佐藤 滋

During female mammal reproductive tract development, epithelial cells of the lower Müllerian duct are committed to become stratified squamous epithelium of the vagina and ectocervix, when the expression of ΔNp63 transcription factor is induced by mesenchymal cells. The absence of ΔNp63 expression leads to adenosis, the putative precursor of vaginal adenocarcinoma. Our previous studies with genetically engineered mouse models have established that fibroblast growth factor (FGF)/mitogen-activated protein kinase (MAPK), bone morphogenetic protein (BMP)/SMAD, and activin A/runt-related transcription factor 1 (RUNX1) signaling pathways are independently required for ΔNp63 expression in Müllerian duct epithelium (MDE). Here, we report that sine oculis homeobox homolog 1 (SIX1) plays a critical role in the activation of ΔNp63 locus in MDE as a downstream transcription factor of mesenchymal signals. In the developing mouse reproductive tract, SIX1 expression was restricted to MDE within the future cervix and vagina. SIX1 expression was totally absent in SMAD4 null MDE and was reduced in RUNX1 null and FGFR2 null MDE, indicating that SIX1 is under the control of vaginal mesenchymal factors: BMP4, activin A and FGF7/10. Furthermore, Six1, Runx1, and Smad4 gene-dose-dependently activated ΔNp63 expression in MDE within the vaginal fornix. Using a mouse model of diethylstilbestrol (DES)-associated vaginal adenosis, we found DES action through epithelial estrogen receptor α (ESR1) inhibits activation of ΔNp63 locus in MDE by transcriptionally repressing SIX1 and RUNX1 in the vaginal fornix.

Background: Various senses and sensory nerve architectures of animals have evolved during adaptation to exploit diverse environments. In craniates, the trunk sensory system has evolved from simple mechanosensory neurons inside the spinal cord (intramedullary), called Rohon-Beard (RB) cells, to multimodal sensory neurons of dorsal root ganglia (DRG) outside the spinal cord (extramedullary). The fish and amphibian trunk sensory systems switch from RB cells to DRG during development, while amniotes rely exclusively on the DRG system. The mechanisms underlying the ontogenic switching and its link to phylogenetic transition remain unknown. Results: In Xenopus, Six1 overexpression promoted precocious apoptosis of RB cells and emergence of extramedullary sensory neurons, whereas Six1 knockdown delayed the reduction in RB cell number. Genetic ablation of Six1 and Six4 in mice led to the appearance of intramedullary sensory neuron-like cells as a result of medial migration of neural crest cells into the spinal cord and production of immature DRG neurons and fused DRG. Restoration of SIX1 expression in the neural crest-linage partially rescued the phenotype, indicating the cell autonomous requirements of SIX1 for normal extramedullary sensory neurogenesis. Mouse Six1 enhancer that mediates the expression in DRG neurons activated transcription in Xenopus RB cells earlier than endogenous six1 expression, suggesting earlier onset of mouse SIX1 expression than Xenopus during sensory development. Conclusions: The results indicated the critical role of Six1 in transition of RB cells to DRG neurons during Xenopus development and establishment of exclusive DRG system of mice. The study provided evidence that early appearance of SIX1 expression, which correlated with mouse Six1 enhancer, is essential for the formation of DRG-dominant system in mice, suggesting that heterochronic changes in Six1 enhancer sequence play an important role in alteration of trunk sensory architecture and contribute to the evolution of the trunk sensory system.

Inner ear mechanosensory hair cells transduce sound and balance information. Auditory hair cells emerge from a Sox2-positive sensory patch in the inner ear epithelium, which is progressively restricted during development. This restriction depends on the action of signaling molecules. Fibroblast growth factor (FGF) signalling is important during sensory specification: attenuation of Fgfr1 disrupts cochlear hair cell formation; however, the underlying mechanisms remain unknown. Here we report that in the absence of FGFR1 signaling, the expression of Sox2 within the sensory patch is not maintained. Despite the down-regulation of the prosensory domain markers, p27(Kip1), Hey2, and Hes5, progenitors can still exit the cell cycle to form the zone of non-proliferating cells (ZNPC), however the number of cells that form sensory cells is reduced. Analysis of a mutant Fgfr1 allele, unable to bind to the adaptor protein, Frs2/3, indicates that Sox2 maintenance can be regulated by MAP kinase. We suggest that FGF signaling, through the activation of MAP kinase, is necessary for the maintenance of sensory progenitors and commits precursors to sensory cell differentiation in the mammalian cochlea.

