Melatonin is a potential drug for the prevention of bone loss during space flight.

Astronauts experience osteoporosis-like loss of bone mass because of microgravity conditions during space flight. To prevent bone loss, they need a riskless and antiresorptive drug. Melatonin is reported to suppress osteoclast function. However, no studies have examined the effects of melatonin on bone metabolism under microgravity conditions. We used goldfish scales as a bone model of coexisting osteoclasts and osteoblasts and demonstrated that mRNA expression level of acetylserotonin O-methyltransferase, an enzyme essential for melatonin synthesis, decreased significantly under microgravity. During space flight, microgravity stimulated osteoclastic activity and significantly increased gene expression for osteoclast differentiation and activation. Melatonin treatment significantly stimulated Calcitonin (an osteoclast-inhibiting hormone) mRNA expression and decreased the mRNA expression of receptor activator of nuclear factor κB ligand (a promoter of osteoclastogenesis), which coincided with suppressed gene expression levels for osteoclast functions. This is the first study to report the inhibitory effect of melatonin on osteoclastic activation by microgravity. We also observed a novel action pathway of melatonin on osteoclasts via an increase in CALCITONIN secretion. Melatonin could be the source of a potential novel drug to prevent bone loss during space flight.

Type of milk feeding affects hematological parameters and serum lipid profile in Japanese infants.

BACKGROUND: Type and volume of infant feeding determines infant growth, hematological parameters, and serum lipids. METHODS: Study subjects consisted of 103 infants who were born vaginally at term, with birthweight >2200 g. Milk feeding amount, type, and anthropometry were measured at 1 and 6 months. Hematological tests and serum lipid profile were assessed at 1 and 6 months. Thirty-four infants were breast-fed and 36 were formula-fed at 6 months. RESULTS: Breast-fed infants demonstrated similar growth patterns compared with partially breast-fed or formula-fed infants, despite the lower milk intake. Infants with higher breast milk intake at 6 months, however, tended to have lower hemoglobin levels. In contrast, higher formula intake at 6 months was related to lower serum total cholesterol. CONCLUSIONS: Japanese breast-fed infants were more likely to be anemic at 6 months, while formula-fed infants were likely to have low serum lipid levels. Iron-fortified infant foods may be useful to prevent anemia in breast-fed infants. Fat quality of infant formulas should be improved to enhance lipid status of formula-fed infants.

Role of gap junctions and protein kinase A during the development of oocyte maturational competence in Ayu (Plecoglossus altivelis).

Meiotic resumption in teleost oocytes is induced by a maturation-inducing hormone (MIH). The sensitivity of oocytes to MIH, also known as oocyte maturational competence (OMC), is induced by LH via mechanisms that are not fully understood. A previous study of Ayu (Plecoglossus altivelis) showed the presence of functional heterologous gap junctions (GJs) between oocytes and their surrounding granulosa cells. The objectives of this study were to determine the role of ovarian GJs and of protein kinase A (PKA) during the acquisition of OMC. We examined the effects of the specific GJ inhibitor carbenoxolone (CBX) and 18alpha-glycyrrhetinic acid (alpha-GA) on the LH-(hCG)-dependent acquisition of OMC and on MIH-(17,20beta-dihydroxy-4-pregnen-3-one)-dependent meiotic resumption; measured the cAMP content of ovarian follicles during the hCG-dependent acquisition of OMC; and determined the effects of PK activators and inhibitors on hCG-dependent OMC. Production of follicular cAMP increased during the hCG-dependent acquisition of OMC. Both GJ inhibitors and the PKA inhibitor H8-dihydrochloride, but not the PKC inhibitor GF109203X, suppressed the hCG-dependent acquisition of OMC in a dose-dependent manner. The PKA activator forskolin induced OMC with a similar potency to hCG. Unlike previous observations with teleosts where disruption of heterologous GJ either blocks or stimulates meiotic resumption, treatment with GJ inhibitors did not affect MIH-dependent meiotic resumption in maturationally competent follicles of Ayu. These observations suggest that ovarian GJs are essential for LH-dependent acquisition of OMC but not for MIH-dependent meiotic resumption, and that the stimulation of OMC by LH is mediated by cAMP-dependent PKA. They are also consistent with the view that a precise balance between GJ-mediated signals (positive or negative) and oocyte maturational readiness is required for hormonally regulated meiotic resumption.

Differential expression and localization of four connexins in the ovary of the ayu (Plecoglossus Altivelis).

