Project Summary Background This research project will explore how pigmentation develops in cichlid fish.

In particular, I intend to focus on the development of the chromatophores from the neural crest throughout embryonic development and into adulthood (time permitting). Similar studies and photographs of Danio rario, more commonly known as zebrafish, homologs of the African cichlids done by Dr. David Parichy of the University of Washington seem to indicate that there are two waves of pigmentation development. First, melanophores develop along the sides of the fish, forming black stripes. As the fish matures, xanthophores and iridophores fill in the spaces between the melanophore stripes, and then a second surge of melanophores matures and reinforces the bands, giving the zebrafish their distinctive coloring patterns. However, preliminary data and pictures of cichlids throughout development show that this is not the case with the African cichlids. Researching cichlid pigmentation patterning and development will provide us with a more thorough understanding of neural crest cell differentiation and multipotency, the effects of sexual and natural selection on the phenotype of a species and how species evolve. As selection and evolution play such an important role in the continued growth and health of the human race, it is extremely important that we understand as much as possible about how our environment and circumstances affect our population and evolution. Evolution, or the idea that a species gradually changes over time, was first proposed by the Greeks over twenty-three hundred years ago1. Early Christians however, believed it was blasphemy and prevented the idea from spreading to the public. It remained an unknown theory until Charles Darwin published On the Origin of Species in 1858. Although Darwin was not the first to propose the theory of evolution, he was the first to come up with the idea of natural selection. He suggested that evolution occurs through random, heritable mutations in a specific individual, which cause the individual to be more or less fit. Should the mutation confer any sort of advantage to the individual, their children will receive it and also be more fit than others in the population. If the trait expressed by the mutation is advantageous enough, eventually it will spread to the entire population. As more and more of these mutations build up in physically and geographically separate populations, the two communities begin to adapt to their specific environment, and eventually speciate. The cichlids in Africa are one of the fastest evolving species on earth. Due to the effects of natural selection and strong sexual selection by the females, in just ten million years, they have evolved into over fifteen hundred distinct species. The diverse environments found in their native lakes in Africa led to two broad categories of cichlids: the rock dwellers and the sand dwellers. Then, cichlids evolved different jaw structures for different methods of feeding, and very distinct and striking pigmentation patterns. Pigment cells ultimately develop from the neural crest, a unique vertebrate characteristic. The neural crest forms shortly after gastrulation when the neural folds come together at the border of the ectoderm and the neural plate to form the neural tube. Neural crest cells from the roof plate transform from epidermal cells, to mesenchymal cells. The mesenchymal cells are a type of undifferentiated connective tissue which can migrate through the periphery before differentiating into glial, cartridge, bone, smooth muscle, pigment or other cells.
1

http://rationalrevolution.net/articles/understanding_evolution.htm

Due to the fact that they are migratory, multipotent and found only in vertebrates, neural crest cells have become the focus of a lot of research lately. In the past decade or so, researchers have discovered that adult neural crest cells can differentiate into a wide variety of cell types, as opposed to cells found in the organ they developed from2 and, that neurogenesis continues into adulthood3. Scientists hope that by understanding neural crest cell migratory patterns and how the cells know when to differentiate as well as what type of cell to become, they can apply this knowledge towards clinical as well as theoretical use. As the cells differentiate into pigment cells, they can be tracked as they migrate throughout the fish. Cichlids are very diverse in their pigmentation, both in color and pattern. This is due to a combination of sexual selection and natural selection. Studies show that in six of nine cichlid species, males have more striking colors than females, in both the visual and ultraviolet light ranges4. Cichlid coloring is clearly an important part of their phenotype, as a regenerated fin or bit of tissue will match the pigmentation of the missing fin both in color and pattern5. I would like to study the development and migration of chromatophores in cichlids. Ectotherms, animals that use their environment to help regulate their body temperature (such as amphibians, reptiles and fish) have three types of chromatophores. Melanophores, which contain eumelanin and show up black or dark brown since they absorb light; xanthophores which appear yellow due to the pteridine they contain; and iridiphores which are stacks of guanine platelets that reflect light depending upon the angle of incidence. Pictures taken of maturing zebrafish seem to indicate that xanthophores and iridophores can inhabit the same space, whereas melanophores create a boundary and do not share space with the other pigment cells6. Cichlid chromatophores, however, seem to do the opposite. Pictures reveal that in cichlids, the iridophores and xanthophores remain separate, while the melanophores are evenly dispersed throughout the fin and scales and that this separation leads to the distinguishing patterns of cichlids. I will study BLANK species of cichlids, the NAMES, WHY. Metriaclima lombardoi is particularly interesting, due to its clear melanophore bars very early on in development. We hypothesize that these bars are the result of pigmentation expressed in multiple stages. Studies done with zebrafish show that there are two phases of pigmentation: embryonic and adult7. Initially, just a few days after fertilization only melanophores are present and visible on the fish. The xanthophores and iridophores develop and give the impression that they push the melanophores into solid stripes, while settling down in between the stripes. Next, the second wave of melanophores matures and solidifies the stripes. The embryonic melanophores are migratory, whereas the adult melanophores are not, and grow only in areas that already have a high melanophore concentration. Similarly, we propose that cichlids also have a multi-step pigmentation process. The distinctive bars of the embryonic M. lombardoi could be a consequence of this developmental
2 3