The Six1 homeobox gene plays critical roles in vertebrate organogenesis. Mice deficient for Six1 show severe defects in organs such as skeletal muscle, kidney, thymus, sensory organs and ganglia derived from cranial placodes, and mutations in human Sal cause branchio-oto-renal syndrome, an autosomal dominant developmental disorder characterized by hearing loss and branchial defects. The present study was designed to identify enhancers responsible for the dynamic expression pattern of Six1 during mouse embryogenesis. The results showed distinct enhancer activities of seven conserved non-coding sequences (CNSs) retained in tetrapod Six1 loci. The activities were detected in all cranial placodes (excluding the lens placode), dorsal root ganglia, somites, nephrogenic cord, notochord and cranial mesoderm. The major Six1-expression domains during development were covered by the sum of activities of these enhancers, together with the previously identified enhancer for the pre-placodal region and foregut endoderm. Thus, the eight CNSs identified in a series of our study represent major evolutionarily conserved enhancers responsible for the expression of Six1 in tetrapods. The results also confirmed that chick electroporation is a robust means to decipher regulatory information stored in vertebrate genomes. Mutational analysis of the most conserved placode-specific enhancer, Six1-21, indicated that the enhancer integrates a variety of inputs from Sox, Pax, Fox, Six, Wnt/Lef1 and basic helix-loop-helix proteins. Positive autoregulation of Six1 is achieved through the regulation of Six protein-binding sites. The identified Six1 enhancers provide valuable tools to understand the mechanism of Six1 regulation and to manipulate gene expression in the developing embryo, particularly in the sensory organs. (C) 2012 Elsevier Inc. All rights reserved.

Muscle satellite cells are essential for muscle growth and regeneration and their morphology, behavior and gene expression have been extensively studied. However, the mechanisms involved in their proliferation and differentiation remain elusive. Six1 and Six4 proteins were expressed in the nuclei of myofibers of adult mice and the numbers of myoblasts positive for Six1 and Six4 increased during regeneration of skeletal muscles. Six1 and Six4 were expressed in quiescent, activated and differentiated muscle satellite cells isolated from adult skeletal muscle. Overexpression of Six4 and Six5 repressed the proliferation and differentiation of satellite cells. Conversely, knockdown of Six5 resulted in augmented proliferation, and that of Six4 inhibited differentiation. Muscle satellite cells isolated from Six4(+/-)Six5(-/-) mice proliferated to higher cell density though their differentiation was not altered. Meanwhile, overproduction of Six1 repressed proliferation and promoted differentiation of satellite cells. In addition, Six4 and Six5 repressed, while Six1 activated myogenin expression, suggesting that the differential regulation of myogenin expression is responsible for the differential effects of Six genes. The results indicated the involvement of Six genes in the behavior of satellite cells and identified Six genes as potential target for manipulation of proliferation and differentiation of muscle satellite cells for therapeutic applications. (C) 2010 Elsevier Inc. All rights reserved.

All cranial sensory organs and sensory neurons of vertebrates develop from cranial placodes. In chick, amphibians and zebrafish, all placodes originate from a common precursor domain, the pre-placodal region (PPR), marked by the expression of Six1/4 and Eya1/2 However, the PPR has never been described in mammals and the mechanism involved in the formation of PPR is poorly defined. Here, we report the expression of Six1 in the horseshoe-shaped mouse ectoderm surrounding the anterior neural plate in a pattern broadly similar to that of non-mammalian vertebrates To elucidate the identity of Six1-positive mouse ectoderm, we searched for enhancers responsible for Six1 expression by in vivo enhancer assays One conserved non-coding sequence. Six1-14, showed specific enhancer activity in the rostral PPR of chick and Xenopus and in the mouse ectoderm These results strongly suggest the presence of PPR in mouse and that it is conserved in vertebrates Moreover, we show the importance of the homeodomain protein-binding sites of Six1-14, the Six1 rostral PPR enhancer, for enhancer activity, and that Dlx5, Msx1 and Pax7 are candidate binding factors that regulate the level and area of Six1 expression, and thereby the location of the PPR Our findings provide critical information and tools to elucidate the molecular mechanism of early sensory development and have implications for the development of sensory precursor/stem cells (C) 2010 Elsevier Inc All rights reserved

