| Fish has the largest number of species in vertebrate.The varieties of body colors,external fertilization and ectogenesis make fish very good materials for research studies on animal's body color growth and evolution.Crucian and carp are two widely bred fishes in fresh water,with advantages of strong anti-disease capability,manifold feeding habits and good adaptability to the changing environment.Thus,many ornamental fishes of vital ornamental and economic values are reproduced through a long-time targeted cultivation.This thesis researches on the body color growth of three types of crucians: white crucian,red crucian and red-white crucian from five different angels.We observed the formation,distribution and morphological structure of chromocytes of these three crucians through a tissue study on the adult skin,scale and fins o fthem.We understand the characteristics of early body color growth of these three types of crucians by observing the early formation of their body colors.We try to further understand and control the hereditary rules of crucian's body color through observing the early body color growth on hybrids between carp and crucian and their parents.We try to research on the genes of several important body colors in cloned crucians and their roles in the formation of body color in different types of crucians.From which all others derive.At last wo explore the adult pigment stem cells and their significance in forming the pigment patterning.The results of this study provide a theoretical basis for further analysising of the developmental mechanism of the formation of fish body color,It can provide theoretical guidance for the selection and improvement of ornamental fish.1 In this paper,we studied the cell and subcellular levels of three kinds of skin cells by means of frozen sections and ultrathin sections.Melanocytes usually have two shapes,a large cell size,dendritic branching;the other is relatively dense clumps,small cell size.Under the electron microscope,there were a large number of melanin bodies with high electron density.Xanthophore and erythrophores were granular under electron microscope,there are pteridine carotenoid vesicles and vesicles,xanthophores contain more Pteridine,erythrophores contain a lot of carotenoid vesicles.We observed iridophore showed light blue and purple by electron microscope.Iridophore contain a lot of crystalline bodies under the electron microscope.There were many xanthophores,erythrophores and iridophore in the skin of red crucian carp.The pigment cells of red skin of red and white crucian carp were the same as those of red crucian carp,and only iridophore was observed in white skin.2 The composition of the pigment cells in different parts of fins of three kinds of crucian carp is consistent with the pigment composition the upper scale in its base.The results showed that the scales of three kinds of crucian carp with different body color and the composition of the pigment cells were consistent with the observation of skin tissue.white crucian carp scales have melanocytes,xanthophores,erythrophores and iridophore.red crucian carp and red carp scalesof red-white crucian carp scales were observed xanthophores,erythrophores and iridophore;white scales of red-white crucian carp contain only iridophore.Fins and scales both are the derivatives of skin.The fins in pigment cells and fish body composition have consistency.3 The early body color formation of three different color crucian carp was observed.Their early body color formation is similar.Melanocytes firstly appear before fertilization,then xanthophores happen and lastly iridophore appear.The body color of larvae of three different color crucian carp are dark grayThen,the black gradually faded in red crucian carp and red-white crucian carp.After 3-month old or so,the body color turned to red-orange or red and white-red.White crucian carp has been maintaining its body color.4 By statistical methods,regression models are constructed to describe the inheritance rule on the red and black body colors in fish.In virtue of the models,some interesting properties in inheritance are revealed for crucian carp and carp:(i)the percentage of the black color in parents generates negative cross-correlation with the red color of offspring for both of crucian carp and carp.(ii)The negative effect on the percentage of the red color in offspring by the black color in parents is stronger for carp than that for crucian carp.5 the full-length cDNAs for Mitfa,Slc7a11 and Pnp4 a were isolated.Sequence analysis revealed that the full-length Mitfa cDNA consists of 1578 bp,with an open reading frame of 1236 nucleotides,which encodes a deduced protein of 412 amino acids.coding for a deduced sequence alignment showed that crucian carp Mitfa shares high levels of identity to those from Cyprinus carpio(95.6%).The full-length Pnp4 a cDNA contains 1535 bp,with an open reading frame of 783 nucleotides,which encodes a deduced protein of 291 amino acids,coding for a deduced sequence alignment showed that crucian carp Mitfa shares high levels of identity to those from Danio rerio(95.1%)..The full-length Slc7a11 cDNA contains 1782 bp,with an open reading frame of 1488 nucleotides,which encodes a deduced protein of 496 amino acids.The amino acid sequence alignment demonstrated that the red crucian carp Slc7a11 protein shared a sequence identity of 93.7% with Danio rerio,Oryzias latipes,6 The expression of Mitfa,Slc7a11 and Pnp4 a in three kinds of different color crucian carp embryos and adult tissues were detected by RT-PCR.The results showed that Mitfa,Slc7a11 and Pnp4 a were expressed in three kinds of color crucian carp embryos.It is worth studying that Mitfa is an important marker gene for melanin stem cells.However,the expression of Mitfa mRNA was detected in the skin of the red crucian carp and crucian carp.These results indicate that Mitfa gene is not only involved in the differentiation and development of melanocytes,but also closely related to the development of non melanoma.7 A light-colored region(LCR)is observed at the heel of caudal fin in juvenile crucian carp.By cutting off the caudal fin,the operated caudal fin can regenerate in accordance with the original pigment pattern from the retained LCR.As markers of stem cells,Oct4 and Sox2 have been found to be highly expressed in the LCR as well as Mitfa,a label of the melanoblasts.In vitro,Mitfa+ melanoblasts are observed in the cells which are derived from the LCR and transfected with Mitfa-EGFP reporter by using Tol2 transposon system.Furthermore,by real-time qPCR,it is shown that the level of sox2 mRNA is gradually decreased from the LCR to proximal and distal caudal fin,and that of Mitfa mRNA in the proximal caudal fin is higher than that in the LCR,while it is the lowest in the distal caudal fin.Hence,we propose that the LCR is a pigment progenitor niche,sending melanocytes to the distal of caudal fin,which gradually emerges as caudal fin grow.We reveal that the LCR of caudal fin might be a niche of pigment progenitors,and contribute to pigment-producing stem cells in crucian carp. |
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