http://www.sciencemag.org/content/288/5471/1660

http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6SYT-45V6XPF3&_user=961305&_coverDate=04/30/2002&_rdoc=1&_fmt=high&_orig=search&_origin=search&_sort=d&_docanchor=&view=c&_acct=C00 0049425&_version=1&_urlVersion=0&_userid=961305&md5=86e4418bb5e4f14da15856d4f71a9ff2&searchtype=a 4 The fish eye view: are cichlids conspicuous?

5 6 7

Figure 16. Regeneration of Metriaclima zebra fin at 2, 4 and 6 days post-amputation. Lecture 4-pigmentation http://protist.biology.washington.edu/dparichy/Movies.htm#wtalb_movies idea http://protist.biology.washington.edu/dparichy/ResearchPage.htm#1 lecture 4

progression, as the embryonic melanophores could develop first, followed by xanthophore and iridophore growth. To test this theory and understand more about cichlid pigmentation patterns, I intend to use a TYPE microscope, equipped with a TYPE camera to photograph NUMBER cichlids throughout their development and into adulthood. This microscope is a BLANK type, developed by BLANK in BLANK. It is accepted as a good quality microscope by researchers and is trusted to give a clear and properly magnified picture up to SIZE. The camera, made by BLANK is from the year BLANK. I will count the number of chromatophores in each picture, carefully marking their position and type. Using a microscope calibration slide I will be able to see each fish with evenly spaced units, so as to correctly follow chromatophore migration during development. Once all the pictures are collected and the cells properly counted and identified, I intend to make a time-lapse movie using BLANK to show the overall migration and developmental patterns of the chromatophores in the different cichlids. This should allow for easy comparison between the various types of cichlids, hopefully allowing us to understand more about their migratory crest cell and chromatophore development. 3d? how to count What exactly counts as xanthophore Cichlid pigmentation scheme follows same pattern as zebrafish, egg spots http://www.ncbi.nlm.nih.gov/pubmed?term=cichlid%20pigmentation%20patterns&itool=Query Suggestion http://protist.biology.washington.edu/dparichy/Movies.htm http://www.annualreviews.org/doi/full/10.1146/annurev.ecolsys.31.1.163 http://zfin.org/cgi-bin/webdriver?MIval=aa-labview.apg&OID=ZDB-LAB-970507-55 http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002845 http://books.google.com/books?id=CtdwxA4NtnQC&pg=PA108&lpg=PA108&dq=endotherm+ pigment&source=bl&ots=AdS1V9xLT4&sig=zxHJ4HLbm0cWEU6N79WUMfB0bFc&hl=en& ei=3fZXTaDzEcq4twe17nYDA&sa=X&oi=book_result&ct=result&resnum=10&ved=0CEoQ6AEwCQ#v=onepage&q &f=true http://books.google.com/books?id=dPG_2in8kIC&pg=PA83&lpg=PA83&dq=endotherms+and+ectotherms+pigment&source=bl&ots=14 OuFqJtM2&sig=EnL95e1TJam-lk7BGhTh9LhnsaE&hl=en&ei=u_RXTaCfD9K_tgfYo8jDA&sa=X&oi=book_result&ct=result&resnum=3&ved=0CCgQ6AEwAg#v=onepage&q=endot herms%20and%20ectotherms%20pigment&f=false http://www.ncbi.nlm.nih.gov/pubmed/10985850 http://www.bettas4all.nl/photos/Scientificarticles/Kelshetal2004.pdf

http://salk.academia.edu/IanQuigley/Papers/353278/Pigment_Pattern_Evolution_by_Differential _Deployment_of_Neural_Crest_and_Post-Embryonic_Melanophore_Lineages_In_Danio_Fishes http://www.ncbi.nlm.nih.gov/pubmed/10404123 Retinoic acid stimulates development of adulttype chromatophores in the flounder http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002845 Zebrafish Endzone Regulates Neural Crest-Derived Chromatophore Differentiation and Morphology http://www.annualreviews.org/doi/full/10.1146/annurev.ecolsys.31.1.163 AFRICAN CICHLID FISHES: Model Systems for Evolutionary Biology http://www.ncbi.nlm.nih.gov/pubmed/15588588 The evolution of genes for pigmentation in African cichlid fishes. http://www.ncbi.nlm.nih.gov/pubmed/20543123 The fish eye view: are cichlids conspicuous?