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. 2012 Sep;8(9):e1002938.
doi: 10.1371/journal.pgen.1002938. Epub 2012 Sep 20.

An essential role of variant histone H3.3 for ectomesenchyme potential of the cranial neural crest

Samuel G Cox  1 , Hyunjung Kim, Aaron Timothy Garnett, Daniel Meulemans Medeiros, Woojin An, J Gage Crump
Affiliations

An essential role of variant histone H3.3 for ectomesenchyme potential of the cranial neural crest

Samuel G Cox et al. PLoS Genet. 2012 Sep.

Abstract

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 skeleton.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
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 h3f3adb1092 mutants at 34 hpf (7/7 mutant; 0/10 wild-type). c, d, Homozygous h3f3adb1092/db1092 embryos 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/+ heterozygous larvae is highly variable between clutches. g, h, At 27 hpf, dct-positive melanophore precursors are selectively missing anterior to the ear (arrowheads) in both homozygous and heterozygous h3f3adb1092 embryos (5/5 mutant; 0/5 wild-type). i, j, At 27 hpf, cranial xdh-positive xanthophore precursors are mildly reduced in both homozygous and heterozygous h3f3adb1092 embryos (6/8 mutant; 0/4 wild-type). k, l, Wild-type and both homozygous and heterozygous h3f3adb1092 embryos have comparable numbers of foxd3-positive glial cells at 24 hpf (4 mutant; 5 wild-type). m-r, D123N h3f3a mRNA-injected but not wild-type h3f3a mRNA-injected embryos lack fli1a:GFP-positive ectomesenchyme (9/19 D123N; 0/12 wild-type), CNC-derived head skeleton (24/45 D123N; 0/21 wild-type) and cranial dct-positive melanophore precursors (anterior to the ear: arrowheads) (7/14 D123N; 0/11 wild-type). fli1a:GFP-positive blood vessels are unaffected (arrows). s, Except for the loss of the majority of the skull (no facial structures below the level of the eye: arrowheads) and mild heart edema (arrows), the overall morphologies of wild-type, homozygous and heterozygous h3f3adb1092, wild-type h3f3a-injected, and D123N h3f3a-injected larvae are indistinguishable at 5 dpf. Melanophores (black) and xanthophores (yellow) are also largely normal. Except for panels e and f, homozygous h3f3adb1092 examples are shown. Scale bars: a, b, m & n, 50 µm; c–l, o–s, 250 µm.
Figure 2
Figure 2. Identification of the h3f3adb1092 lesion.
a, The db1092 allele was crossed to the highly polymorphic WIK strain for linkage analysis. As the db1092 mutation is semi-dominant, we enriched for putative heterozygotes by selecting for partial head skeletal loss, and events were scored as recombination only if both chromosomes displayed the wild-type WIK polymorphism. Using a set of microsatellite ‘Z’ markers spanning the zebrafish genome, we placed db1092 on linkage group 3 near Z3725 and Z20058, and subsequent linkage analysis placed it between Z63643 and Z66457. Recombinants per 1065 meioses are listed above each marker. Sequencing of 3′ UTRs identified single nucleotide polymorphisms (SNPs) that created or destroyed restriction sites between the mutant and WIK chromosomes. These SNPs (identified by their position in millions of base pairs) and Z48485 were then used to map db1092 to a 464 kb interval. b, Electrophoretograms show a G to A transition in the h3f3a gene of db1092 homozygotes. c, Schematic of the H3.3 variant histone protein encoded by h3f3a. The db1092 mutation results in a D123N substitution near the C-terminus of the core domain.
Figure 3
Figure 3. h3f3a is ubiquitously expressed throughout embryogenesis.
