Overall, this study demonstrates how histone
variants can determine epigenetic states through
direct modulation of chromatin-modifying enzyme activity. Further, the ability of ATXR5/6 to
discriminate between the variants H3.3 and H3.1
provides a mechanism for the mitotic inheritance
and genome-wide distribution of H3K27me1 in
plants. According to this model, ATXR5/6 are
recruited to the replication fork during S phase
through their interaction with PROLIFERATING
CELL NUCLEAR ANTIGEN (PCNA) (2), where
they specifically monomethylate K27 at newly
incorporated, CAF-1–dependent H3.1 to rapidly
restore this epigenetic mark (Fig. 4D) and prevent overreplication. This model does not rule
out the possibility that some H3.1 might escape
DNA replication–coupled K27 monomethylation
(fig. S5). The inability of ATXR5/6 to methylate H3.3 may contribute to the protection of
transcriptionally active, H3.3-enriched regions
against H3K27me1 and repression during DNA
References and Notes
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Acknowledgments: We thank J. Calarco for critically reading
the manuscript and J. Goodrich, C. Kohler, S. E. Jacobsen,
D. J. Patel, and T. Schalch for materials and advice. Y.J.
was supported by a Louis-Berlinguet postdoctoral fellowship
(Fonds Québécois de la Recherche en Santé/Génome
Québec). P.V. is supported by fellowships from the Deutsche
Akademie der Naturforscher Leopoldina (LPDS 2009-5)
and the Empire State Training Program in Stem Cell
Research (NYSTEM, contract no. C026880). Work in the
Reinberg laboratory is supported by grants from the NIH
(GM064844 and R37GM037120) and the Howard Hughes
Medical Institute. S.D.M. is supported by a grant from the
NIH (GM075060). J.F.C. is supported by grants from the
Canadian Institute of Health Research (BMA-355900) and
the Natural Sciences and Engineering Research Council of
Canada (Discovery Grant 191666) and acknowledges an
Ontario Early Research Award and a Canada Research
Chair in Structural Biology and Epigenetics. This work was
supported by the Howard Hughes Medical Institute–Gordon
and Betty Moore Foundation and by grants from the NSF
(DBI-1025830) and the NIH (GM067014) to R.A.M. R.A.M.
acknowledges a Chaire Blaise Pascal (Region Ile-de-France)
at IBENS, Paris. The Protein Data Bank (PDB) accession
number for the RcATXR5-H3.1-AdoHcy ternary structure is
Materials and Methods
Figs. S1 to S12
11 November 2013; accepted 12 February 2014
Vertebrate Limb Bud Formation
Is Initiated by Localized
Jerome Gros* and Clifford J. Tabin†
Vertebrate limbs first emerge as small buds at specific locations along the trunk. Although a fair
amount is known about the molecular regulation of limb initiation and outgrowth, the cellular
events underlying these processes have remained less clear. We show that the mesenchymal limb
progenitors arise through localized epithelial-to-mesenchymal transition (EMT) of the coelomic
epithelium specifically within the presumptive limb fields. This EMT is regulated at least in part
by Tbx5 and Fgf10, two genes known to control limb initiation. This work shows that limb buds
initiate earlier than previously thought, as a result of localized EMT rather than differential
In 1971, Searls and Janners found that, at early limb stages (Hamburger-Hamilton stage 17 to 18 in the chick), there is a substantial decrease
in proliferation of the flank mesoderm, whereas
higher rates are maintained within the emerging
vertebrate limb buds. Accordingly, they proposed
that localized regulation of proliferation at spe-
cific levels along the body axis is responsible
for limb initiation (1). However, the cellular
properties of the somatopleural lateral plate
cells that give rise to the limb bud have not
During gastrulation, the mesodermal germ layer
is formed through the generation of mesenchy-
mal cells from the epithelial epiblast. However,
shortly after gastrulation a reepithelization occurs
such that essentially the entire embryo is epithelial,
as defined by apical (F-actin) and basal (laminin)
epithelial markers: Not only are the ectoderm,
neural tube, and endoderm epithelial, but also the
notochord, the somites, the intermediate meso-
derm and the lateral plate mesoderm (i.e.,
splanchnopleural and somatopleural mesoderm,
Fig. 1, A and D). At stage 13 in the chick, before
any signs of limb bud formation, the somatopleure
displays epithelial rather than mesenchymal char-
acteristics. Molecular characterization revealed
that, at this stage, F-actin and N-cadherin, as well
as b-catenin and atypical protein kinase C (aPKC),
localize at the apical end of somatopleure cells
(Fig. 1, A and D, and fig. S1, A and D). On the other
hand, vimentin is localized at the basal end of
somatopleural cells, and laminin is deposited only
on the basal side (Fig. 1, A and D, and fig. S1A),
demonstrating that at early stages the somatopleure
is a single cell layer and highly polarized, pseudo-
stratified columnar epithelium. These observations
differ from the previous assumption that limbs
originate from a preexisting mesenchymal pop-
ulation. Forelimb bud mesenchyme first becomes
apparent at stage 14 to 15, whereas the more
posterior hindlimb mesenchyme can be first
observed only at stage 15 to 16, as revealed by
enrichment of vimentin expression and a con-
comitant loss of polarized localization of N-cadherin,
b-catenin, F-actin, and aPKC within somatopleural
cells and basement membrane of laminin break-
down (Fig. 1, B and E, and fig. S1, B, C, E, and
F). Furthermore, mesenchyme in the trunk region
is only seen at stage 17, long after forelimb and
hindlimb mesenchymes have emerged (Fig. 1, C
and F, and fig. S2), and thus out of order relative
to the general rostral-caudal wave of development
Department of Genetics, Harvard Medical School, 77 Avenue
Louis Pasteur, Boston, MA 02115, USA.
*Present address: Department of Developmental and Stem
Cell Biology, Institut Pasteur, 25, 28 rue du Docteur Roux,
75724 Paris, Cedex 15, France.
†Corresponding author. E-mail: email@example.com.