Eya1 is a homolog of eyes absent in Drosophila, and essential for various organ formations in vertebrates. Mouse and chick Eya1 shows dynamic expression pattern in early development. We identified ten independent Eya1 enhancers by screening evolutionarily conserved sequences. They exhibited enhancer activities in Hensen's node, neural tube, migrating neural crest cells, otic vesicle, olfactory placode, cranial ganglia, and somites at HH6-17 of chick embryo. The sum of the enhancer activities of the enhancers covers the endogenous expression domains of Eya1 common to chick and mouse. Enhancer activities were also observed in species-specific expression domains such as trigeminal ganglia and brain. Mutational study of one of the enhancers revealed that the enhancer is composed of positive and negative cis-regulatory elements. Thus, we successfully identified a comprehensive group of enhancers around Eya1 locus, which are probably involved in the control of the complex expression pattern of Eya1 in vivo. Developmental Dynamics 237.3142-3156, 2008. (C) 2008 Wiley-Liss, Inc.

Eyes absent is essential for compound eye formation in Drosophila. Its mammalian homologues of Eya are involved in the development of sensory organs, skeletal muscles and kidneys. Mutations of EYA1 in human cause branchio-oto-renal syndrome, with abnormalities in branchial derivatives, ear and kidney. For an insight into the function of Eya1 and Eya2 in early development, we performed whole-mount in situ hybridization and compared the expression patterns of these two genes in the developing chick embryos. Eya1 was first expressed in the primitive streak at Hamburger and Hamilton stage 4 (HH4) and appeared in the ectoderm and head mesenchyme distinct from migrating neural crest cells at HH6-HH11. At HH15 and HH17, the olfactory, otic and vagal/nodose placodes and cranial ganglia were positive for Eya1. In contrast, Eya2 was already expressed in the endoderm at HH4, and appeared in the endoderm and prospective placodal region at HH6-HH11. Eya2 expression was observed in pharyngeal clefts and pouches as well as cranial placodes at HH15 and HH17. These results indicate differential expression of Eya1 and Eya2, both spatially and temporally, in chick during early development. The expression patterns are somewhat different from those of other species such as Xenopus, zebrafish and mouse. The results suggest distinct and unique functions for Eya1 and Eya2 in early chick development. (c) 2008 Elsevier B.V. All rights reserved.

The olfactory epithelium (OE) is derived from the olfactory placode (OP) during mouse development. At embryonic day (E) 10.0-E10.5, "early neurogenesis" occurs in the OE, which includes production of pioneer neurons that emigrate out of the OE and other early-differentiated neurons. Around E12.5, the OE becomes organized into mature pseudostratified epithelium and shows "established neurogenesis," in which olfactory receptor neurons (ORNs) are differentiated from basal progenitors. Little is known about the molecular pathway of early neurogenesis. The homeodomain protein Six1 is expressed in all OP cells and neurogenic precursors in the OE. Here we show that early neurogenesis is severely disturbed despite the unaltered expression of Mash1 at E10.5 in the Six1-deficient mice (Six1(-/-)). Expression levels of neurogenin1 (Ngn1) and NeuroD are reduced and those of Hes1 and Hes5 are augmented in the OE of Six1(-/-) at E10.5. Pioneer neurons and cellular aggregates, which are derived from the OP/OE and situated in the mesenchyme between the OE and forebrain, are completely absent in Six1(-/-). Moreover, ORN axons and the gonadotropin-releasing hormone-positive neurons fail to extend and migrate to the forebrain, respectively. Our study indicates that Six1 plays critical roles in early neurogenesis by regulating Ngn1, NeuroD, Hes1, and Hes5. (C) 2007 Elsevier Inc. All rights reserved.

Mitf is a central regulator of pigment cell development that is essential for the normal development of the melanocyte and retinal pigment epithelium (RPE) lineages. To understand better the role of Mitf, we have used the Xenopus laevis experimental system to allow a rapid examination of the role of Mitf in vivo. Here, we report the function of XlMitfalpha-M on melanophore development and melanization compared with that of Slug that is expressed in neural crest cells. Overexpression of XlMitfalpha-M led to an increase in melanophores that was partly contributed by an increase in Slug-positive cells, indicating that XlMitfalpha-M is a key regulator of melanocyte/melanophore development and melanization. Moreover, overexpression of a dominant-negative form of XlMitfalpha led to a decrease in the number of melanophores and induced abnormal melanoblast migration. We also observed an induction of ectopic RPE and extended RPE by overexpression of XlMitfalpha-M and possible interactions between XlMitfalpha and several eye-related genes essential for normal eye development.

