An Essential Role of Variant Histone H3.3 for
Ectomesenchyme Potential of the Cranial Neural Crest
Samuel G. Cox1, Hyunjung Kim2, Aaron Timothy Garnett3, Daniel Meulemans Medeiros3, Woojin An2,
J. Gage Crump1*
1Department of Cell and Neurobiology, Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research, University of Southern California Keck School of
Medicine, Los Angeles, California, United States of America, 2Department of Biochemistry, Norris Comprehensive Cancer Center, University of Southern California Keck
School of Medicine, Los Angeles, California, United States of America, 3Department of Ecology and Evolutionary Biology, University of Colorado, Boulder, Colorado,
United States of America
The neural crest (NC) is a vertebrate-specific cell population that exhibits remarkable multipotency. Although derived from
the neural plate border (NPB) ectoderm, cranial NC (CNC) cells contribute not only to the peripheral nervous system but also
to the ectomesenchymal precursors of the head skeleton. To date, the developmental basis for such broad potential has
remained elusive. Here, we show that the replacement histone H3.3 is essential during early CNC development for these
cells to generate ectomesenchyme and head pigment precursors. In a forward genetic screen in zebrafish, we identified a
dominant D123N mutation in h3f3a, one of five zebrafish variant histone H3.3 genes, that eliminates the CNC–derived head
skeleton and a subset of pigment cells yet leaves other CNC derivatives and trunk NC intact. Analyses of nucleosome
assembly indicate that mutant D123N H3.3 interferes with H3.3 nucleosomal incorporation by forming aberrant H3
homodimers. Consistent with CNC defects arising from insufficient H3.3 incorporation into chromatin, supplying exogenous
wild-type H3.3 rescues head skeletal development in mutants. Surprisingly, embryo-wide expression of dominant mutant
H3.3 had little effect on embryonic development outside CNC, indicating an unexpectedly specific sensitivity of CNC to
defects in H3.3 incorporation. Whereas previous studies had implicated H3.3 in large-scale histone replacement events that
generate totipotency during germ line development, our work has revealed an additional role of H3.3 in the broad potential
of the ectoderm-derived CNC, including the ability to make the mesoderm-like ectomesenchymal precursors of the head
Citation: Cox SG, Kim H, Garnett AT, Medeiros DM, An W, et al. (2012) An Essential Role of Variant Histone H3.3 for Ectomesenchyme Potential of the Cranial
Neural Crest. PLoS Genet 8(9): e1002938. doi:10.1371/journal.pgen.1002938
Editor: David W. Raible, University of Washington, United States of America
Received April 10, 2012; Accepted July 18, 2012; Published September 20, 2012
Copyright: ? 2012 Cox et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This research was supported by a CIRM Training Fellowship to SGC and a CIRM New Faculty Award to JGC. The skeletal mutagenesis screen was
conducted at the University of Oregon with funding by an NIH P0 grant. The funders had no role in study design, data collection and analysis, decision to publish,
or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
The development of multipotent, migratory NC cells was a key
step in the evolution of many of the vertebrate-specific features of
the head . CNC cells generate the skeleton of the face and
anterior skull, as well as supporting development of the brain and
sense organs. NC arises from NPB ectoderm and gives rise to
neurons, glia, and pigment cells along nearly the entire anterior-
posterior extent of the embryo, yet CNC has a greater potential
than trunk NC to generate ectomesenchymal cell types such as
facial skeletal precursors [2–5]. Whereas we know more about how
neuroglial and pigment cell types are specified, how the CNC is
able to generate ectomesenchymal derivatives has remained
unclear . In particular, the ectomesenchyme derives from the
ectoderm yet shares both gene expression signatures and
skeletogenic potential with the embryonic mesoderm, suggesting
that there may be a large-scale fate transition during its
The post-translational modification of histones is emerging as an
important mechanism for regulating cell potential during devel-
opment. In the embryo, early progenitors exhibit a broad potential
with cis-regulatory elements for developmental genes existing in a
poised state characterized by bivalent histone modifications [7–9]
and open permissive chromatin structure . A number of
proteins that regulate chromatin structure are known to play
essential roles in NC development, including CHD7-PBAF
[11,12] and jmjD2A . The CHD7-PBAF chromatin remo-
deler complex has been shown to bind H3K4me1-positive
enhancers of some early CNC genes, and histone demethylase
jmjD2A was found to bind regulatory regions proximal to similar
early NC genes and was associated with demethylation of
repressive H3K9me3 marks. Disruption of CHD7-PBAF and
jmjD2A function similarly resulted in reduced expression of early
CNC gene expression and defects in NC derivatives, with no effect
on upstream NPB/dorsal-neural-tube factors. However, these
types of chromatin remodeling complexes also have more general
roles in embryonic development ; for example CHD7
depletion also results in neural and placodal defects [11,15]. In
contrast, our genetic studies reveal a more selective role of the
replacement histone H3.3 in CNC development.
