RESUMO
Solitary ascidian tadpole larvae develop two types of black pigment cells in the major sensory organs of the brain. Such pigment cells have been demonstrated to express the melanogenic genes, tyrosinase and Tyrp/TRP (tyrosinase-related protein). To understand the genetic and developmental mechanisms underlying the differentiation of chordate pigment cells, we examined the function of the promoter region of Tyrp/TRP gene, an ascidian (Halocynthia roretzi) tyrosinase family gene. The expression of the gene in pigment cell lineage starts at the early-mid gastrula stages. To identify the transcriptional regulatory region of the gene allowing cell-type-specific expression, a deletion series of the HrTyrp 5' flanking region fused to a lacZ reporter gene was constructed and microinjected into ascidian fertilized eggs. The region of 73 bp in HrTyrp was identified as sufficient for expression in pigment cell-precursors of tailbud stage embryos. It is noteworthy that there is no M-box element highly conserved in the promoters for vertebrate tyrosinase family genes such as tyrosinase, Tyrp1/TRP-1 and Tyrp2/TRP-2 (Dct). Although the regulatory system of ascidian pigment-cell development is likely to contain most factors critical to vertebrate pigment-cell development, there might be critical differences in the mode of regulation, such as the developmental timing of interactions of factors, proteins and genes, involved in pigment cell differentiation and pigmentation.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Melanócitos/metabolismo , Oxirredutases , Proteínas/genética , Urocordados/genética , Região 5'-Flanqueadora/genética , Animais , Sequência de Bases , Linhagem da Célula , Ciona intestinalis/genética , DNA/química , DNA/genética , Embrião não Mamífero/enzimologia , Embrião não Mamífero/metabolismo , Evolução Molecular , Regulação Enzimológica da Expressão Gênica , Oxirredutases Intramoleculares/genética , Melanócitos/citologia , Dados de Sequência Molecular , Monofenol Mono-Oxigenase/genética , Pigmentação/genética , Regiões Promotoras Genéticas/genética , Análise de Sequência de DNA , Sítio de Iniciação de Transcrição , Urocordados/embriologia , Urocordados/enzimologiaRESUMO
The microphthalmia-associated transcription factor (Mitf) is a basic-helix-loop-helix-leucine zipper (bHLH-ZIP) transcription factor essential for the development and function of all melanin-producing pigment cells in vertebrates. To elucidate the evolutionary history of Mitf and the antiquity of its association with pigment cells, we have isolated and characterized HrMitf, a sole member of the Mitf-TFE bHLH-ZIP subfamily in the ascidian Halocynthia roretzi. Maternal HrMitf mRNA is detected in the fertilized egg and in the animal hemisphere from 4-cell stage through the gastrula stage. From the neurula through the early tailbud stage, HrMitf is preferentially expressed in the pigment-lineage cells that express the lineage-specific melanogenesis genes tyrosinase (HrTyr) and Tyrp. Overexpression of HrMitf induced ectopic expression of HrTyr enzyme activity in mesenchymal cells where the same enzyme activity was induced by overexpression of HrPax3/7, suggesting that a part(s) of the Pax3-Mitf-tyrosinase gene regulatory cascade seen in vertebrate melanocytes is operative during ascidian embryogenesis. We also show HrMitf and mouse Mitf-A, a Mitf isoform abundantly expressed in pigmented epithelial cells, share similar functional characteristics. These results suggest antiquity of the association of the Mitf-TFE subfamily with pigment cells and may support the idea that acquisition of multiple promoters (isoforms) by an ancestral Mitf gene has allowed the evolution of multiple pigment cell types.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Evolução Molecular , Melanócitos/metabolismo , Pigmentos Biológicos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Urocordados/embriologia , Urocordados/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Sequência Conservada/genética , Proteínas de Ligação a DNA/química , Embrião não Mamífero/embriologia , Embrião não Mamífero/enzimologia , Embrião não Mamífero/metabolismo , Gástrula/citologia , Gástrula/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Melanócitos/citologia , Camundongos , Fator de Transcrição Associado à Microftalmia , Modelos Genéticos , Dados de Sequência Molecular , Monofenol Mono-Oxigenase/genética , Monofenol Mono-Oxigenase/metabolismo , Filogenia , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Transcrição/química , Urocordados/enzimologia , Urocordados/metabolismo , VertebradosRESUMO
During the early stages of embryogenesis of the ascidian Halocynthia roretzi the test cells creep exclusively on the inner surface of the chorion. Concomitant with elongation of the embryonic tail, however, the test cells begin to gather around the embryo and finally cover the whole embryo. The time at which the test cells surround the embryo almost coincides with that of initiation of larval tunic formation. Scanning electron microscope observations revealed that the test cells extend numerous cytoplasmic processes or pseudopodia. During larval tunic formation, the test cells compose a net by intertwining their filopodia, and the cell net covers the whole embryo.
RESUMO
In the swimming phase of the larvae of the ascidian, Halocynthia roretzi, changes in the activity of acid phosphatase (AcP-ase) were studied cytochemically with respect to the appearance of metamorphosing potencies. The AcP-ase activity in the larvae before and soon after hatching is weakly visible only in and around the nuclei of the epithelial, muscular and notochordal cells. 6 hr after hatching the enzyme activity begins t o appear weakly in the microblasts around the proximal end of the notochordal sheath, whereas the activities which were found in the previous stages disappear. In the larvae which passed for 12 hr after hatching, the activity of AcP-ase is distinctly shown in the microblasts and also in the other 2 mesodermal cells, meso- and macro- blasts. The microblasts of this stage are closely attached to the notochordal sheath at the proximal end. At the same time, many large granules which appear similar to lysosomes are found in the microblast by an electron microscopy. The 6th hour's larvae after hatching can be induced slowly to resorb its tail by the treatment with a nile blue solution, but the time which it takes for tail resorption is gradually shortened depending on the age of the larva up until 12 hr after hatching. From these results, i t was concluded that the appearance of the AcP-ase activity in the microblasts was parallel with the appearance of the potency of metamorphosis of the larvae after hatching. Possible roles of the microblasts at onset of meta- morphosis would seem to play a role in the rupture of the notochordal sheath and in the succeeding regression of the tail tissues.
