Endoplasmic reticulum (ER)-plasma membrane (PM) junctions form functionally active microdomains that connect intracellular and extracellular environments. While the key role of these interfaces in maintenance of intracellular Ca levels has been uncovered in recent years, the functional significance of ER-PM junctions in non-excitable cells has remained unclear. Here, we show that the ER calcium sensor protein STIM1 (stromal interaction molecule 1) interacts with the plasma membrane-localized adenylyl cyclase 6 (ADCY6) to govern melanogenesis. The physiological stimulus α-melanocyte-stimulating hormone (αMSH) depletes ER Ca stores, thus recruiting STIM1 to ER-PM junctions, which in turn activates ADCY6. Using zebrafish as a model system, we further established STIM1's significance in regulating pigmentation STIM1 domain deletion studies reveal the importance of Ser/Pro-rich C-terminal region in this interaction. This mechanism of cAMP generation creates a positive feedback loop, controlling the output of the classical αMSH-cAMP-MITF axis in melanocytes. Our study thus delineates a signaling module that couples two fundamental secondary messengers to drive pigmentation. Given the central role of calcium and cAMP signaling pathways, this module may be operative during various other physiological processes and pathological conditions.

Macroautophagy/autophagy is a dynamic and inducible catabolic process that responds to a variety of hormonal and environmental cues. Recent studies highlight the interplay of this central pathway in a variety of pathophysiological diseases. Although defective autophagy is implicated in melanocyte proliferation and pigmentary disorders, the mechanistic relationship between the 2 pathways has not been elucidated. In this study, we show that autophagic proteins LC3B and ATG4B mediate melanosome trafficking on cytoskeletal tracks. While studying melanogenesis, we observed spatial segregation of LC3B-labeled melanosomes with preferential absence at the dendritic ends of melanocytes. This LC3B labeling of melanosomes did not impact the steady-state levels of these organelles but instead facilitated their intracellular positioning. Melanosomes primarily traverse on microtubule and actin cytoskeletal tracks and our studies reveal that LC3B enables the assembly of microtubule translocon complex. At the microtubule-actin crossover junction, ATG4B detaches LC3B from melanosomal membranes by enzymatic delipidation. Further, by live-imaging we show that melanosomes transferred to keratinocytes lack melanocyte-specific LC3B. Our study thus elucidates a new role for autophagy proteins in directing melanosome movement and reveal the unconventional use of these proteins in cellular trafficking pathways. Such crosstalk between the central cellular function and housekeeping pathway may be a crucial mechanism to balance melanocyte bioenergetics and homeostasis.

Tanning response and melanocyte differentiation are mediated by the central transcription factor MITF. This involves the rapid and selective induction of melanocyte maturation genes, while concomitantly the expression of other effector genes is maintained. In this study, using cell-based and zebrafish model systems, we report on a pH-mediated feed-forward mechanism of epigenetic regulation that enables selective amplification of the melanocyte maturation program. We demonstrate that MITF activation directly elevates the expression of the enzyme carbonic anhydrase 14 (CA14). Nuclear localization of CA14 leads to an increase of the intracellular pH, resulting in the activation of the histone acetyl transferase p300/CBP. In turn, enhanced H3K27 histone acetylation at selected differentiation genes facilitates their amplified expression via MITF. CRISPR-mediated targeted missense mutation of CA14 in zebrafish results in the formation of immature acidic melanocytes with decreased pigmentation, establishing a central role for this mechanism during melanocyte differentiation in vivo. Thus, we describe an epigenetic control system via pH modulation that reinforces cell fate determination by altering chromatin dynamics.

In the neural crest lineage, progressive fate restriction and stem cell assignment are crucial for both development and regeneration. Whereas fate commitment events have distinct transcriptional footprints, fate biasing is often transitory and metastable, and is thought to be moulded by epigenetic programmes. Therefore, the molecular basis of specification is difficult to define. In this study, we established a role for a histone variant, H2a.z.2, in specification of the melanocyte lineage from multipotent neural crest cells. H2a.z.2 silencing reduces the number of melanocyte precursors in developing zebrafish embryos and from mouse embryonic stem cells in vitro. We demonstrate that this histone variant occupies nucleosomes in the promoter of the 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. Thereby, our study establishes the role of a histone variant upstream of the core gene regulatory network in the neural crest lineage. This epigenetic mark is a key determinant of cell fate and facilitates gene activation by external instructive signals, thereby establishing melanocyte fate identity.

