Organogenesis

1. Organogenesis Some things to think about: 1) Cell Fate Specification -Where do cells for an organ come from and how many different cell types are involved? (Fate map) -How many different cell types are involved? -How are they specified? -How do inductive interactions control their identity? 2) Morphogenesis -Where do cells for an organ come from and how do they get to the site of organ formation? -How do different cell types recognize one another? (Adhesion, signaling) -How does individual cell shape contribute to tissue shape and function? -How do these cells generate the proper organ architecture? 3) Terminal Differentiation -What changes in gene expression are required to generate organ-specific cellular functions? -How are these specialized cellular functions controlled? 4) Homeostasis -How is the function of the organ maintained over time? (stem cells?) -How does adult homeostasis resemble organ development?

2. Organogenesis I:The Mesoderm

3. Chick The Mesoderm After Gastrulation Reminder: The mesoderm from the organizer gives rise to “axial” mesoderm or notocord Frog

4. Fate Map of The Mesoderm Xenopus V D Chick Zebrafish

5. The Mesoderm After Gastrulation Chick

6. Gut, Lungs, Liver, Pancreas, Gall Bladder, Thymus, Thyroid… Epidermis, CNS, PNS, Melanocytes Notocord, Muscle, Bone, Dermis, Heart, Kidney, Somatic Gonad, Vaculature, Blood Major Derivatives of the Germ Layers

7. The Mesoderm After Gastrulation axial Not Much Heart Vasculature Body Wall Vertebrae Dermis Limb and Back Muscle

8. Somitogenesis Segmentation Somite Formation Somite Patterning Somite Differentiation

9. (Growth Zone) Somite formation is species specific in timing and number Chick: 50 Somites (90 minutes/somite) Mouse: 65 Somites (120 minutes/somite) Snakes: 400-500 Somites

10. Clock and Wavefront Model (Cooke and Zeeman, 1976) Clock: Inherent timekeeper or oscillator in a tissue Wavefront: Progression of maturation or determination across a tissue A regular, segmented structure can be formed if cells experiencing a particular point in “wave” at a particular time on “clock” exhibit a particular behavior (e.g. somite boundary formation)

11. Molecular Evidence for the “Clock”

12. The Segmentation Clock

13. A cell-autonomous “Hes Clock” created by negative feedback Hes genes can even cycle in tissue culture cells A molecular model for the segmentation clock

14. Other autonomous cellular “clocks” exist The ≈24hr circadian clock

15. “Cycling Genes”: hairy/HES, Delta, Lunatic Fringe, Axin 2 All related to Notch or Wnt Signaling Inhibit Notch=inhibit clock and inhibit somitogenesis -still have Axin2 oscillations Mutate wnt3a - block both Axin 2 and Lunatic Fringe oscillations

16. Segmentation Defects in Humans

17. FGF8 Inhibits Determination Front and Blocks Somitogenesis Inhibiting FGF “advances” the Determination Front hairy FGF8 Defines the Determination Front A gradient of FGF8 in the PSM Dubrulle, McGrew and Pourquie, 2001

18. fgf8 is only transcribed in the tailbud A model Morphogen diffusion: RNA Inheritence: Exon Probe Intron Probe Proliferation + Decay = Gradient Fgf8 Gradient Formation by RNA Inheritance New transcription is not required for gradient formation

19. Molecular Basis for “Clock and Wavefront” Clock = cyclic expression of N and wnt targets Wavefront (gradient) = combination of reciprocal fgf/wnt and Retinoic Acid Gradients RADH wnt3a fgf8

20. Steps of Somite Formation A/P Patterning

21. Somite Boundary Formation Next Boundary Wt and integrin alpha-5 mutant stained for fibronectin Signaling somite boundaries: Ephs are tyrosine kinase receptors for cell-surface attached Ephrin ligandsCell-ECM: Integrins and fibronectin have also been implicated in boundary formation

22. MET EMT Somite Development Gastrulation (EMT)

23. Individual Somite Identity

24. Drosophila Hypothetical common ancestor Amphioxus Mouse

25. 5/6 mutant Triple homozygote A/a C/c D/c X A/a C/c D/c 1/64 pups = aaccdd Hox Genes Specify A/P Identity in Vertebrates Wellik and Capecchi, 2003

26. “Co-linearity” of HOX gene expression both responds to A/P position and helps DETERMINE A/P position Expression of Hox genes along the A/P axis is determined prior to somitogenesis Hox gene expression can also DETERMINE a cell’s position along the A/P axis

27. The Segmentation Clock Also Regulates HOX Gene Expression -FGF8 slows determination front -Get more somites that are smaller -Same somite # is found at more anterior position -HOX code reflects somite # (# oscillations of clock) rather than actual A/P position Dubrulle, McGrew and Pourquie, 2001

28. Derivatives of the Somite Dermamyotome Back Muscles (Epaxial) Body Wall and Limb Muscles (Hypaxial) (Vertebrae)

29. Derivatives of the Somite

30. D/V Patterning in the Somite D 4, 6,7 V

31. Limb Development Chick Mouse

32. Question 1: Where to form limbs and what kind?

33. FGFs Determine Where the Limb Buds Will Form Ectopic FGF10 can induce FGF8 and a complete ectopic limb Ohuchi et al. Development 1997

34. Tbx Genes Help Specify Forelimb vs. Hindlimb Tbx5 Wing Tbx4 Leg Of course, like other aspects of patterning along the A/P axis, the position of the limb buds and the type of limbs that form are regulated by the HOX genes

35. Question 2: How is the limb formed and patterned? Limb outgrowth (proximal-distal axis) is regulated by FGFs secreted from the the Apical Ectodermal Ridge (AER) FGF4, 8,9, and 17 all expressed in AER

36. Limb truncation observed with AER removal at designated stage The AER is Also Critical For Patterning the P/D Axis P D Chick Mouse

37. The “Zone of Polarizing Activity” (ZPA) Patterns the A/P Axis of the Limb

38. Sonic hh is the Morphogen Secreted by the ZPA (Repressor of hh signal)

39. Cell Movement and “Time of Exposure” Also Contribute to Patterning by Shh Shh in situ Shh fate map Shh promoter-CRE + loxP loxP 2004 General Promoter STOP lacZ

40. Integrating the P/D, A/P and D/V Axes of the Limb RA Niswander 2003

41. Limb Patterning and Evolutionary Change Chicken Behringer and Niswander Labs Fish vs. tetrapods Fish--only stylopod and zeugopod Tetrapod--now see autopod Lizards vs. Snakes -first lose forelimb (hox code change) -then lost hindlimb (loss of Shh) Duck Merino et al., 1999