Page 72 - Biennial Report 2018-20 Jun 2021

embryos had lower ratiometric values of BCECF (490/440), suggesting an acidic intracellular pH
in melanocytes. A chemical rescue approach was also attempted, by subjecting the mutant
embryos to embryo water buffered at various pH between 24 hpf to 36 hpf, when the
melanocyte maturation commenced. While the acidic pH 5 did not show a rescue, but rather
showed a marginal increase in deformities; alkaline pH 10 rescued the phenotype marginally and
animals with small eyes now had substantially high melanin content. The extent of pH-mediated
rescue was comparable when the animals were injected with mouse Ca14 mRNA. It is interesting
to note that in the ca14fs003-/- mutant number of melanocytes remains unaltered, but the
expression of differentiation effectors is relatively low. Therefore, in the absence of ca14,
melanocytes would still respond to external cues and activate Mitf, however, the extent of
pigmentation would be severely curtailed. The mutant melanophores clearly showed decreased
melanin content, confirming its role in melanocyte maturation.

In the neural crest lineage, progressive fate-restriction and stem cell assignment are critical for
both development and regeneration. While the fate-commitment events have distinct
transcriptional footprints, fate- biasing is often transitory and metastable, and is thought to be
molded by epigenetic programs. Hence the molecular basis of specification is difficult to define.
In this study, a role of a histone variant H2a.z.2 was established in specification of melanocyte
lineage from multipotent neural crest cells. Silencing of H2a.z.2 reduces the number of
melanocyte precursors in developing zebrafish embryos, and from mouse embryonic stem cells
in vitro. It was demonstrated that this histone variant occupies nucleosomes in the promoter of
key melanocyte determinant Mitf and enhances its induction. CRISPR-Cas9 based targeted
mutagenesis of this gene in zebrafish drastically reduces adult melanocytes, as well as their
regeneration. This study, thus, established a histone-based specification code upstream to the
core gene regulatory network in the neural crest lineage of melanocytes.

MITOCHONDRIAL DYNAMICS IN PIGMENTATION

The model system for studying mitochondrial remodeling during pigmentation was established
earlier. Using siRNAs and shRNAs, it was demonstrated that MFN2 (key mitochondrial fusion
regulating protein) negatively regulates pigmentation. It was further shown that the expression
of MFN2 decreases with increase in the cellular pigmentation. More recently, the molecular
mechanisms connecting MFN2 silencing to the enhanced pigmentation have been delineated.
Extensive studies aimed at understanding the signaling cascades involved in increasing
pigmentation upon MFN2 knockdown were carried out. The data suggested that MFN2 silencing
leads to increased ROS levels and scavenging of ROS with antioxidant (NAC), results in rescue of
hyperpigmentary phenotype observed upon MFN2 silencing. Further, it was demonstrated that
melanosome biogenesis and maturation increases upon MFN2 knockdown. Currently, TEM
studies are being performed for further validation of these observations. Excitingly, the MFN2
expression and functional studies in the primary human melanocytes were carried out as well. A
similar trend was observed in primary melanocytes, that is MFN2 silencing led to a significant
increase in the pigment levels in these cells. Further, upon comparison of MFN2 expression in
lightly pigmented melanocytes (Caucasian origin) and darkly pigmented melanocytes (African
origin), lower MFN2 levels were detected in darkly pigmented melanocytes, suggesting that
indeed MFN2 could be a critical negative regulator of pigmentation in humans. For detailed

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