a–i, Lack of h3f3a expression at the one-cell stage shows that h3f3a mRNA is not maternally provided. From 4–14.5 hpf, h3f3a is expressed ubiquitously throughout the embryo. By 16.5 hpf and 27 hpf, h3f3a expression is still widespread, with higher levels apparent in the anterior part of the embryo, including the CNC-derived ectomesenchyme of the pharyngeal arches (arrows) at 27 hpf. Scale bars = 250 µm.
Figure 4
Figure 4. The dominant D123N mutation prevents chromatin incorporation and promotes the formation of aberrant H3 homodimers.
a, Western blots show α-FLAG immunostaining of nuclear extracts or purified mononucleosome fractions from HEK cells transfected with vector alone or FLAG-tagged H3.3 (f:H3.3) vectors. Wild-type and D123N f:H3.3 proteins are expressed at equal levels in total extract, but wild-type f:H3.3 is present at much higher levels in the nucleosome fraction (consistent over three replicate experiments). α-FLAG immunoprecipitation from purified nucleosomes shows that wild-type but not D123N f:H3.3 is incorporated into nucleosomes containing H2A, H2B, H3, and H4 (consistent over three replicate experiments). b, Confocal images from H2A.F/Z:GFP embryos expressing wild-type and D123N versions of mCherry(m)H3.3 and mCherry(m)H3.2 fusion proteins. Merged images show that whereas all H3 proteins are nuclear localized in surrounding non-mitotic cells, wild-type mH3.3 and mH3.2, but not D123N mH3.3 and mH3.2, co-localize with H2A.F/Z:GFP in the chromosomes of metaphase/anaphase cells (arrowheads) after nuclear envelope breakdown (wild-type mH3.3, 11/11 cells in 2 embryos; D123N mH3.3, 0/25 cells in 3 embryos; wild-type mH3.2, 21/21 cells in 3 embryos; D123N mH3.2, 0/16 cells in 2 embryos). c, α-FLAG, α-H3 and α-H4 western blots for samples immunoprecipitated by α-FLAG from nuclear extracts of f:H3.3-transfected HEK cells. Recombinant octamer is used as a reference. Whereas both endogenous H3 and H4 co-immunoprecipitate with the wild-type f:H3.3 protein (*), H3 but not H4 co-immunoprecipitates with D123N f:H3.3 (asterisk marks the larger recombinant f:H3.3 protein). Results were consistent over three replicate experiments. d, mRNA injection of D123N mH3.3 (8/17), but not wild-type mH3.3 (0/26), wild-type mH3.2 (0/19), or D123N mH3.2 (0/18), results in loss of the CNC-derived head skeleton at 4 dpf. Scale bars: b, 10 µm; d, 250 µm.
Figure 5
Figure 5. Injection of wild-type H3.3 RNA and reduction of mutant H3.3 levels both rescue craniofacial skeletal development in h3f3adb1092 mutants.
a, b, Results from scoring the severity of craniofacial losses in 5 dpf larval head skeletons from h3f3adb1092 siblings injected with 450 ng/µl mRNA encoding wild-type H3.3 or a control kikGR fluorescent protein. The scoring system ranges from Grade 0 (wild-type phenotype) to Grade 5 (complete loss of CNC derivatives); see Materials and Methods for more detail. H3.3-mRNA-injected h3f3adb1092 homozygotes (a) and heterozygotes (b) exhibited a decrease in the severity of h3f3adb1092 craniofacial phenotypes over kikGR-RNA-injected controls (significant by Fisher's exact test: homozygotes, p = 7.7E-05; heterozygotes, p = 1.0E-04). c, An antisense morpholino oligonucleotide was designed to inhibit splicing at the exon 3/intron 3–4 boundary (green arrowhead) of the h3f3a transcript. Morpholinos were injected into one-cell-stage h3f3adb1092 embryos at 400 µM. d, Morpholino efficacy was demonstrated by PCR amplification between exons flanking the targeted splice junction from 10 hpf cDNA from 20 pooled embryos (position of primers shown as red arrows in c). Compared to the sample from uninjected (un) embryos, the morpholino-treated sample (MO) exhibited a partial decrease in PCR product representing spliced transcript (295 bp) and a concomitant increase in un-spliced PCR product (390 bp). e, f, Compared to uninjected siblings, both morpholino-injected h3f3adb1092 homozygotes (e) and heterozygotes (f) exhibited a decrease in the severity of h3f3adb1092 craniofacial phenotypes (significant by Fisher's exact test: homozygotes, p = 2.0E-04; heterozygotes, p = 6.3E-06). (g) Craniofacial development in wild-type embryos was unaffected by morpholino injection.
