It is becoming increasingly appreciated that changes in chromatin states are intimately tied to changes in cellular potential. During differentiation of many stem/progenitor cells, combinatorial histone marks associated with the promoters and enhancers of poised developmental genes are progressively resolved to active or inactive states in a lineage-specific manner 
. In the germ line, rather than progressive enzymatic modifications of histone marks, the global replacement of existing histones with H3.3 and other variant histones is thought to contribute to a dramatic alteration of lineage potential 
. The unusual developmental history of ectomesenchyme may explain why it too is uniquely sensitive to defects in the incorporation of H3.3 replacement histones. CNC ectomesenchyme derives from the ectoderm yet expresses a gene repertoire and gives rise to derivatives (e.g. skeleton) in common with the mesoderm. One possibility then is that an H3.3-dependent histone replacement event in the NPB, which by its widespread nature is particularly sensitive to defective H3.3 incorporation, endows ectoderm-derived CNC cells with their exceptionally broad lineage potential, including the ability to generate mesoderm-like derivatives (). The requirement for histone replacement (as opposed to enzymatic histone modifications) could reflect a need to overcome a particularly entrenched level of silencing during this fate transition, or alternatively to actively maintain mesoderm-like potential from earlier developmental stages 
. In contrast, the progressive fate restriction of most other embryonic lineages may depend more on an incremental refinement of chromatin structure, such as the post-translational modification of histone tails and re-positioning of existing nucleosomes, and thus be less sensitive to defective H3.3 incorporation.
Model for the role of H3.3-dependent histone replacement during CNC development.
The open permissive chromatin structure at poised regulatory regions is characterized by low nucleosome occupancy and high H3.3 levels, although it remains unclear whether H3.3 simply fulfills a neutral replacement function in such areas of high turnover or has a facilitative destabilizing role 
. An important question is which genes, and in particular which regulatory regions, are targets of histone replacement in the early NPB/CNC. Early CNC genes such as sox10
, whose expression is dramatically perturbed in the presence of mutant D123N H3.3, could be direct targets, although it is unclear why their activation would uniquely depend on H3.3. Alternatively or in parallel, the regulatory regions of a larger set of lineage-specific genes, in particular those associated with later ectomesenchyme development, could be primed by H3.3-dependent histone replacement during early CNC specification. Future chromatin immunoprecipitation studies, currently not feasible due to the rarity of CNC precursors in zebrafish embryos, will clearly be critical for determining which types of genes are direct targets of histone replacement during CNC specification.
Another unresolved question is how H3.3 incorporation is specifically targeted to CNC genes/enhancers. Several putative H3.3 chaperones have been identified, including Hira 
, Daxx 
, and Dek 
. However, effective morpholino-mediated reduction of zebrafish hira
, or dek
gene products, either alone or in combination, failed to cause CNC-specific defects (Figure S6
mouse mutants also do not display CNC-specific defects 
, whereas Daxx−/−
embryos die around E8.5 before CNC can be extensively analyzed 
. Whereas evidence for Hira, Daxx, or Dek mediating H3.3 incorporation in CNC is inconclusive, an intriguing alternative candidate is the chromatin remodeling complex CHD7-PBAF. Losses of CHD7-PBAF components disrupt early CNC specification in a manner similar to mutant D123N H3.3, with reduced expression of early CNC gene expression but not upstream NPB factors 
, and CHD7 genomic localization coincides with H3K4me1 marks which are particularly enriched in H3.3 
. It is less clear to what extent CHD7-PBAF, as well as the histone demethylase jmjD2A, share similar requirements with H3.3 for CNC lineage potential. As in h3f3adb1092
mutants, inhibition of CHD7 function in Xenopus laevis
embryos disrupts craniofacial cartilage development, yet the effects on other NC lineages were not examined. In contrast, depletion of jmjD2A in avians affected development of the CNC-derived ganglia, a structure that is specifically spared in h3f3adb1092
mutants. Whether CHD7-PBAF and jmjD2A act together with H3.3 histone replacement at similar regulatory regions of early CNC genes, or whether these complexes have distinct roles in CNC specification and subsequent lineage diversification, will be fertile areas for future research.
mutants display an initial delay in the expression of all CNC genes examined, which then translates into a more restricted loss of CNC-derived ectomesenchyme. How then does this specific loss of ectomesenchyme relate to the earlier delay in CNC specification? In one model, early-forming CNC cells encounter local environmental cues that promote ectomesenchymal fates 
, with a delay in CNC formation causing cells to miss such cues. Alternatively, the delay in CNC appearance, and its later inability to form the normal range of derivatives, may both result from defects in H3.3-dependent epigenetic remodeling during early CNC stages. Indeed, studies in zebrafish indicate that the future lineage potential of CNC may be determined at very early stages 
. In addition, the cranial pigment lineage defects of h3f3adb1092
mutants indicate more general roles for H3.3 histone replacement in CNC lineage potential. In contrast to trunk NC 
, the existence of a multipotent precursor for all CNC lineages has yet to be demonstrated in vivo. Evidence in avians suggests that the ectomesenchyme arises from a temporally and spatially distinct subpopulation of CNC from precursors of other derivatives 
, and lineage tracing studies in zebrafish show that CNC cells are largely unipotent at early stages of development (13 hpf) 
. Hence, despite individual cultured avian CNC cells being able to generate all derivatives 
, the embryonic CNC may be heterogeneous from initial stages. H3.3 histone replacement could therefore be selectively required for ectomesenchyme and pigment cell potential, but not neuroglial potential, within a common multipotent CNC precursor, or alternatively within distinct subpopulations of ectomesenchyme and pigment cell precursors (). In the future, techniques to more severely perturb H3.3 histone replacement should help reveal whether the apparent lack of CNC neuroglial and trunk NC defects in h3f3adb1092
mutants reflects a fundamentally different mode of chromatin remodeling in the development of these NC populations, as well as other embryonic cell types, or merely a lower sensitivity to loss of H3.3 function.