Six genes are homologs of Drosophila sine oculis and encode transcription factors that are characterized by a conserved Six domain and homeodomain. Of the six family members (Six1-Six6) in mice, Six1 and Six4 show similar expression patterns during embryogenesis. Six1(-/-) mice show defective formation of various organs such as inner ear, nose, skeletal muscle, kidney and thymus, whereas Six4(-/-) mice show little anomaly in organogenesis. To understand the molecular basis for the differential function of Six1 and Six4 in vivo, we screened target genes of Six1 and Six4 and found that Six1 and Six4 differentially regulated a set of target genes. Gel-retardation assays indicated that the promoter region of one of the targets, sodium-potassium-chloride cotransporter 1 (Slcl2a2), contains multiple Six1-binding sites and one common binding site of Six1 and Six4, suggesting that the DNA-binding specificity of Six1 is distinct from that of Six4. This underlies the differential regulation of common target genes by Six1 and SiA. Furthermore, in situ hybridization demonstrated that the expression of Slc12a2 was reduced in the developing dorsal root ganglia of Six1(-/-)/Six4(-/-) mice, suggesting that Six1 and Six4 regulate S1c12a2 in vivo.

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 by 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. (C) 2004 Elsevier B.V. All rights reserved.

Mitf (gene for microphthalmia-associated transcription factor) encodes a transcription factor of the basic/helix-loop-helix/leucine-zipper family and is a key regulator during the development of two different types of melanin-producing cell lineages, namely neural crest-derived melanocytes/melanophores, and the retinal pigment epithelium (RPE) differentiated from the outer layer of the eye cup. Mitf-deficient mice show a lack of melanocytes and small eyes caused by abnormal RIPE development. An interesting feature of Mitf is the existence of multiple isoforms with different amino termini and their functions in the development of these melanin-producing pigment cells. In this study, we isolated two Mitf homologues (XlMitfalpha and XlMitfbeta) and their isoforms from Xenopus laevis. Alignment analysis of the amino acid sequences of the N-termini suggests that these isoforms are homologues of mouse Mitf-M (expressed specifically in the melanocyte lineage) and Mitf-A (strongly expressed in the RPE, although this expression is ubiquitous). In Xenopus, XlMitfalpha is strongly expressed in the melanophore lineage (especially in premigratory melanoblasts) and the presumptive RPE and the epiphysis, in which melanin-producing cells differentiate in some vertebrates. Conservation of the Mitf isoforms expected to possess specific functions in the development of melanin-producing cells and of the expressions in such cell types in Xenopus suggest that XlMitf plays a central role in the development of melanin-producing cell lineages, and that, as in mice and humans, most of the signaling molecules or transcription factors implicated genetically in the development of melanin-producing cell lineages affect either Mitf expression or its function (Goding [2000] Genes Dev. 14:1712-1728). (C) Wiley-Liss, Inc.

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.

The tyrosinase family of genes in vertebrates consists of three related members encoding melanogenic enzymes, tyrosinase (Tyr), tyrosinase-related protein-1 (TRP-1, Tyrp1) and tyrosinase-related protein-2 (Dct, TRP-2, Tyrp2). These proteins catalyze melanin production in pigment cells and play important roles in determining vertebrate coloration. This is the first report examining melanogenic gene expression in pigment cells during embryonic development of amphibians. Xenopus provides a useful experimental system for analyzing molecular mechanisms of pigment cells. However, in this animal little information is available not only about the developmental expression but also about the isolation of pigmentation genes. In this study, we isolated homologues of Tyr, Tyrp1 and Dct in Xenopus laevis (XlTyr, XlTyrp1, and XlDct). We studied their expression during development using in situ hybridization and found that all of them are expressed in neural crest-derived melanophores, most of which migrate through the medial pathway, and in the developing diencephalon-derived retinal pigment epithelium (RPE). Further, XlDct was expressed earlier than XlTyr and XlTyrp1, which suggests that XlDct is the most suitable marker gene for melanin-producing cells among them. XlDct expression was detected in migratory melanoblasts and in the unpigmented RPE. In addition, the expression of XlDct was detected in the pineal organ. The sum of these studies suggests that expression of the tyrosinase family of genes is conserved in pigment cells of amphibians and that using XlDct as a marker gene for pigment cells will allow further study of the developmental mechanisms of pigment cell differentiation using Xenopus.

