Together with the lineage analysis, these observations strongly suggest that the limb bud initiates
earlier than stage 17 to 18, through EMT and independent of proliferation rate changes. When the
limb bud mesenchyme is generated, it induces a
source of fibroblast growth factor (Fgf ) activity in
the overlying ectoderm, the apical ectoderm ridge,
which serves to maintain the limb’s high level of
proliferation (3). This does not occur in the trunk
region, which we hypothesize is the reason for
its relative decrease in mitotic activity as mesenchyme is generated. We suggest that the difference in proliferation of trunk versus limb bud
mesenchyme is a result of limb bud initiation, as
opposed to being a cause of it.
To verify that EMT of the somatopleure represents a necessary step of limb initiation, we
blocked EMT in the presumptive limb region by
RhoA overexpression, which abrogates EMT by
inducing strong interaction of cells with extracellular matrix components (4). We electroporated
RhoA with GFP within the epithelial somatopleure.
In control embryos electroporated with GFP alone,
limb buds formed normally (Fig. 2A), the basement membrane of laminin broke down, and cells
underwent EMT (Fig. 2, B to D). In contrast,
coelectroporation of RhoA with GFP completely
abrogated limb formation (Fig. 2, E and I).
Sections revealed that RhoA electroporated cells
were stuck in the epithelial somatopleure and
failed to undergo proper EMT (Fig. 2, F to H).
These cells were attached to aggregates of laminin
(Fig. 2, K and L) and did not exhibit enrichment in
vimentin staining (fig. S5, G to L). We found that
RhoA-overexpressing cells overexpressed F-actin,
maintained adherens junction as revealed by
N-cadherin and b-catenin stainings (fig. S5, A
to L), and failed to relocalize aPKC from their
apical cortex to the cytoplasm (fig. S5, A to F) as
observed in control GFP-electroporated cells
(fig. S4, N, O, R, and S). RhoA overexpression
did not lead to dramatic reduction of proliferation (fig. S6, A to C) or increase in apoptosis in
the electroporated cells (fig. S6, D to G), confirming that RhoA acts to prevent epithelial-to-mesenchymal cell state change. Thus as a
consequence of this failure to undergo EMT, the
electroporated cells did not participate in the initiation and formation of the limb primordium.
To understand how EMT is regulated in this
context, we explored the possibility that Snail1,
a transcription factor upstream of EMT in other
contexts (5), plays a similar role here, but (per-
haps because of redundancy) we failed to find
such a connection. We therefore turned to factors
known to be involved in establishing the forma-
tion of a limb bud. Ectopic Fgf protein, applied
up to stage 17, is sufficient to induce the forma-
tion of an entire additional limb from trunk tissue
(6). To test whether Fgf10 promotes limb forma-
tion by inducing EMT, we coelectroporated Fgf10
and GFP into the trunk somatopleure of stage-13
to -14 chick embryos. As expected, 36 hours after
electroporation Fgf10 induced swelling of the
trunk, indicating ectopic limb initiation (Fig. 3A,
red arrowheads). Sectioning through the electro-
porated region showed that most of the GFP-
positive cells had left the epithelium and had
acquired a mesenchymal phenotype (81%, Fig. 3,
B to D), showing that indeed EMT had taken
place. The trunk is only competent to form an
ectopic limb up to stage 16 to 17 (7, 8). This was
previously interpreted as the time at which the
trunk mesenchyme becomes determined and is
no longer capable of being redirected to a limb
fate. However, our data show that this is precisely
the time at which the trunk mesenchyme is first
generated. Thus, we would reinterpret those re-
sults as indicating that ectopic Fgf activity can
induce limb bud formation from epithelial trunk
somatopleure cells but not from mesenchymal
cells of the same rostrocaudal level.
