Vertebrate Limb Development

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Vertebrate Limb Development

• Carolyn Hedley
• March 25, 2002

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Introduction
Ant.

• develop from limb buds
• 3 developmental axes proximo-distal,
  antero-posterior, and dorso-ventral
• early limb bud progress zone, apical ectodermal
  ridge, polarizing region

Vent.
Prox.
Dist.
Dors.
Post.
Progress zone
Apical ectodermal ridge
Polarizing region
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Apical Ectodermal Ridge (AER)

• 1 of 2 organizing regions.
• length of ridge controls width of bud.
• essential for both outgrowth and proximo-distal
  patterning truncated limbs result after removal.

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AER continued

• positioning determined by gene Radical Fringe
  (r-Fng) develops at boundary of r-Fng-expressing
  and non-expressing cells.
• AER signals to underlying mesenchyme.
• signals include proteins of fibroblast growth
  factor (Fgf) gene family
• FGF8 expressed throughout ridge
• FGF4 in posterior region - can act as a
  functional substitute for apical ridge

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Progress Zone (PZ)

• lies beneath AER.
• produces initial outgrowth of limb bud rapidly
  proliferating mesenchymal cells.
• cells begin to differentiate only after leaving
  the progress zone.
• differentiation proceeds distally as limb extends
  - cell fate determined by time spent in progress
  zone.

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Zone of Polarizing Activity (ZPA)

• second organizing region.
• diffusible morphogen specifies position of cells
  along antero-posterior (A/P) axis by
  concentration of morphogen, or short-range
  signals.
• Sonic Hedgehog (SHH) protein key component.

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ZPA, PZ and AER signals

• ZPA and PZ signals are necessary for survival and
  function of AER FGFs from AER maintain ZPA and
  PZ.
• positive feedback loop between SHH protein in
  mesoderm and FGF4 expression in ridge, via bone
  morphogenetic proteins (BMPs).

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Dorso-ventral (D/V) axis

• specified by ectoderm
• Wnt-7a gene identified in mice - if mutated,
  dorsal tissues adopt ventral fates.

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Forelimbs vs Hindlimbs

• same signals interpreted differently.
• 23 different Hox genes expressed Hoxa-d gene
  clusters
• Hoxa d - expressed in both forelimbs and
  hindlimbs
• Hoxb c - restricted to either forelimb or
  hindlimb bud

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Cartilage

• elements can develop in absence of polarizing
  region.
• reaction-diffusion mechanism - establishes a
  prepattern by periodic peaks in a morphogen.
• acquires positional information in PZ.

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Muscle

• muscle cells in early embryo are all equivalent
  migrate into the limb bud from somites.
• dorsal and ventral blocks of muscle initially.
  Divisions give rise to individual muscles
  pattern determined by connective tissue.

dorsal muscle mass
ulna
ventral muscle mass
radius
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Apotosis

• programmed cell death
• key role in formation of digits - separation
  depends on death of cells between cartilaginous
  elements.
• mesoderm determines cell fate

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Normal limb development in conditional mutants of
Fgf4

• Moon, A. M., Boulet, A. M., and M. R. Capecchi.
  2000. Development. 127 989-996.

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Objective

• To produce a conditional mutant of Fgf4 for
  assessing the role of FGF4 during limb
  development in living murine embryos.

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Fibroblast Growth Factors

• expression strongest in the posterior AER at
  E10.5-11.0.
• hypothesized to mediate AER activity in vivo.
• FGF4 - may provide a proliferative signal to the
  posterior PZ to support limb outgrowth.
• evidence for SHH-FGF4 feedback loop hypothesis
  lack of Fgf4 expression is associated with i)
  failure to maintain Shh expression and, ii)
  abnormal patterning of distal limb elements.

