A number of recent studies have documented scRNA-seq profiles from zebrafish NC-derived cells
[60][79][80][62,81,82]. In general, these have confirmed observations made by standard ISH that premigratory NCCs show early expression of pigment cell markers, indicating early fate specification of these lineages. For example, a recent characterization of 24 hpf trunk NCCs highlighted expression of xanthophore markers
[60][62], confirming old observations of readily-detectable expression in premigratory and migratory trunk NCCs for multiple xanthophore genes; it is worth noting that whilst these cells are likely premigratory, their location adjacent to the epidermis dorsal to the neural tube is also a postmigratory location for xanthophores and melanocytes, so that early differentiation in this region may not be that surprising. However, it has become clear in recent years that these cells, and also migrating NCCs, are often specified to >1 fate, and the scRNA-seq data imply that they may be expressing markers of 2 fates at significant levels
[60][79][80][62,81,82]. Detection of such a signature is very much limited by technical sensitivity to low-level expression in these studies, although that is rarely commented upon. The identified cell clusters also show low-level expression (defined as a low proportion of cells showing positive expression) of key markers of other fates too, hinting at the possibility that these cells retain very broad multipotency; thus, the difference between the scRNA-seq and Nanostring data is most likely one of sensitivity of detection of low-level expression, rather than fundamental differences in the biology observed. Indeed, sensitivity of detection appears to be an issue, since the 24 hpf scRNA-seq dataset does not identify
ltk expression in the putative pigment cell progenitors, despite the fact that this has been repeatedly reported by whole-mount ISH
[39][40][41][41,42,43]. This conclusion is reinforced by Current observations using an optimized RNAscope approach, where researchers see distinct, but low level, expression of
phox2bb, a classic sympathetic/enteric neuron fate specification factor, in unexpected locations, including in numerous glial cells, likely Schwann cell precursors, even in 72 hpf larvae
[81][16]. Researchers would argue that this expression clearly indicates the
potential of these cells to adopt one of these sympathetic and enteric neuronal fates, regardless of whether this is realized in vivo. (A similar situation is observed for pigment cell markers—although the authors do not comment on this, their quantitative Hybridization Chain Reaction ISH shows wide heterogeneity of expression levels of xanthophore markers
aox5, slc2a15b, and
gjb8, with some cells in their figures clearly showing just a handful of mRNAs
[60][62]). Researchers note too that work in mouse and lamprey has recently provided evidence for a contribution of Schwann cell precursors to the enteric neuron population
[83][84][84,85]; Researchers would predict that the same is likely in zebrafish. Finally, researchers note how Current observations support the growing awareness of the breadth of multipotency of Schwann cell precursors
[31][85][86][87][88][89][33,86,87,88,89,90].
3.3. Broad Multipotency of Melanocyte Stem Cells
In mammals, hair pigmentation is derived from melanocytes within the hair bulb, with melanosomes being transferred to the keratinocytes that generate the hair shaft (reviewed in
[47][90][91][92][49,91,92,93]). At each hair molt cycle, the hair shaft is regenerated from new keratinocytes derived from keratinocyte stem cells, and melanin is supplied de novo by new melanocytes generated from melanocyte stem cells (MSCs), retained in the hair bulge. In addition, it has been shown in mammals that cells with the stem cell properties of multipotency and self-renewal and capable of forming neurospheres, can be isolated from diverse embryonic (including the original neural crest stem cells (NCSCs) isolated by the Anderson lab from rodent NC) and adult locations including the skin and the gut (reviewed in
[46][93][94][95][48,94,95,96]). Although sharing the properties noted, these NCSCs show differences in their apparent fate biases, reflecting their source location (e.g.,
[96][97]). MSCs and NCSCs have been shown to be NC derived; MSCs are probably best thought of as another, perhaps highly specialized, type of NCSC, since they have been shown to be multipotent too
[48][50].
Fish are characterized by distinct embryonic/early larval and adult pigment patterns, with the former widely conserved and likely involved in camouflage and protection of the germline against UV damage, and the latter highly divergent, and important for diverse aspects of adult biology
[97][98][99][100][101][102][98,99,100,101,102,103]. Early studies in zebrafish revealed that ablation of pigment cells in the early larva leads to their regeneration, apparently dependent upon a cryptic source of pigment cell precursors
[103][104][104,105]. Subsequently it was shown that the majority of adult pigment cells are generated de novo during metamorphosis, with the likely exception of an unknown proportion of the adult xanthophores which result from a dedifferentiation-proliferation-differentiation process of the embryonically-derived skin xanthophores
[105][106][107][108][109][110][106,107,108,109,110,111]. The source of the de novo pigment cells is hypothesized to be a progenitor cell, likely a stem cell, equivalent to the MSC, and initially named as such. Early studies showed that these cells are NC derived, and that ErbB signaling plays a key role in their being set aside as a quiescent source of pigment cells
[105][106].
However, the identity, number and location of the progenitor cells remain unclear. Pioneering studies indicated that the stem cells are either associated with the peripheral nervous system and/or use the peripheral nerves to deliver their progeny to the skin
[111][112]. A set of cells within the dorsal root ganglia marked with an
mitfa:gfp transgene became the first well-defined source, and were named MSCs
[112][113], but subsequent work indicates there may well be other such sources associated with other parts of the PNS
[113][114]; indeed it may be that Schwann cell precursors throughout the PNS are (or include) these stem cells
[85][111][114][115][86,112,115,116]. Study of the MSCs soon showed that, in addition to melanocytes, they generate xanthophores and iridophores, but also neurons and glia of the PNS
[116][117]. Thus, they appear to be broadly multipotent, and hence equivalent to the diverse NC-derived neural crest stem cells (NCSCs) identified in mammals, although their strict fulfilment of the stem cell property of self-renewal has not been formally tested. From Current perspective here, the key observation is the retention even in the adult of a subset of NC-derived cells that generally sit in a quiescent state, maintained by the local niche, but that retain a clear multipotency when activated, whether at metamorphosis, during regeneration or in tissue maintenance.