Histone H3 proteins contribute to the fundamental packing unit
of chromatin, the nucleosome, which consists of two molecules
PLOS Genetics | www.plosgenetics.org1 September 2012 | Volume 8 | Issue 9 | e1002938
each of histones H2A, H2B, H3 and H4, wrapped by two turns of
double-stranded DNA. Whereas canonical H3 histones (H3.1 and
H3.2) are incorporated into chromatin predominantly during
replication, H3.3 is also incorporated outside of replication ,
which has implicated it in various histone replacement events
including gene regulation. In mammals, H3.3 has been associated
with large-scale histone replacement during the specification of
primordial germ cells  and the inactivation of meiotic sex
chromosomes . However, a developmental requirement for
H3.3 outside the germ line has yet to be described. Flies lacking
both H3.3 genes are infertile yet largely adult viable with no
specific developmental abnormalities [19,20]. Similarly, mice
hypomorphic for H3.3A display growth reduction and infertility
but no specific developmental defects . However, the presence
of multiple, identical copies of histone genes, such as H3.3, has
complicated loss-of-function studies, particularly in vertebrates.
Through genetic studies in zebrafish, we have identified a D123N
mutant form of H3.3 that allows us to dominantly interfere with
H3.3 chromatin incorporation during development. In so doing,
we have found that the formation of CNC cells, and their
subsequent lineage potential, are particularly sensitive to defects in
A dominant H3.3 mutation specifically disrupts CNC
In an ethylnitrosourea mutagenesis screen, we identified a
dominant zebrafish mutant, db1092, that exhibited severe reduc-
tions in fli1a:GFP-labeled ectomesenchyme  at 36 hours-post-
fertilization (hpf) (Figure 1a, 1b). As in other vertebrates, the facial
skeleton and anterior skull of the larval zebrafish derive from CNC
ectomesenchyme, with the posterior skull being of mesoderm
origin [23,24]. In db1092 homozygous mutants, nearly all of the
CNC-derived cartilage, bone, and teeth were lost at 5 days-post-
fertilization (dpf), leaving only the mesoderm-derived skull
(Figure 1c, 1d). These skeletal phenotypes were very reminiscent
of those seen in foxd3; tfap2a compound mutants that completely
lack CNC, again confirming the CNC specificity of the head
skeletal defects in db1092 mutants . db1092 homozygous larvae
die by around 7 dpf, presumably due to an inability to feed.
Whereas some db1092 heterozygotes survived to adulthood, others
(Figure 1e, 1f). Due to the shared phenotypes of db1092
homozygous and heterozygous embryos, ‘‘db1092 mutants’’ will
refer to both genotypes unless otherwise stated.
We next examined whether other NC derivatives, such as
pigment cells, glia, and neurons, were affected in db1092 mutants.
of the jaw-supportskeleton
Melanophore pigment cells and their dct-positive precursors were
reduced in the cranial but not trunk regions of db1092 mutants,
and to a lesser extent so were xanthophore pigment cells and their
xdh-positive precursors (Figure 1h, 1j and Figure S1). In contrast,
foxd3-positive peripheral glia (Figure 1l), neurons of the cranial
ganglia (Figure S1), and the dorsal root ganglia and sympathetic
neurons derived from trunk NC (data not shown) were unaffected.
db1092 mutants also displayed mild heart edema, consistent with a
known CNC contribution to the heart , but had an otherwise
remarkably normal morphology at 5 dpf (Figure 1s). In summary,
db1092 mutants have highly specific reductions of CNC deriva-
tives, in particular the ectomesenchymal/skeletal components of
We next used microsatellite polymorphism mapping to place
db1092 within a 464 kb region on linkage group 3 which contained
h3f3a, one of five genes encoding identical H3.3 proteins (Figure 2).
Sequencing of h3f3a revealed a G to A transition in db1092 that
converts aspartic acid 124 to asparagine (referred to as D123N due
to cleavage of the amino-terminal methionine). Given the semi-
dominant nature of db1092, we reasoned that the D123N
mutation might result in a dominantly acting version of H3.3.
To test this, we separately injected mRNAs encoding wild-type
and D123N forms of H3.3 into one-cell-stage zebrafish embryos.