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Methods Conditional mutagenesis

• technique used to conditionally inactivate Fgf4
  in a developmentally regulated, tissue-restricted
  manner.
• used the Cre/loxP system - two mouse lines
  required
• 1) vital Fgf4 coding sequences are flanked by
  loxP sites
• 2) conventional transgenic mouse line with Cre
  targeted to a specific tissue or cell type

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Methods contd

• 3 components of Fgf4 conditional mutagenesis
• 1) mice carrying either a conditional or a null
  allele of Fgf4 were created by gene targeting in
  embryonic stem cells ? transgenic switch
  generated to inactivate the conditional allele
• 2) Southern analysis used for identification of
  offspring with Fgf4 mutant alleles
• 3) animals genotyped using PCR technique

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Evaluation of Cre-mediated combination

• the human alkaline phosphatase gene (AP) is
  expressed in the conditional allele following
  Cre-mediated inactivation of Fgf4.
• stained for AP AP activity labels cells in which
  recombination has occurred

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Results phenotypic

• appearance of AER and limb bud initiation,
  location, or size were indistinguishable between
  embryonic conditional mutants and littermates at
  E10.0-11.5.
• patterning and outgrowth of forelimbs of Ffg4
  conditional mutants were normal in E15.5 embryos,
  newborns, and adults. Normal external appearance
  and function in living animals.

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Skeletal phenotypes of newborns
Wild-type controls
Fgf4 conditional mutants
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Results Shh, Bmp2 and Fgf8 expression

• no detected changes in Shh expression in
  forelimbs of Fgf4 conditional mutants.
• normal expression of Fgf8, Bmp2.

RNA in situ hybridization
E10.5 embryos
E11.5 embryos
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Discussion

• may be other in vivo AER factors involved
• compensation of another FGF for absence of FGF4.
• FGF1, FGF2 and FGF8 can each support limb
  outgrowth in absence of AER.
• FGF8 can induce and maintain SHH expression in
  the absence of the AER.

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Discussion

• the normal skeletal and molecular phenotypes of
  conditional mutants are unlikely a function of
  early or persistent Fgf4 gene function
• no detection of Fgf4 transcripts in forelimb AER
  at E 10.0-11.5.

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Summary Implications

• Fgf4 conditional mutants had normal limb
  outgrowth development.
• in vivo results indicate that FGF4 is not
  required for normal limb development or Shh
  expression in the ZPA.
• some of the functions currently attributed to
  FGF4 may be performed by other AER factors.

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Future directions

• determine role of Fgf8 in limb development by
  conditional mutagenesis.
• most likely possible to disrupt multiple genes
  simultaneously in conditional mutagenesis
• eliminate more than one FGF in the AER

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Additional research

• Sun et al. (May 2000) - upregulation of Fgf8 and
  Fgf9 did not occur in Fgf4 conditional mutants.
• Moon Capecchi (Dec. 2000) - conditional
  mutagenesis of FGF8.
• Fgf8 absolutely required in the anterior AER, and
  to maintain Shh expression.
• Fgf4 expression increased at E11.5 in Fgf8
  conditional mutants possible compensation.

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References

• Cohn, M. J., and P. E. Bright. 1999. Molecular
  control of vertebrate limb development,
  evolution and congenital malformations. Cell
  Tissue Research. 296 3-17.
• Moon, A. M., Boulet, A. M., and M. R. Capecchi.
  2000. Normal limb development in conditional
  mutants of Fgf4. Development. 127 989- 996.
• Moon, A. M., and M. R. Capecchi. 2000. Fgf8 is
  required for outgrowth and patterning of the
  limbs. Nature Genetics. 26 455-459.
• Sun, X., Lewandoski, M., Meyers, E. N., Liu, Y.,
  Maxson, R. E., and G. R. Martin. 2000.
  Conditional inactivation of Fgf4 reveals
  complexity of signaling during limb bud
  development. Nature Genetics. 25 83-86.
• Wolpert, L. 1998. Principles of Development.
  Oxford University Press, New York, NY.

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Questions?