute…

For the sake of my quickly declining eyesight, pleaseat least 1.5 space everything (I still have no vision plan…)

POLYDACTYLY

(aka “digital enhancement”)

The “normal” number of digits variesgreatly between vertebrate species:

humans, mice, cats, dogs, elephants - 5

pigs - 4camels - 2horses - 1

Relatively common for extra digits to occur:

1:3000 Europeans (usually thumbs)

1:300 Africans (usually pinkies)

extra pinkie finger extra pinkie toe

Same guy, by the way…

POLYDACTYLY

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 usingsurgically manipulated chicken embryos

For correct limb and digit development, three specialized cell clusters are of primary importance: the apical ectodermal ridge (AER), the progress zone (PZ), andthe zone of polarizing activity (ZPA).

limb bud

Saunders and Gasseling

The result was a palindromic duplication of the wing and digits mirror image polydactyly

Postulated that the ZPA secreted a polarizing morphogen

They named this area of mesodermal tissue the Zone of Polarizing Activity

morphogen intensity

numerous potential candidates for the morphogen that dictates digit number and polarity

Numerous mouse models for polydactyly usedto investigate possible morphogen candidates

Doublefoot

Sasquatch

Extra Toes

Crick, A. et al., Developmental mechanisms underlying polydactyly in the mouse mutant Doublefoot. J. Anat., 202: 21-26, 2003.

skeletal phenotype of right hindlimbs in WT andDoublefoot mutant mice at embryonic day 17.5

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.

Doublefoot

The sonic hedgehog signaling pathway

SHH

SHH receptor(Patched - PTC)

CELL

NucleusCytoplasm

is activated by the binding of SHH to PTC

activated smoothened signals for the expression of SHH target genes

Smoothened

Crick, A. et al., Developmental mechanisms underlying polydactyly in the mouse mutant Doublefoot. J. Anat., 202: 21-26, 2003.

Patched expression in embryonic day 10.5 embryos

SHH expression in embryonic day 10.5 limb buds

Doublefoot

Blanc, I. et al., Unusual pattern of sonic hedgehog expressionin the polydactylous mouse mutant Hemimelic extra-toes. Int. J. Dev. Biol. 46: 969-974, 2002.

Mouse extra-toes mutant stained for SHH expression at embryonic day 11.5

WTforelimb

Hx +/-forelimb

Hx +/-hindlimb

Extra toes

Blanc, I. et al., Unusual pattern of sonic hedgehog expressionin the polydactylous mouse mutant Hemimelic extra-toes. Int. J. Dev. Biol. 46: 969-974, 2002.

forelimb paw (7 digits) hindlimb paw (6 digits) hindlimb skeleton

Appearance of newborn polydactylic limbs in mouse extra-toes mutant

Extra toes

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.

Sharpe et al., Identification of Sonic hedgehog as a candidate gene responsible for the polydactylous mouse mutant Sasquatch. Current Biology. 9: 97-100.

WT Ssq/-strong weak

variable polydactyly phenotypesassociated with the Sasquatch mutation (all pictures are of the hindlimb)

Ssq/-

adult

embryo

Sasquatch

Sharpe et al., Identification of Sonic hedgehog as a candidate gene responsible for the polydactylous mouse mutant Sasquatch. Current Biology. 9: 97-100.

forelimb

hindlimb

Sasquatch

Shh

ShhFgf8

Hoxd13

WT WT+/- +/- -/-

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.

forelimb-------------------hindlimb------------------

Sasquatch mutation induces ectopic expression of Shh, elevated expression of FGF8, and ectopic expression of Hoxd13

Sasquatch

Sonic hedgehog seems to be a good candidate for theZPA 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 theZPA 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 pathwaysmake it complicated to determine exactly whatcontrols 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 activityof the AER

one function of the AER is to maintain and shapeShh 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 numbersin tissues that undergo cell turnover:

liverblood cells (~5 x 1011 killed daily)

also used as a defense mechanism to protect against virusesand 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 connectionswith their target cells cause those cells to secrete growth factors whichblock 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 displayedon the cell surface (certain lipid moieties, for example,phophatidylserine)

Apoptosis is different than accidental cell deathfrom injury - those cells will swell and burst, releasing their contents into the extracellularspace 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

the lobe-finned fishes seem to have cognatesof our humerus, radius and ulna.

even have some small bones that could becognates of our digits

made of the right kind of bone…

Pros:

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 timeand over a short range

in limb buds, Hoxd13 stays on much longer and reachesall 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-dwellingvertebrates had more digits than what we now have and that Hox geneshave 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