Myotonic dystrophy 1 (DM1) is the most common inherited neuromuscular disease in adults. The disorder, characterized by myotonia, muscle wasting and weakness, cataract, insulin resistance, and mental Impairment, Is caused by the expansion of an unstable CTG repeat located in the 3' untranslated region of DMPK. The repeat expansion suppresses the expression of the homeobox gene SIX5. We describe here an experimental system to identify downstream transcriptional targets of mouse Six5 in order to elucidate the role of SIX5 in the pathogenesis of DM1 and development. By overexpressing a constitutively active Six5 (VP16-Six5wt) using adenovirus-mediated gene transfer in P19 cells and subsequent expression profiling using cDNA arrays, 21 genes, whose expression level increased by the treatment, were identified as potential target genes. Genes expressed in the somites, skeletal muscles, brain and meninges comprised the majority, suggesting the role of Six5 in the development and function of mesodermal tissues and brain. We provide evidence that lgfbp5 encoding a component of IGF signaling is a direct Six5-target. Moreover, the overall expression level of lgfbp5 was decreased in Six5 deficient mouse fibroblasts, and the response of human IGFBP5 to MyoD-induced muscle conversion was altered in cells of DM1 patients. Our results not only identify Six5 as an activator that directs lgfbp5 expression but also suggest that reduced SIX5 expression in DM1 might contribute to specific aspects of the DM1 phenotype.

In vertebrates, melanins produced in specialized pigment cells are required for visual acuity, camouflage, sexual display and protection from ultra violet (UV) radiation. There are three pigment cell types that are classified based on their distinct embryonic origins. Retinal pigment epithelium (RPE) cells originate from the outer layer of the optic cup. Pigment cells of the pineal organ are formed from the developing diencephalon. Melanocytes are derived from the neural crest unique to vertebrate embryos. Some of these pigment cells also play roles that are independent of the activity of tyrosinase, the key melanogenesis enzyme, or melanin: production of substrate(s) for catecholamine synthesis, maintenance of endolymph composition in the cochlea, maintenance of photoreceptor cells in the retina and retinoid metabolism essential for the visual cycle. To deduce the evolutionary origins of vertebrate pigment cells and a possible archetypal genetic circuitry, which may have been modified and utilized to generate multiple pigment cell types, comparison of developmental mechanisms of pigment cells between vertebrates and closely related invertebrate ascidians are proposed to provide useful information. The tadpole-type larva of ascidians possesses two melanin-containing pigment cells, termed the otolith and ocellus pigment cells, in the brain that are believed to be required for photo- and geotactic responses during swimming. In this review, current knowledge on the development of the two ascidian pigment cells is summarized, i.e. complete cell lineage, structure and expression of genes encoding two melanogenesis enzymes, and molecular developmental mechanisms involving BMP-CHORDIN antagonism, and possible evolutionary relationships between ascidian and vertebrate pigment cells are discussed.

Tyrosinase is the key enzyme for synthesizing melanin pigments, which primarily determine mammalian skin coloration. Considering the important roles of pigments in the evolution and the adaptation of vertebrates, phylogenetic changes in the coding and flanking regulatory sequences of the tyrosinase gene are particularly intriguing. We have now cloned cDNA encoding tyrosinase from Japanese quail and snapping turtle. These nonmammalian cDNA are highly homologous to those of the mouse and human tyrosinases, whereas the 5' flanking sequences are far less conserved except for a few short sequence motifs. Nevertheless, we demonstrate that the 5' flanking sequences from the quail or turtle tyrosinase genes are capable of directing the expression of a fused mouse tyrosinase cDNA when introduced into cultured mouse albino melanocytes. This experimental method, which reveals the functional conservation of regulatory sequences in one cell type (the melanocyte), may be utilized to evaluate phylogenetic differences in mechanisms controlling specific gene expression in many other types of cells. We also provide evidence that the 5' flanking sequences from these nonmammalian genes are functional in vivo by producing transgenic mice. Phylogenetic changes of vertebrate tyrosinase promoters and the possible involvement of conserved sequence motifs in melanocyte-specific expression of tyrosinase are discussed.

