Page 73 - Biennial Report 2018-20 Jun 2021

undertraining of the molecular pathways perturbed upon MFN2 silencing, Unilever team
decided to carry out microarrays. Therefore, experiments were performed for these arrays and
RNA was isolated from them. Post initial QC, these samples were shared with the Unilever team
for carrying out the microarray studies. The initial data suggested the presence of melanophages
in the dermal section of these samples with significant differences in the number of
melanosomes in these samples. Further, detailed characterization of the samples is going on.

Extensive studies aimed at understanding the signaling cascades involved in increasing
pigmentation upon MFN2 knockdown were carried out. It was established that MFN2 silencing
leads to increase in reactive oxygen species (ROS) levels and ROS in turn augments pigmentation
downstream of MFN2 silencing. Further, it was demonstrated that ROS scavenging with
antioxidant (NAC) results in rescue of hyperpigmentary phenotype observed upon MFN2
knockdown. Moreover, the data showed that melanosome biogenesis and maturation is
enhanced significantly upon MFN2 silencing. Further these observations were corroborated by
performing ultrastructural studies, which showed a significant increase in the number of stage 3
and 4 melanosomes upon MFN2 silencing. Next the role of MFN2 in primary human melanocytes
was examined. MFN2 expression and functional analysis was carried out in the primary human
melanocytes of two independent origin i.e. lightly pigmented melanocytes (Caucasian origin) and
darkly pigmented melanocytes (African origin). It was observed that MFN2 silencing in these
primary melanocytes led to a significant increase in the pigment levels. Further, upon
comparison of MFN2 expression in lightly pigmented melanocytes (Caucasian origin) and darkly
pigmented melanocytes (African origin), we detected lower MFN2 levels in darkly pigmented
melanocytes suggesting that indeed MFN2 could be a critical negative regulator of pigmentation
in humans. Further, for detailed investigation of the molecular pathways perturbed upon MFN2
silencing, microarrays were carried out. Experiments were performed for these arrays and
isolated RNA from them. Post QC, these samples were shipped to Unilever team for carrying out
the microarray studies. The data collected from these arrays is currently under analysis. Finally,
TEM studies were performed on Melasma biopsies and patient matched peri-lesional biopsies.
A detailed quantitation of epidermal features revealed comparable numbers of melanosomes
and mitochondria between the two skin sets. Interestingly, a high number of melanophages
were also observed in the dermis in skin tissues from both sites.

DECIPHERING THE ROLE OF GUIDANCE CUES ON MELANOCYTE MIGRATION,
PATTERNING AND SURVIVAL: TOWARDS REGENERATIVE APPROACH FOR
VITILIGO

This study constituted the following objectives. First, prioritizing chemotactic and
chemorepulsive factors involved in melanocyte migration in zebrafish early embryonic
development. Second, investigating the role specific semaphorin – plexin signalling axis in
zebrafish melanophore patterning. Third, identification of downstream components of the
pathway using biochemical approaches that couple ligand receptor interaction to cell motility.
Fourth, cell biological investigation of the coupling of signaling mechanisms to the migration of
melanocytes. Fifth, set up of a regeneration model system in adult zebrafish to study melanocyte
regeneration and investigate candidate signaling pathways in this system.

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