Page 74 - Biennial Report 2018-20 Jun 2021

To phenotypically screen for molecular signaling events essential for directed melanocyte
migration in -vivo, the role of guidance cues is being investigated with respect to melanocyte
migration during the early developmental stages and further during regeneration. Different
classes of proteins have been classified as guidance cues: netrins, slits, ephrin- Eph signaling
complexes and semaphorin-plexin signaling complexes. The expression signatures of these
factors (guidance cues) were analyzed in vitiligo array.

Further, to test the role of differentially expressed cues (semaphorin –plexin signaling axis)
following strategies were employed. For developmental melanocyte migration, in-vivo
knockdown of plexin receptors in zebrafish embryos was planned. To study melanocyte
migration during regeneration, a melanophore regeneration model in zebrafish would be
established. For studying the effect on metamorphic and regenerative melanocytes, CRISPR-KO
of plexin receptors was planned.

The preliminary observations suggested that sema3e - plexin D1 signaling has a significant role
in melanocyte patterning during early pigment pattern formation in zebrafish embryos. There is
also evidence suggesting that PlexinD1 knock down causes a loss of migration property in
metamorphic and regenerative pools of melanocyte precursors. To investigate the possibility,
PlxnD1 CRISPR-KO is being generated.

To understand the differences in migration potential of melanocytes as they attain functional
maturity: Terminally differentiated melanocytes have a limited turn-over rate and are not known
to have any tendency toward migration. These cells, however, are fully functional and produce
the pigment melanin. Interestingly, melanocytes originate from neural crest cells that are known
to be one of the most migratory populations of cells.

Prioritising chemotactic factors for their role in melanocyte migration: Transgenic melanocyte
reporter lines in Zebrafish were generated and maintained in the institutional fish facility.
Melanocytes were sorted out from zebrafish embryos during various stages of its early
development using FACS. Extensive gene expression analysis from these sorted populations
revealed the varying ability of these cells to respond to the environmental cues. Interestingly,
the most prominent guidance pathway that showed differential expression pattern across
various stages of melanocyte differentiation happened to be of Semaphorin-plexin signaling
mechanism. Other classical guidance cues, such as eph, ephrin, slits and netrins were poorly
represented across all the stages. Further, it was also observed that the expression profiles of
these guidance cues did significantly vary between the lesions (v) and normal epidermal tissue
(N) of patients with Vitiligo (Chronic melanocyte degeneration condition). This indicated towards
a possible role of semaphorin-plexin cues in maintenance of melanocytes in epidermis. An in-
vitro pigment oscillator model was set-up to understand melanogenesis and required cellular
adaptation(s) in detail. The expression profiles of plexin receptors during the course of
pigmentation revealed that they are reversely correlated with genes involved in melanin
synthesis. The gene expression analyses, therefore clearly indicated that Sema- Plxn signaling is
differentially regulated across various aspects of melanocyte life and since all these aspects are
dependent on inter- cellular crosstalk possibly such guidance helps in establishing specific
cellular connections.
Semaphorin-plexin signaling in early melanocyte development: Knock-down of plexin receptors
with morpholino resulted in various defects as previously reported by other studies especially
those pertaining to vasculature. However, even at much lower dosage of PlxnD1 morpholino

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