Figure 6
Figure 6. H3.3 functions at the NPB–CNC transition.
a–f, Expression of msxb, pax3a, zic2a, and tfap2a at 10 hpf and msxb and pax3a at 11 hpf is indistinguishable between wild types and both homozygous and heterozygous h3f3adb1092 mutants (mut) (n≥10 for each). g–k, At 11 hpf, both homozygous and heterozygous h3f3adb1092 mutants have severe reductions in the expression of snai2 (4/4 mut; 0/4 wt), sox10 (10/12 mut; 0/6 wild-type), foxd3 (9/9 mut; 0/5 wt), tfap2a (7/7 mut; 0/4 wt), and sox9b (8/8 mut; 0/3 wt). l, sox10 expression is also lost in embryos injected with D123N (10/12) but not wild-type (0/16) h3f3a mRNA. m, In both homozygous and heterozygous h3f3adb1092 embryos, sox10 expression partially recovers by 16.5 hpf yet is specifically reduced in presumptive CNC ectomesenchyme domains (arrows) (6/6 mut; 0/4 wt). An increase in sox10-positive cells is evident in the mutant dorsal neural tube (insert) between the sox10-positive otic placodes (arrowheads) which are unaffected in mutants. n, At 16.5 hpf, dlx2a expression in three streams of migrating ectomesenchyme is reduced in both homozygous and heterozygous h3f3adb1092 mutants (6/7 mut; 0/5 wt). o, The 16.5 hpf ectomesenchyme expression (arrows) of twist1a is reduced in both homozygous and heterozygous h3f3adb1092 mutants yet paraxial mesoderm expression is unaffected (white arrowheads) (8/8 mut; 0/5 wt). In all panels, homozygous h3f3adb1092 examples are shown. All images are dorsal views with anterior to the left. Scale bars: 250 µm.
Figure 7
Figure 7. Trunk NC is largely unaffected in h3f3adb1092 mutants.
a–c, crestin expression at 11.7 hpf shows similar amounts of trunk NC in wild-type and both homozygous and heterozygous h3f3adb1092 embryos (n = 5 for each genotype). d–i, Trunk views of sox9b expression at 11.7 hpf show that trunk NC specification is largely normal in both homozygous and heterozygous h3f3adb1092 mutants (n = 10 for each genotype). Cranial views of the same embryos show reduced amounts of sox9b-expressing CNC. Arrows show the sox9b-positive otic placodes that are unaffected in mutants. Scale bars = 250 µm.
Figure 8
Figure 8. H3.3 function is required tissue- and cell-autonomously for CNC development.
a, Wild-type cells were transplanted unilaterally into the CNC precursor domain of h3f3adb1092/db1092 homozygous mutants at 6 hpf. b, Compared to the non-recipient control side (bottom), expression of the early CNC marker snai2 is restored in the recipient side at 11hpf (top) (n = 17/29 with rescue). c–e, fli1a:GFP (green) marks CNC ectomesenchyme of the pharyngeal arches at 30 hpf and facial skeletal elements at 5 dpf. The red fluorescent dye, Alexa568, marks transplanted wild-type cells, whereas both donor and host cells harbor the fli1a:GFP transgene. When transplanted into an h3f3adb1092/db1092 homozygous host, wild-type Alexa568+ CNC precursors contribute to pharyngeal arch ectomesenchyme and rescue arch size (c) and form wild-type cartilage and bone (e). In contrast, the non-recipient control side (d) has reduced pharyngeal arch ectomesenchyme. Whereas wild-type donor cells appear yellow due to red Alexa568 and green fli1a:GFP, mutant host cells have only fli1a:GFP and hence appear green. Rescue of arch size was observed in 8/11 cases. f, Alcian staining at 5 dpf shows that cartilage is restored