The post-vitellogenic oocytes of teleost fish are generally unresponsive to maturation-inducing hormone (MIH) until a luteinizing hormone (LH) surge stimulates sensitivity via the acquisition of oocyte-maturational competence (OMC). Heterologous gap junctions (GJs) between granulosa cells and the oocyte have been previously implicated in the regulation of oocyte maturation in various vertebrate species. Although heterologous GJ are present in ovarian follicles of ayu (Plecoglossus altivelis), their role in maturation remains unclear. In the present study, we cloned and characterized complementary DNAs for GJ protein connexin (Cx), and examined the expression pattern of Cx messenger RNAs in the ayu ovary. Four Cx cDNAs with predicted molecular masses of 32.1 (Cx32.1), 34.9 (Cx34.9), 44.1 (Cx44.1), and 44.2 (Cx44.2) kDa, respectively, were cloned. Northern blot analysis revealed that the levels of Cx44.1 and Cx44.2 transcripts were similar during the vitellogenic and ovulatory stages. Cx32.1 transcripts were more abundant during the vitellogenic stage, but their levels declined thereafter. Cx34.9 transcript levels increased during the vitellogenic stage and remained high during the acquisition of OMC. In situ hybridization revealed that Cx44.1 and Cx44.2 signals were restricted to the oocyte, whereas the Cx32.1 and Cx34.9 signals were detected in both cellular fractions. Furthermore, a dye-transfer assay revealed the presence of functional GJs between the oocytes and follicle cells. These results suggest that Cx34.9 contributes to the formation of heterologous GJs between oocytes and granulosa cells. Moreover, GJs formed by Cx34.9 may function during the LH-dependent acquisition of OMC and the MIH-dependent resumption of meiosis in ayu.

Generation of viable fish from cryopreserved primordial germ cells.

An increasing number of wild fish species are in danger of extinction, often as a result of human activities. The cryopreservation of gametes and embryos has great potential for maintaining and restoring threatened species. The conservation of both paternal and maternal genetic information is essential. However, although this technique has been successfully applied to the spermatozoa of many fish species, reliable methods are lacking for the long-term preservation of fish eggs and embryos. Here, we describe a protocol for use with rainbow trout (Oncorhynchus mykiss) primordial germ cells (PGCs) and document the restoration of live fish from gametes derived from these cryopreserved progenitors. Genital ridges (GRs), which are embryonic tissues containing PGCs, were successfully cryopreserved in a medium containing 1.8 M ethylene glycol (EG). The thawed PGCs that were transplanted into the peritoneal cavities of allogenic trout hatchlings differentiated into mature spermatozoa and eggs in the recipient gonads. Furthermore, the fertilization of eggs derived from cryopreserved PGCs by cryopreserved spermatozoa resulted in the development of fertile F1 fish. This PGC cryopreservation technique represents a promising tool in efforts to save threatened fish species. Moreover, this approach has significant potential for maintaining domesticated fish strains carrying commercially valuable traits for aquaculture purposes.

A novel transforming growth factor-beta superfamily member expressed in gonadal somatic cells enhances primordial germ cell and spermatogonial proliferation in rainbow trout (Oncorhynchus mykiss).

Our understanding of the molecular mechanisms of primordial germ cell (PGC) proliferation in fish is rudimentary, but it is thought to be controlled by the surrounding somatic cells. We assumed that growth factors that are specifically involved in PGC proliferation are expressed predominantly in the surrounding genital ridge somatic cells. In order to isolate these growth factors, we compiled a complementary DNA (cDNA) subtractive library using cDNA from the genital ridges of 40-dpf rainbow trout embryos as the tester and cDNA from embryos without genital ridges as the driver. This approach identified a novel cytokine, designated gonadal soma-derived growth factor (GSDF), which is a member of the transforming growth factor (TGF)-beta superfamily. GSDF was expressed in the genital ridge somatic cells surrounding the PGCs during embryogenesis, and in both the granulosa and Sertoli cells at later stages. Inhibition of GSDF translation by antisense oligonucleotides suppressed PGC proliferation. Moreover, isolated testicular cells that were cultured with recombinant GSDF demonstrated dose-dependent proliferation of type-A spermatogonia; this effect was completely blocked by antiserum against GSDF. These results denote that GSDF, a novel member of the TGF-beta superfamily, plays an important role for proliferation of PGC and spermatogonia.

Expression of masu salmon delta5-desaturase-like gene elevated EPA and DHA biosynthesis in zebrafish.

Farmed fish could substitute for marine capture fish as a source of fatty acids such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) beneficial for human health; however, they require these compounds in their diets. In the present study on a model fish species, we modified the EPA/DHA biosynthesis pathway by overexpression of masu salmon Delta5-desaturase-like gene in zebrafish to increase its ability to synthesize EPA and DHA. Expression of this gene in transgenic fish fed a commercial diet and Artemia helped to improve their EPA content by 1.21-fold and DHA by 1.24-fold. In similar fish that were fed only Artemia the increments were 1.14-fold for EPA and 1.13-fold for DHA, compared with nontransgenic fish. In contrast, eicosatetraenoic acid content decreased, as it is a substrate of Delta5-desaturase, while the total lipid remained constant. The results demonstrated that masu salmon Delta5-desaturase is functional in zebrafish and can modify its fatty acid metabolic pathway. The technique could be applied to farmed fish to generate a nutritionally richer product for human consumption.

Testicular germ cells can colonize sexually undifferentiated embryonic gonad and produce functional eggs in fish.