Tyrosinase is the key enzyme required for the synthesis of melanin pigments. Sequence comparison and functional analysis of the 5' upstream legions of vertebrate tyrosinase genes have revealed the importance of conserved E-box motifs in regulating their specific expression in pigment cells, optic cup-derived retinal pigment epithelium (RPE) and neural crest-derived melanocytes. In ascidians (more basal protochordates), two pigment cells that resemble vertebrate RPE cells are formed and specifically express the orthologous tyrosinase gene (HrTyr) in the cerebral vesicle located at the anterior end of the neural tube. To define regulatory sequences required for pigment cell-lineage-specific expression of HrTyr during embryogenesis, a series of mutations of the 5' upstream legion of HrTyr were fused to the lacZ reporter gene and were microinjected into fertilized eggs. We found that the -152 bp upstream of the translational start site is essential for expression in pigment cell precursors of tailbud-stage embryos. Further, additional positive and unique restriction elements were identified in the region up to -1.8 kb. Surprisingly, in the - 152 bp minimal promoter or in other legions with regulatory activities, there are no E-box motifs or sequences correlating with other conserved elements regulating vertebrate tyrosinase promoters. The possibility that Pax proteins regulate HrTyr expression is also discussed. (C) 2000 Elsevier Science B.V. All rights reserved.

The members of the Six gene family were identified as homologues of Drosophila sine oculis which is essential for compound-eye formation. The Six proteins are characterized by the Six domain and the Six-type homeodomain, both of which are essential for specific DNA binding and for cooperative interactions with Eya proteins. Mammals possess six Six genes which can be subdivided into three subclasses, and mutations of Six genes have been identified in human genetic disorders. Characterization of Six genes from various animal phyla revealed the antiquity of this gene family and roles of its members in several different developmental contexts. Some members retain conserved roles as components of the Pax-Six-Eya-Dach regulatory network, which may have been established in the common ancestor of all bilaterians as a toolbox controlling cell proliferation and cell movement during embryogenesis, Gene duplications and cia-regulatory changes may have provided a basis for diverse functions of Six genes in different animal lineages. (C) 2000 John Wiley & Sons, Inc.

Drosophila sine oculis and eyes absent genes synergize in compound-eye formation, The murine homologues of these genes, Six and Eya, respectively, show overlapping expression patterns during development. We hypothesized that Six and Eya proteins cooperate to regulate their target genes. Cotransfection assays were performed with various combinations of Six and Eya to assess their effects on a potential natural target, myogenin promoter, and on a synthetic promoter, the thymidine kinase gene promoter fused to multimerized Six4 binding sites, A clear synergistic activation of these promoters was observed in certain combinations of Six and Eya, To investigate the molecular basis for the cooperation, we first examined the intracellular distribution of Six and Eya proteins in transfected COS7 cells, Coexpression of Six2, Six4, or Six5 induced nuclear translocation of Eya1, Eya2, and Eya3, which were otherwise distributed in the cytoplasm, In contrast, coexpression of Six3 did not result in nuclear localization of any Eya proteins. Six and Eya proteins were coimmunoprecipitated from nuclear extracts prepared from cotransfected COS7 cells and from rat liver. Six domain and homeodomain, two evolutionarily conserved domains among various Six proteins, were necessary and sufficient for the nuclear translocation of Eya, In contrast, the Eya domain, a conserved domain among Eya proteins, was not sufficient for the translocation, A specific interaction between the Six domain and homeodomain of Six4 and Eya2 was observed by yeast two-hybrid analysis. Our results suggest that transcription regulation of certain target genes by Six proteins requires cooperative interaction with Eya proteins: complex formation through direct interaction and nuclear translocation of Eya proteins. This implies that the synergistic action of Six and Eya is conserved in the mouse and is mediated through cooperative activation of their target genes.

The tyrosinase family in vertebrates consists of three related melanogenic enzymes: tyrosinase, tyrosinase-related protein-1 (TRP-1), and TRP-2, These proteins control melanin production in pigment cells and play a crucial role in determining vertebrate coloration. We have isolated a gene from the ascidian Halocynthia roretzi which encodes a tyrosinase-related protein (HrTRP) with 45-49% identity with vertebrate TRP-1 and TRP-2, The expression of the HrTRP gene in pigment lineage a8.25 cells starts at the early-mid gastrula stage, which coincides with the stage when these cells are determined as pigment precursor cells; therefore, it provides the earliest pigment lineage-specific marker, which enables us to trace the complete cell lineage leading to two pigment cells in the larval brain. In addition, the expression pattern of the HrTRP gene appears to share similar characteristics with the mouse TRP-2 gene although structurally the HrTRP gene is more closely related to mammalian TRP-I genes. Based on these observations and on results from molecular phylogenetic and hybridization analyses, we suggest that triplication of the tyrosinase family occurred during the early radiation of chordates. Initially, duplication of an ancestral tyrosinase gene produced a single TRP gene before the urochordate and cephalochordate-vertebrate divergence, and a subsequent duplication of the ancestral TRP gene in the vertebrate lineage gave rise to two TRP genes before the emergence of teleost fishes. Evolution of the melanin synthetic pathway and possible phylogenetic relationships among chordate pigment cells that accommodate the metabolic process are discussed, (C) 1999 Wiley-Liss, Inc.