Bone is an evolutionary novelty of vertebrates, likely to have first emerged as part of ancestral dermal armor that consisted of osteogenic and odontogenic components. Whether these early vertebrate structures arose from mesoderm or neural crest cells has been a matter of considerable debate. To examine the developmental origin of the bony part of the dermal armor, we have performed in vivo lineage tracing in the sterlet sturgeon, a representative of nonteleost ray-finned fish that has retained an extensive postcranial dermal skeleton. The results definitively show that sterlet trunk neural crest cells give rise to osteoblasts of the scutes. Transcriptional profiling further reveals neural crest gene signature in sterlet scutes as well as bichir scales. Finally, histological and microCT analyses of ray-finned fish dermal armor show that their scales and scutes are formed by bone, dentin, and hypermineralized covering tissues, in various combinations, that resemble those of the first armored vertebrates. Taken together, our results support a primitive skeletogenic role for the neural crest along the entire body axis, that was later progressively restricted to the cranial region during vertebrate evolution. Thus, the neural crest was a crucial evolutionary innovation driving the origin and diversification of dermal armor along the entire body axis.

22 differentiation, epigenetics and zebrafish 23 24 Running title: pH regulates melanocyte maturation 25 pH regulates melanocyte maturation 2 Abstract 26 Tanning response and melanocyte differentiation are mediated by the central transcription factor MITF. 27Enigmatically, these involve rapid and selective induction of melanocyte maturation genes, while 28 concomitantly maintaining the expression of other effectors. In this study using cell-based and zebrafish 29 model systems, we elucidate a pH mediated feed-forward mechanism of epigenetic regulation that enables 30 selective amplification of melanocyte maturation program. We demonstrate that MITF activation directly 31 elevates the expression of Carbonic Anhydrase 14 (Ca14) enzyme. Nuclear localized Ca14 increases the 32 intracellular pH, resulting in the activation of histone acetyl transferase activity of p300/CBP. In turn 33 enhanced H3K27 histone acetylation marks of select differentiation genes facilitates their amplified 34 expression by MITF. CRISPR-mediated targeted missense mutation of CA14 in zebrafish results in 35 immature acidic melanocytes with decreased pigmentation, establishing the centrality of this mechanism 36 in rapidly activating melanocyte differentiation. Thereby we reveal a novel epigenetic control through pH 37 modulation that reinforces a deterministic cell fate by altering chromatin dynamics. 38 39 pH regulates melanocyte maturation 102 pH regulates melanocyte maturation 6 Results 103 Alkaline intracellular pH induces pigmentation by enhancing the melanogenesis gene expression 104During the culture of B16 cells in DMEM medium CO 2 -HCO 3 buffering system maintains media pH. 105Hence we resorted to establish the pH-pigmentation link by modulating the prevailing CO 2 levels. We set 106 up progressive pigmentation using the B16 cells as described earlier (Natarajan et al, 2014). In this set up 107 the cells progressively activate the melanogenesis gene expression program and increase pigmentation 108 over a course of 8 to 12 days. To alter the pH, 10% CO 2 levels were tested. We measured the extracellular 109 pH (pH e ) using the standard pH electrode under controlled conditions of temperature and CO 2 saturation. 110While the cells grown in 5% CO 2 showed an increase in pH e from around 7.4 to 7.8 on day 4 after 111 induction of the pigmentation program, the 10% CO 2 sustained a constant pH of around 7.4 112( Supplementary Fig 1A). This trend was reflected in the intracellular pH (pH i ), which is close to 7.9 on 113 day 4 under the routine 5% CO 2 condition. However, under the 10% CO 2 the pH remained close to 7.0, 114 similar to the day 0 where the cells are depigmented (Fig 1A). When the cells were assessed for the 115 cumulative accumulation of melanin on day 8, under the 10% CO 2 condition we observed depigmented 116 cells (Fig 1B). Melanin content assay performed using synthetic melanin standard confirmed that the 117 level of melanin is significantly low (Supplementary Fig S1B). Further electron microscopic evaluation 118 of day...

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