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Transcript
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? Organogenesis I: The Mesoderm The Mesoderm After Gastrulation Reminder: The mesoderm from the organizer gives rise to “axial” mesoderm or notocord Frog Chick Fate Map of The Mesoderm Xenopus V Zebrafish D Chick The Mesoderm After Gastrulation Chick Major Derivatives of the Germ Layers Epidermis, CNS, PNS, Melanocytes Gut, Lungs, Liver, Pancreas, Gall Bladder, Thymus, Thyroid… Notocord, Muscle, Bone, Dermis, Heart, Kidney, Somatic Gonad, Vaculature, Blood The Mesoderm After Gastrulation axial Not Much Vertebrae Heart Vasculature Limb and Dermis Back Muscle Body Wall Somitogenesis 1) 2) 3) 4) Segmentation Somite Formation Somite Patterning Somite Differentiation (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 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) Molecular Evidence for the “Clock” The Segmentation Clock 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 Other autonomous cellular “clocks” exist The ≈24hr circadian clock “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 Segmentation Defects in Humans FGF8 Defines the Determination Front A gradient of FGF8 in the PSM Inhibiting FGF “advances” the Determination Front hairy FGF8 Inhibits Determination Front and Blocks Somitogenesis Dubrulle, McGrew and Pourquie, 2001 Fgf8 Gradient Formation by RNA Inheritance fgf8 is only transcribed in the tailbud A model Morphogen diffusion: Exon Probe Intron Probe RNA Inheritence: New transcription is not required for gradient formation Proliferation + Decay = Gradient 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 Steps of Somite Formation A/P Patterning 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 ligands Cell-ECM: Integrins and fibronectin have also been implicated in boundary formation Somite Development Gastrulation (EMT) MET EMT Individual Somite Identity Drosophila Hypothetical common ancestor Amphioxus Mouse Hox Genes Specify A/P Identity in Vertebrates 5/6 mutant Triple homozygote A/a C/c D/c X A/a C/c D/c 1/64 pups = aaccdd Wellik and Capecchi, 2003 “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 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 Derivatives of the Somite Dermamyotome Body Wall and Limb Muscles (Hypaxial) Back Muscles (Epaxial) (Vertebrae) Derivatives of the Somite D/V Patterning in the Somite D 4, 6,7 V Limb Development Chick Mouse Question 1: Where to form limbs and what kind? FGFs Determine Where the Limb Buds Will Form Ectopic FGF10 can induce FGF8 and a complete ectopic limb Ohuchi et al. Development 1997 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 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 The AER is Also Critical For Patterning the P/D Axis P D Chick Limb truncation observed with AER removal at designated stage Mouse The “Zone of Polarizing Activity” (ZPA) Patterns the A/P Axis of the Limb Sonic hh is the Morphogen Secreted by the ZPA (Repressor of hh signal) Cell Movement and “Time of Exposure” Also Contribute to Patterning by Shh Shh in situ Shh fate map Shh promoter-CRE + loxP General Promoter loxP STOP lacZ 2004 Integrating the P/D, A/P and D/V Axes of the Limb RA Niswander 2003 Limb Patterning and Evolutionary Change Chicken Behringer and Niswander Labs Duck Merino et al., 1999 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)