Current Biology 20, 1993–2002, November 23, 2010 ª2010 Elsevier Ltd All rights reserved DOI 10.1016/j.cub.2010.09.063
Article
WNT5A/JNK and FGF/MAPK Pathways
Regulate the Cellular Events
Shaping the Vertebrate Limb Bud
Jerome Gros,
1
Jimmy Kuang-Hsien Hu,
1
Claudio Vinegoni,
2
Paolo Fumene Feruglio,
2,3
Ralph Weissleder,
2
and Clifford J. Tabin
1,
*
1
Department of Genetics, Harvard Medical School, 77 Avenue
Louis Pasteur, Boston, MA 02115, USA
2
Center for Systems Biology, Massachusetts General
Hospital, Harvard Medical School, 185 Cambridge Street,
Boston, MA 02114, USA
3
Department of Neurological, Neuropsychological,
Morphological and Movement Sciences, University of Verona,
Strada Le Grazie 8, 37134 Verona, Italy
Summary
Background: The vertebrate limb is a classical model for
understanding patterning of three-dimensional structures
during embryonic development. Although decades of research
have elucidated the tissue and molecular interactions within
the limb bud required for patterning and morphogenesis of
the limb, the cellular and molecular events that shape the
limb bud itself have remained largely unknown.
Results: We show that the mesenchymal cells of the early limb
bud are not disorganized within the ectoderm as previously
thought but are instead highly organized and polarized. Using
time-lapse video microscopy, we demonstrate that cells move
and divide according to this orientation. The combination of
oriented cell divisions and movements drives the proximal-
distal elongation of the limb bud necessary to set the stage for
subsequent morphogenesis. These cellular events are regu-
lated by the combined activities of the WNT and FGF pathways.
We show that WNT5A/JNK is necessary for the proper orienta-
tion of cell movements and cell division. In contrast, the FGF/
MAPK signaling pathway, emanating from the apical ecto-
dermal ridge, does not regulate cell orientation in the limb bud
but instead establishes a gradient of cell velocity enabling
continuous rearrangement of the cells at the distal tip of the limb.
Conclusions: Together, these data shed light on the cellular
basis of vertebrate limb bud morphogenesis and uncover
new layers to the sequential signaling pathways acting during
vertebrate limb development.
Introduction
The vertebrate limb bud forms as a mound of cells slightly
elongated along the rostrocaudal axis of the embryo. As it
grows, the early limb bud rapidly transforms into a paddle
shape with an extended proximal-distal axis. Attaining this
shape of the progenitor field is critical for producing limb
segments and skeletal elements of the correct size and shape.
One previous model proposed to account for the proximal-
distal directional elongation of the early limb mesenchyme
based on differential proliferation rates between the proximal-
and distalmost ends of the limb bud. This view posits that
a higher proliferation rate at the distal end of the limb bud could
act to ensure a proximal-distal oriented outgrowth [1].
Although a number of computational models have suggested
that this mechanism could in principle account for observed
changes in the shape of the limb bud [1–3], several studies
have reported that proliferation is uniform throughout the
mesenchyme during limb development and that only at late
stages can an increase in proliferation be seen in the digital
tips (from stage 23–25 in the chick and from E12.5 in the mouse)
[4–6]. At these stages, the limb has already acquired its overall
elongated shape. Thus, because proliferation appears to be
largely isotropic at early stages, it cannot account for the
dramatic changes observed in the shape of the limb bud.
An alternative hypothesis to explain how the limb acquires
its shape involves oriented rearrangements of mesenchymal
cells. This hypothesis was proposed in the early 1970s by
Hornbruch and Wolpert when they were unable to identify
differential proliferation within the developing limb bud [4].
However, at the time, tools were not available to test the
veracity of this hypothesis in the context of the limb bud.
Here, we brought powerful imaging methods to bear on the
question of how the early limb primordium attains the shape
required to serve as a substrate for patterning and to poten-
tiate limb morphogenesis.
Results
Characterization of Chick Limb Elongation
To understand the mechanisms that might be involved in limb
elongation, we first characterized it at the tissue level. Using
optical projection tomography, we were able to accurately
measure all three axes of the limb (anterior-posterior [A-P],
dorsal-ventral [D-V], and proximal-distal [P-D]). Axis measure-
ments were performed on 3D reconstructed limbs of chick
embryos at Hamburger-Hamilton (HH) stages 18, 20, 21, and
23, which cover w24–30 hr of development (Figures 1A–1H;
see also Movie S1 available online). As expected, we found
that during this time window, the P-D axis length increased
dramatically (about three times). Surprisingly, we found that
the D-V axis length did not increase much, and the A-P axis
length actually decreased (Figure 1I). Because cells in the
limb mesenchyme have been previously shown to uniformly
proliferate at these stages [1, 4, 6], one would have expected
all three axes to increase in length. The fact that the P-D axis
is
the only one to dramatically increase in length suggests
that differential rates in isotropic proliferation cannot explain
limb shape. Cell death has been extensively studied in this
context and has been shown to play a role in refining the
limb shape at later stages of this process. Although cell death,
known to be present in the proximal anterior and posterior part
of the limb bud, can explain the decrease in length of the A-P
axis [6], it cannot account for the absence of major growth of
the D-V axis. Thus, this analysis strongly suggests that other
oriented mechanisms within the limb bud must act to accen-
tuate its growth preferentially along the P-D axis.
Mesenchymal Cells of the Limb Bud Are Oriented
The early limb bud is generally conceptualized as an ecto-
dermal bag containing a mound of uniformly distributed and
*Correspondence: tabin@genetics.med.harvard.edu