to half the face in an h3f3adb1092/db1092 larvae that received an unilateral wild-type CNC precursor transplant. Compare the recipient side (left) to the control side that forms little facial cartilage (right). Skeletal rescue was observed in 21/30 cases. g, Individual cells of 32-cell stage sox10:GFP embryos were injected with mRNA encoding mCherry-tagged versions of wild-type or D123N H3.3 to generate mosaic mCherry-H3.3 expression at later stages. h, Soon after the appearance of GFP-labeled CNC at approximately 11 hpf, mosaic embryos were assessed for incorporation of mCherry-H3.3-expressing cells (red) into the sox10:GFP-positive CNC domain (green). i/i′/i″ and j/j′/j″, Confocal images from sox10:GFP embryos with mosaic expression of wild-type (i/i′/i″) and D123N (j/j′/j″) versions of mCherry-H3.3. Cells doubly-positive for wild-type mCherry-H3.3 (red) and GFP (green) (arrowheads) were observed within the CNC domain (7/14 cells over 4 embryos), whereas mutant D123N mCherry-H3.3 cells within the CNC domain failed to up-regulate sox10:GFP (arrowheads) (0/18 cells over 4 embryos). Only cells with strong mCherry-H3.3 were used in the analysis. hs: hyosymplectic cartilage, pq: palatoquadrate cartilage, ch: ceratohyal cartilage, op: opercular bone. Scale bars: b, f, 250 µm; c–e, 50 µm; i and j,10 µm.
Figure 9
Figure 9. Cell death in h3f3adb1092 embryos.
a–c, Lysotracker Red staining marks similar amounts of dying cells in wild-type (n = 2), h3f3adb1092/+ heterozygous (n = 5), and h3f3adb1092/db1092 homozygous (n = 3) embryos at 12.5 hpf. The bright staining in the bottom of each panel is the yolk. d–f, At 16 hpf, increased Lysotracker Red staining (arrows) was evident in the dorsal neural tube of h3f3adb1092/+ heterozygotes (2/2) and h3f3adb1092/db1092 homozygotes (3/3) but not wild types (0/4). These dying cells were located in a similar position to where CNC forms in wild-type embryos. Scale bar = 50 µm.
Figure 10
Figure 10. Model for the role of H3.3-dependent histone replacement during CNC development.
At the early embryonic blastula stage, cells have a broad potential with cis-regulatory elements for developmental genes existing in a “poised” chromatin state. After gastrulation occurs to form the three major germ layers (ectoderm, mesoderm, and endoderm), genes associated with a particular germ layer are activated or maintained in a poised state, whereas genes for other layers are strongly repressed at the chromatin level. The cranial neural crest (CNC) is unusual in that it is derived from ectoderm yet can give rise to mesoderm-like derivatives such as skeleton. H3.3-dependent histone replacement could thus be required to remodel the enhancers of mesodermal genes needed for ectomesenchymal fates, with the distinctive role of H3.3 in CNC correlating with the need to derepress mesodermal enhancers that have been previously silenced in the ectoderm germ layer (1). Alternatively H3.3 incorporation could act to maintain mesoderm-like potential in the CNC ectoderm from an earlier time in development (2). It also remains unresolved the extent to which ectomesenchyme derivatives (e.g. head skeleton) and non-ectomesenchyme derivatives (e.g. pigment, glia, and neurons) derive from a common multipotent precursor. Hence, the cranial pigment and ectomesenchyme defects of h3f3adb1092 mutants could arise from altered histone replacement in a common multipotent precursor, or alternatively from independent defects in different subsets of heterogeneous CNC with more limited potential.
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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.

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