Mutations at the mouse locus encoding microphthalmia-associated transcription factor (Mitf) affect the development of many cell types, including retinal pigment epithelium (RPE), melanocytes, mast cells, and osteoclasts. Here we have identified a novel Mitf isoform, Mitf-a, and its human homologue MITF-A by cDNA cloning. MITF-A consists of 520 amino acid residues and differs in the amino-terminus from authentic melanocyte-type MITF (MITF-M). MITF-A mRNA is widely expressed and represents a predominant MITF isoform in cultured RPE cells, whereas MITF-M mRNA is exclusively expressed in melanocytes and melanoma cells. In situ hybridization analysis suggested that Mitf-a mRNA is enriched in the prospective RPE of mouse embryo. Moreover, transient cotransfection assays suggested that MITF-A activated transcription of the tyrosinase and tyrosinase-related protein 1 genes. MITF-A/Mitf-a therefore may play an important role in melanogenesis in RPE. (C) 1998 Academic Press.

The solitary ascidian Halocynthia roretzi possesses three types of muscle: the larval tail striated muscle, the adult heart striated muscle, and the adult body wall smooth muscle, The troponin complex is observed in ail types of muscle, and the isoform sequences and expression patterns of two of the three troponin components, troponins C and T, have been reported, In this study, we have determined cDNA sequences of the three TnI isoforms from H. roretzi. One of the three isoforms (adult TnI), expressed in adult body wall smooth muscle and heart muscle, was composed of 173 amino acids, being similar to vertebrate fast and slow skeletal TnIs in length, The other two isoforms (larval TnI alpha and TnI beta) were isolated from a cDNA library of larvae. Both larval TnIs were composed of 142 amino acids, with truncation amounting to ca, 30 amino acid residues at the C-termini, These larval TnIs are the smallest known TnIs. The position of the last intron of these TnIs was also determined. When compared with vertebrate TnI genes, the last intron of the ascidian adult TnI gene is located at 6 nucleotides downstream, and the introns of the two larval TnIs are positioned at 9 nucleotides upstream, These results suggest that H. roretzi TnI is encoded by at least three genes.

The origin and elaboration of the central nervous system played an important role in chordate and vertebrate history. All chordates possess a dorsal tubular central nervous system, but elaboration of dorsoventral and segmental pattern is far more pronounced in cephalochordates and vertebrates than in the more basal urochordates. Analysis of the urochordates, therefore, should allow deduction of the neural organization and neuronal patterning mechanisms that predated overt dorsoventral and segmental complexity. Here we report functional studies of the ascidian Pax gene (HrPax-37). The spatiotemporal expression pattern of HrPax-37 has suggested involvement in two distinct developmental processes: specification of dorsal cell fates of ectoderm during neurulation, and regional differentiation of the neural tube in later stages. Here we show that HrPax-37 is descendent from the precursor of the Pax-3 and Pax-7 genes implicated in specification of dorsal fate in the vertebrate neural tube. We also demonstrate that injection of HrPax-37 RNA into fertilized eggs causes ectopic expression of the dorsal neural marker tyrosinase gene in neurulae, confirming a regulatory role in dorsal patterning of the neural tube comparable to its vertebrate homologues. These results suggest that dorsal specification in the neural tube by Pax-3/7 subfamily genes was established in the ancestors of extant chordates during emergence of the dorsal tubular nervous system. (C) 1997 Academic Press.

