Pigment patterns in the integument have long-attracted attention from both scientists and nonscientists alike since their natural attractiveness combines with their excellence as models for the general problem of pattern formation. Pigment cells are formed from the neural crest and must migrate to reach their final locations. In this review, we focus on our current understanding of mechanisms underlying the control of pigment cell migration and patterning in diverse vertebrates. The model systems discussed here -chick, mouse, and zebrafish -each provide unique insights into the major morphogenetic events driving pigment pattern formation. In birds and mammals, melanoblasts must be specified before they can migrate on the dorsolateral pathway. Transmembrane receptors involved in guiding them onto this route include EphB2 and Ednrb2 in chick, and Kit in mouse. Terminal migration depends, in part, upon extracellular matrix reorganization by ADAMTS20. Invasion of the ectoderm, especially into the feather germ and hair follicles, requires specific signals that are beginning to be characterized. We summarize our current understanding of the mechanisms regulating melanoblast number and organization in the epidermis. We note the apparent differences in pigment pattern formation in poikilothermic vertebrates when compared with birds and mammals. With more pigment cell types, migration pathways are more complex and largely unexplored; nevertheless, a role for Kit signaling in melanophore migration is clear and indicates that at least some patterning mechanisms may be highly conserved. We summarize the multiple factors thought to contribute to zebrafish embryonic pigment pattern formation, highlighting a recent study identifying Sdf1a as one factor crucial for regulation of melanophore positioning. Finally, we discuss the mechanisms generating a second, metamorphic pigment pattern in adult fish, emphasizing recent studies strengthening the evidence that undifferentiated progenitor cells play a major role in generating adult pigment cells.

Production of high-value recombinant proteins in transgenic seeds is an attractive and economically feasible alternative to conventional systems based on mammalian cells and bacteria. In contrast to leaves, seeds allow high-level accumulation of recombinant proteins in a relatively small volume and a stable environment. We demonstrate that single-chain variable fragment (scFv)-Fc antibodies, with N-terminal signal sequence and C-terminal KDEL tag, can accumulate to very high levels as bivalent IgG-like antibodies in Arabidopsis thaliana seeds and illustrate that a plant-produced anti-hepatitis A virus scFv-Fc has similar antigen-binding and in vitro neutralizing activities as the corresponding full-length IgG. As expected, most scFv-Fc produced in seeds contained only oligomannose-type N-glycans, but, unexpectedly, 35-40% was never glycosylated. A portion of the scFv-Fc was found in endoplasmic reticulum (ER)-derived compartments delimited by ribosomeassociated membranes. Additionally, consistent with the glycosylation data, large amounts of the recombinant protein were deposited in the periplasmic space, implying a direct transport from the ER to the periplasmic space between the plasma membrane and the cell wall. Aberrant localization of the ER chaperones calreticulin and binding protein (BiP) and the endogenous seed storage protein cruciferin in the periplasmic space suggests that overproduction of recombinant scFv-Fc disturbs normal ER retention and proteinsorting mechanisms in the secretory pathway.glycosylation ͉ molecular farming ͉ recombinant antibody ͉ subcellular localization T ransgenic plants for the production of high-value recombinant proteins are a promising alternative to conventional recombinant protein production systems, such as bacteria, yeast, animal, and insect cell cultures (1). One of the most important factors driving research in this field is yield improvement, because of its significant impact on economic feasibility (2). Strategies to increase recombinant protein yield in plants include development of better expression cassettes, improvement of protein stability and accumulation by using specific subcellular targeting signals, and development of downstream processing technologies (3). In this perspective, seed-based platforms are particularly attractive because they allow recombinant proteins to stably accumulate at a relatively high concentration in a compact biomass, which is beneficial for extraction and downstream processing (4). By using a seed-specific expression cassette based on the regulatory signals of seed storage proteins of common bean (Phaseolus vulgaris), and by targeting the recombinant protein to the endoplasmic reticulum (ER), we obtained the highest yields of recombinant proteins in plants described so far: a single-chain variable fragment (scFv) accumulated to levels in excess of 36% of total soluble protein (TSP) in homozygous Arabidopsis seeds, while retaining its antigen-binding activity and affinity (5).For some applications, fusion of the scFv with the Fc doma...

Using immunogold electron microscopy, we have investigated the relative distribution of two types of vacuolar sorting receptors (VSR) and two different types of lumenal cargo proteins, which are potential ligands for these receptors in the secretory pathway of developing Arabidopsis embryos. Interestingly, both cargo proteins are deposited in the protein storage vacuole, which is the only vacuole present during the bent‐cotyledon stage of embryo development. Cruciferin and aleurain do not share the same pattern of distribution in the Golgi apparatus. Cruciferin is mainly detected in the cisand medial cisternae, especially at the rims where storage proteins aggregate into dense vesicles (DVs). Aleurain is found throughout the Golgi stack, particularly in the transcisternae and transGolgi network where clathrin‐coated vesicles (CCVs) are formed. Nevertheless, aleurain was detected in both DV and CCV. VSR‐At1, a VSR that recognizes N‐terminal vacuolar sorting determinants (VSDs) of the NPIR type, localizes mainly to the transGolgi and is hardly detectable in DV. Receptor homology‐transmembrane‐RING H2 domain (RMR), a VSR that recognizes C‐terminal VSDs, has a distribution that is very similar to that of cruciferin and is found in DV. Our results do not support a role for VSR‐At1 in storage protein sorting, instead RMR proteins because of their distribution similar to that of cruciferin in the Golgi apparatus and their presence in DV are more likely candidates. Aleurain, which has an NPIR motif and seems to be primarily sorted via VSR‐At1 into CCV, also possesses putative hydrophobic sorting determinants at its C‐terminus that could allow the additional incorporation of this protein into DV.

Subscribe to PCMR and stay up-to-date with the only journal committed to publishing basic research in melanoma and pigment cell biology As a member of the IFPCS or the SMR you automatically get online access to PCMR. Sign up as a member today at www.ifpcs.org or at www.societymelanomaresarch.org SummarySmall molecules complement genetic mutants and can be used to probe pigment cell biology by inhibiting specific proteins or pathways. Here, we present the results of a screen of active compounds for those that affect the processes of melanocyte and iridophore development in zebrafish and investigate the effects of a few of these compounds in further detail. We identified and confirmed 57 compounds that altered pigment cell patterning, number, survival, or differentiation. Additional tissue targets and toxicity of small molecules are also discussed. Given that the majority of cell types, including pigment cells, are conserved between zebrafish and other vertebrates, we present these chemicals as molecular tools to study developmental processes of pigment cells in living animals and emphasize the value of zebrafish as an in vivo system for testing the on-and off-target activities of clinically active drugs.