Transcript Organogenesis I: Somites and Limb Formation

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)