The Microphthalmia basic-Helix-loop-Helix-Leucine Zipper (bHLH-LZ) transcription factor (Mi) plays a crucial role in the genesis of melanocytes; mice deficient for a functional (Microphthalmia) gene product lack all pigment cells, We show here that the Mi activation domain resides N-terminal to the DNA-binding domain and that as little as 18 amino acids are sufficient to mediate transcription activation, The minimal activation region of Mi is highly conserved in the related transcription factor TFE3 and is predicted to adopt an amphipathic alphahelical conformation, This region of Mi is also highly conserved with a region of E1A known to be essential for binding the CBP/p300 transcription cofactor, Consistent with these observations, the Mi activation domain can interact in vitro with CBP specifically through a region of CBP required for complex formation with E1A, P/CAF and c-Fos, and anti p300 antibodies can co-immunoprecipitate Mi from both melanocyte and melanoma cell lines, In addition, co-transfection of a vector expressing CBP2 (aas 1621-1891) fused to the VP16 activation domain potentiated the ability of Mi to activate transcription, confirming the significance of the CBP-Mi interaction observed in vitro. These data suggest that transcription activation by Mi is achieved at least in part by recruitment of CBP, The parallels between transcription regulation by Microphthalmia in melanocytes and MyoD in muscle cells are discussed.

Two distinct cDNAs encoding troponin C (TnC) isoforms were isolated from the ascidian, Halocynthia roretzi, One is expressed in adult body wall smooth muscle and heart muscle, and the other in larval striated muscle, The H. rorezti gene is composed of 7 exons separated by B introns, and Southern blot analysis showed that TnC is a single copy gene product. The two isoforms of TnC were derived through the alternative splicing of the third exon.

Tadpole larvae of ascidians have two sensory pigment cells in the brain, One is the otolith cell that functions as a gravity receptor, the other pigment cell is part of a primitive photosensory structure termed the ocellus. These sensory cells, like vertebrate pigment cells, contain membrane-bounded melanin granules and are considered to reflect a crucial position in the evolutionary process of this cell type, To investigate the molecular changes accompanying the evolution of pigment cells, we have isolated from Halocynthia roretzi a gene encoding tyrosinase, a key enzyme in melanin biosynthesis, The cDNA has an open reading frame (ORF) of 596 amino acids, which is 36-39% identical in amino acid sequence to vertebrate tyrosinases, In addition, the sequence analysis of both cDNA and genomic clones reveals an unusual organization of the tyrosinase gene, an extraordinary 3' untranslated region of the transcripts with significant homology to the coding sequence, and a single short intron in the sequence encoding a cytoplasmic domain, Expression of the gene is detected first in two pigment precursor cells positioned in the neural plate of early neurulae, and later in two melanin-containing pigment cells within the brain of late tailbud embryos, Its expression pattern correlates well with the appearance of tyrosinase enzyme activity in the developing brain, These results provide the first description of pigment cell differentiation at the molecular level in the ascidian embryo, and also will contribute to a better understanding of the evolution of chordate pigment cells. (C) 1997 Wiley-Liss, Inc.

Several nuclear factors that interact with sequences in the 5' flanking region of the mouse tyrosinase gene were identified using band shift and methylation interference assays. One of these factors bind to an AT-rich sequence, TATCAATTAG, located at -183 base pairs upstream of the transcription start site. To isolate cDNA clone encoding this DNA binding protein, we have screened a lambda gtll cDNA expression library prepared from mouse melanocyte cell line with a labeled oligonucleotide probe containing its binding site. Complementary DNA clones encoding mouse high mobility group protein HMG-I and its isoform HMG-Y were obtained. HMG-I(Y) is a low molecular size, basic nuclear protein that binds specifically to AT-rich region of double-stranded DNA in vitro. In Northern blot analysis the level of HMG-I(Y) mRNA expression did not correlate with that of tyrosinase or TRP-1. Although the amount of HMG-I(Y) transcripts has no apparent influence on the mouse tyrosinase gene expression, it is possible that HMG-I(Y) binds to the 5' flanking sequence of the tyrosinase gene as an auxiliary factor, and facilitates the binding and activity of other transcription factors.

Highly homologous DNA elements were found to be shared by the upstream regions of the mouse tyrosinase and tyrosinase related protein (TRP-1) genes. Several nuclear proteins were shown to bind to both of these upstream regions. Shared homologous DNA elements were also found in the 5' flanking sequences of Japanese quail and snapping turtle tyrosinase genes. Shared homologous nucleotide sequences were found to be scattered like an archipelago in the 5' upstream regions of mouse and human tyrosinase genes. Comparisons between Japanese quail and snapping turtle tyrosinase genes gave similar results. On the contrary, mammalian (mouse and human) and nonmammalian (quail and snapping turtle) tyrosinase genes did not show significant homology in their 5' upstream regions. In contrast, coding sequences in the first exons of vertebrate tyrosinase genes and their deduced amino acid sequences were found to be highly conserved except for their putative leader sequence-coding regions.