Whereas wild-type H3.3 had no effect on CNC development,
D123N H3.3 caused nearly identical losses of fli1a:GFP-positive
(Figure 1p), and cranial melanophore precursors (Figure 1r) as
seen in the db1092 mutant, confirming D123N H3f3a as the
causative mutation. As reported for other H3.3 genes in zebrafish
, we found that h3f3a was ubiquitously expressed starting at 4
hpf and continuing through 14.5 hpf when CNC has been
specified (Figure 3). At 16.5 and 27 hpf, h3f3a expression remained
largely ubiquitous but was more prominent in the anterior
embryo. As both the endogenous h3f3adb1092gene product, and
in particular the mRNA-injected D123N H3.3, are present
uniformly throughout the embryo at CNC specification stages,
the remarkable specificity of the ectomesenchyme defect is not due
to a preferential expression of this particular h3f3a gene in CNC
precursors. Instead, our data indicate that CNC and ectome-
senchyme development are uniquely sensitive to altered H3.3
Mutant D123N H3.3 dominantly interferes with H3.3
function through aberrant homodimer formation
We next investigated the effect of the D123N substitution on
H3.3 function. When human embryonic kidney cells were
transfected with FLAG-tagged wild-type or D123N H3.3, we
found D123N H3.3 to be under-enriched in purified nucleosomes
compared to wild-type H3.3 (Figure 4a). The D123N mutation
also prevented the incorporation of H3.3 into chromatin in
zebrafish embryos. Whereas mCherry-tagged forms of both wild-
type and D123N H3.3 were nuclear localized during interphase,
during metaphase/anaphase, when the nuclear membrane breaks
down and condensed chromosomes are easily distinguished, wild-
type but not D123N H3.3 co-localized with chromatin marked by
a GFP-tagged H2A.F/Z histone . The failure of D123N H3.3
to associate with chromatin was observed both in the eye
(Figure 4b) and in the pax3a:GFP-positive NPB precursors of
CNC (Figure S2). Time-lapse recordings showed that mCherry-
D123N-H3.3 nuclear fluorescence immediately returned upon
resumption of interphase, indicating that the diffuse metaphase
fluorescence of D123N H3.3 was due to a lack of chromatin
incorporation and not degradation (Figure S3).
The evolution of the vertebrate head was made possible in
large part by the emergence of a new cell population, the
cranial neural crest. These cells contribute to diverse
structures of the head, including most of the skull, yet how
neural crest cells acquire such broad potential during
development has remained a mystery. By studying mutant
zebrafish that lack the neural-crest-derived skull, we find
that the unusual potential of these cells depends on an
‘‘H3.3’’ version of one of the histone proteins that package
their DNA. We propose then that a dramatic change in the
packaging of DNA is a key step in allowing crest cells to
make a wide range of new cell types in the vertebrate
Histone H3.3 Promotes Ectomesenchyme Potential
PLOS Genetics | www.plosgenetics.org2 September 2012 | Volume 8 | Issue 9 | e1002938
Figure 1. A dominant H3.3 mutation results in losses of CNC–derived head skeleton and pigment cells. a, b, fli1a:GFP-labeled arch
ectomesenchyme (arrowheads) is greatly reduced, yet fli1a:GFP-positive endothelial cells (top) are unaffected, in both homozygous and
heterozygous h3f3adb1092mutants at 34 hpf (7/7 mutant; 0/10 wild-type). c, d, Homozygous h3f3adb1092/db1092embryos specifically lack the CNC-
derived head skeleton at 5 dpf (36/71 complete loss; 35/71 partial loss). Diagrams show the CNC-derived cartilage (blue) and bone and teeth (red),
mesoderm-derived cartilage (green), pectoral fin cartilage (black), and eyes (yellow). e, f, h3f3adb1092/+heterozygous larvae exhibit a wide range of
craniofacial defects. In some cases, no defects are observed in the facial skeleton and heterozygotes are adult viable (not shown). In mild cases (e),
dorsal cartilage and bone of the first and second arches are preferentially reduced, including the dorsal hyosymplectic cartilage and opercular bone
of the second arch (arrow). In more severe cases (f), the cartilage and bone of the first arch and dorsal second arch are greatly reduced, with the
anterior neurocranium and the posterior ceratobranchial cartilages being less affected. The frequency of skeletal phenotypes in h3f3adb1092/+
Histone H3.3 Promotes Ectomesenchyme Potential
PLOS Genetics | www.plosgenetics.org3 September 2012 | Volume 8 | Issue 9 | e1002938
teeth). Craniofacial development is unaffected in surviving hira (l),
daxx (n) and dek (p) morpholino-treated individuals and uninjected
controls (k, m, o) (n$35 for each). hira morpholino-injected
embryos did exhibit a high level of death after 24 hpf but prior to 5
dpf (morpholino injected, 55.1%; uninjected, 0%) and a curved/
kinked tail phenotype in surviving 5 dpf larvae (morpholino
injected, 48.6%; uninjected, 0%). q–t, Compared to uninjected
controls (q & s), embryos injected with a combination of all three
morpholinos at 200 mM had no defects in CNC sox10 expression
(r) (n$9 for each) or development of the larval head skeleton (t)
(n$66 for each). un, uninjected; MO, morpholino. Scale
Fish care was provided by Gabriel Finch, Megan Matsutani, Pablo Castillo,
and Corey Gingerich. We thank Cheng-Ming Chuong, Martin Pera, Judd
Rice, and Frank Stellabotte for helpful comments on the manuscript.
Conceived and designed the experiments: SGC HK JGC. Performed the
experiments: SGC HK JGC. Analyzed the data: SGC JGC WA.
Contributed reagents/materials/analysis tools: ATG DMM. Wrote the
paper: SGC JGC.
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