MUTANTS genetic variation in human development Lecture 7 Fall 2006 Bennington College

1. MUTANTSgenetic variation in human developmentLecture 7Fall 2006Bennington College

2. 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…)

3. POLYDACTYLY (aka “digital enhancement”) The “normal” number of digits varies greatly between vertebrate species: humans, mice, cats, dogs, elephants - 5 pigs - 4 camels - 2 horses - 1

4. Relatively common for extra digits to occur: 1:3000 Europeans (usually thumbs) 1:300 Africans (usually pinkies)

5. extra pinkie toe POLYDACTYLY extra pinkie finger Same guy, by the way…

6. POLYDACTYLY bonus thumbs

7. POLYDACTYLY usually genetic generally dominant runs in families over many generations

8. What governs the formation of the “correct” number of digits? John Saunders and Mary Gasseling addressed this question using surgically manipulated chicken embryos

9. 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).

10. Saunders and Gasseling

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

12. They named this area of mesodermal tissue the Zone of Polarizing Activity Postulated that the ZPA secreted a polarizing morphogen

13. morphogen intensity

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

15. Numerous mouse models for polydactyly used to investigate possible morphogen candidates Doublefoot Sasquatch Extra Toes

16. 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.

17. 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.

18. SHH activated smoothened signals for the expression of SHH target genes The sonic hedgehog signaling pathway SHH receptor (Patched - PTC) is activated by the binding of SHH to PTC Smoothened Cytoplasm Nucleus CELL

19. 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.

20. Mouse extra-toes mutant stained for SHH expression at embryonic day 11.5 WT forelimb Hx +/- forelimb Hx +/- hindlimb Extra toes 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.

21. Extra toes Appearance of newborn polydactylic limbs in mouse extra-toes mutant hindlimb skeleton forelimb paw (7 digits) hindlimb paw (6 digits) 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.

22. 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.

23. Sasquatch variable polydactyly phenotypes associated with the Sasquatch mutation (all pictures are of the hindlimb) Ssq/- strong weak Ssq/- WT 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.

24. 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.

25. Sasquatch Sasquatch mutation induces ectopic expression of Shh, elevated expression of FGF8, and ectopic expression of Hoxd13 forelimb -------------------hindlimb------------------ +/- +/- +/- WT +/- 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.

26. 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

27. 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

28. 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

29. 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

30. The role of Programmed Cell Deathin 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

31. 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

32. 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…)

33. Apoptosis - the distinct series of cellular changes that occurs during Programmed Cell Death

34. 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)

36. 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.

37. 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)

38. What can Hox genes tell us about our origins? are our limbs ≈ fins? are our fingers ≈ fin rays?

39. lungfish coelacanth very different type of bone… what about “closer: relatives, the lobe-finned fishes?

40. 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

41. 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

42. 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

43. Some contenders for this common ancestor (from the Devonian era ~360 million years ago) Acanthostega 8 digits

44. Some contenders for this common ancestor (from the Devonian era ~360 million years ago) Icthyostega 6 or 7 digits