Targeted mutation of Fgf10 and Tbx5 in mice
have demonstrated that these genes are necessary
to initiate limb bud formation (9–12). Transverse
sections of embryonic day 9.5 Fgf10−/− and Tbx5−/−
embryos confirmed that neither Tbx5 nor Fgf10
mutant embryos exhibit swellings characteristic
of limb bud initiation (Fig. 3, E, H, and K). Both
FGF10 and Tbx5 mutant embryos showed the
presence of mesenchyme in the forelimb region;
however, the proportion of mesenchymal cells
compared to the proportion of epithelial cells was
significantly lower than that of a wild-type (WT)
sibling embryo (Fig. 3N), with a stronger phenotype observed in Tbx5−/− embryos (only 12% of
cells were epithelial in WT embryos, whereas 21%
and 51% were epithelial in Fgf10- and Tbx5-
deficient embryos, respectively). In Tbx5 mutant
embryos, the epithelium appeared separated from
the mesenchyme (Fig. 3K, asterisks). aPKC and
b-catenin staining revealed hyperplasia of the
somatopleure epithelium, in support of failure of
these cells to undergo EMT (Fig. 3, K and L).
Last, in Fgf10−/− as well as in Tbx5−/− embryos,
the basement membrane of laminin did not properly break down and appeared overstabilized, as
opposed to WT embryos (Fig. 3, F, G, I, J, L, and
M). Taken together, these data show that Tbx5
and Fgf10 act on the somatopleure epithelium to
regulate, at least partially, the early induction of
EMT in the limb fields, the process that is at the
heart of limb bud initiation.
References and Notes
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2. A. Mauger, J. Embryol. Exp. Morphol. 28, 313–341
3. H. Ohuchi et al., Development 124, 2235–2244 (1997).
4. Y. Nakaya, E. W. Sukowati, Y. Wu, G. Sheng, Nat. Cell
Biol. 10, 765–775 (2008).
5. J. P. Thiery, H. Acloque, R. Y. J. Huang, M. A. Nieto, Cell
139, 871–890 (2009).
6. M. J. Cohn, J. C. Izpisúa-Belmonte, H. Abud, J. K. Heath,
C. Tickle, Cell 80, 739–746 (1995).
7. H. Ohuchi et al., Biochem. Biophys. Res. Commun. 209,
8. A. Vogel, C. Rodriguez, J. C. Izpisúa-Belmonte,
Development 122, 1737–1750 (1996).
9. P. Agarwal et al., Development 130, 623–633 (2003).
10. C. Rallis et al., Development 130, 2741–2751 (2003).
11. H. Min et al., Genes Dev. 12, 3156–3161 (1998).
12. K. Sekine et al., Nat. Genet. 21, 138–141 (1999).
Acknowledgments: We thank F. Constantini for help
rederiving the Fgf10 mutant mouse line, B. Bruneau for kindly
providing Tbx5 mutant embryos, and J. deMelo and P. Tschopp
for help with mouse work. J.G. was a fellow of the Human
Frontier Science Program. This work was supported by NIH
grant R01-HD045499 to C.J. T.
Materials and Methods
Figs. S1 to S6
7 November 2013; accepted 7 February 2014
The stum Gene Is Essential
for Mechanical Sensing
in Proprioceptive Neurons
Bela S. Desai, Abhishek Chadha, Boaz Cook*
Animal locomotion depends on proprioceptive feedback, which is generated by mechanosensory
neurons. We performed a genetic screen for impaired walking in Drosophila and isolated a
gene, stumble (stum). The Stum protein has orthologs in animals ranging from nematodes to
mammals and is predicted to contain two transmembrane domains. Expression of the mouse
orthologs of stum in mutant flies rescued their phenotype, which demonstrates functional
conservation. Dendrites of stum-expressing neurons in legs were stretched by both flexion and
extension of corresponding joints. Joint angles that induced dendritic stretching also elicited
elevation of cellular Ca2+ levels—not seen in stum mutants. Thus, we have identified an
evolutionarily conserved gene, stum, which is required for transduction of mechanical stimuli in
a specific subpopulation of Drosophila proprioceptive neurons that sense joint angles.
Animal locomotion is achieved by coor- dination of motor activity according to proprioceptive mechanosensory inputs. In Drosophila, mechanosensation is mediated either by ciliated or multidendritic receptor neu- rons. Multidendritic neurons can respond to direct