Understanding the mechanisms that regulate germ-cell development is crucial to reproductive medicine and animal production. Animal gametes originally derive from sexually undifferentiated primordial germ cells (PGCs), which develop into mitotic germ cells (oogonia or spermatogonia) before proceeding to meiosis [Wylie, C. (1999) Cell 96, 165-174]. Spermatogonia are thought to include a population of cells with stem cell activity, which proliferate throughout the lifespan of male animals and produce spermatozoa [Zhao, G. Q. & Garbers, D. L. (2002) Dev. Cell 2, 537-547]. However, the functional differences between PGCs and spermatogonial stem cells are poorly understood. Here we show that transplanted adult testicular germ cells can colonize sexually undifferentiated embryonic gonads and resume gametogenesis. Testicular germ cells containing spermatogonial stem cells isolated from adult male rainbow trout (Oncorhynchus mykiss) were transplanted into the peritoneal cavity of newly hatched embryos of both sexes, and the behavior of the donor cells was observed. The testicular germ cells differentiated into spermatozoa in male recipients and fully functional eggs in female recipients. Furthermore, the donor-derived spermatozoa and eggs obtained from the recipient fish were able to produce normal offspring. These findings indicate that fish testicular germ cells, probably spermatogonial stem cells, possess a high level of developmental plasticity and sexual bipotency, even after the animal reaches maturity. Furthermore, our results suggest that spermatogonial stem cells are at least partly functionally similar to PGCs.

Segmental and full paternal isodisomy for chromosome 14 in three patients: narrowing the critical region and implication for the clinical features.

We report on segmental and full paternal isodisomy for chromosome 14 in three previously unreported Japanese patients. Patient 1 was a 5(6/12)-year-old girl, Patient 2 was a male neonate, and Patient 3 was a -year-old girl. Physical examination at birth showed various somatic features characteristic of paternal uniparental disomy for chromosome 14 (upd(14)pat) such as hairy forehead, protruding philtrum, micrognathia, small thorax, and abdominal wall defects in Patients 1-3, and the constellation of somatic features was persistently observed in Patients 1 and 3. Radiological studies at birth delineated unique bell-shaped thorax with coat-hanger appearance of the ribs in Patients 1-3, but the thoracic deformity ameliorated in Patients 1 and 3 by mid childhood. Chromosome analysis showed a 46,XX karyotype in Patients 1 and 3 and was not performed in Patient 2. Microsatellite analysis indicated full paternal isodisomy for chromosome 14 in Patients 1 and 2 and segmental paternal isodisomy for chromosome 14 distal to D14S981 at 14q23.3 in Patient 3. Methylation specific PCR assay for the differentially methylated region (DMR) of GTL2 at 14q32 yielded positive products with methylated allele specific primers and no products with unmethylated allele specific primers in Patients 1-3. Since clinical phenotype was similar between Patient 3 with segmental upd(14)pat and Patients 1 and 2 with full upd(14)pat, the results are keeping with the 14q32 localized imprinted genes as the critical components of the phenotype observed in upd(14)pat and help narrow the search for additional genes to the approximately 40 Mb region distal to D14S981. Furthermore, it is likely that the characteristic thoracic deformity ameliorates with age.

High-efficiency gene knockdown using chimeric ribozymes in fish embryos.

We report an effective gene knockdown technique in rainbow trout embryos using additional RNA components combined with ribozymes (R(z)s). Chimeric R(z)s (tR(z)Cs) containing tRNA(Val), R(z) against GFP, and a constitutive transport element were microinjected into transgenic embryos. tR(z)Cs induced greater gene interference than R(z)s alone. Control tR(z)Cs did not affect unpaired bases of target RNA, and the tR(z)C did not interfere with non-relevant gene expression, suggesting that the tR(z)C-mediated gene-interference effects were sequence-specific. Furthermore, the tR(z)C-containing expression vector specifically suppressed target GFP expression in transgenic trout. tR(z)Cs enhance R(z) cleavage and could therefore be powerful tools for studying unknown gene function in vertebrates.

Enhancement of EPA and DHA biosynthesis by over-expression of masu salmon delta6-desaturase-like gene in zebrafish.

The n - 3 polyunsaturated fatty acids, especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have important nutritional benefits in humans. Farmed fish could serve as promising sources of EPA/DHA, but they need these fatty acids or their precursors in their diets. Here we transferred masu salmon delta6-desaturase-like gene in zebrafish to increase its ability for synthesizing EPA and DHA. Expression of this gene in transgenic fish elevated their EPA content by 1.4-fold and DHA by 2.1-fold. On the other hand, the alpha-linolenic acid (ALA) content decreased, it being a substrate of delta6-desaturase, while the total lipid remained constant. This achievement demonstrates that fatty acid metabolic pathway in fish can be modified by the transgenic technique, and perhaps this could be applied to tailor farmed fish as even better sources of valuable human food.

Green fluorescent protein labeling of primordial germ cells using a nontransgenic method and its application for germ cell transplantation in salmonidae.

Transplanting primordial germ cells (PGCs) has a number of potential applications in fish bioengineering. Previously, we established a system to visualize live PGCs in the rainbow trout by introducing the green fluorescent protein (Gfp) gene driven by rainbow trout vasa gene regulatory regions. However, for PGC transplantation to be practically useful in aquaculture, visualization of PGCs using a nontransgenic technique is required. In this study, we demonstrate a method for labeling PGCs from various fish species by introducing chimeric RNAs composed of the Gfp coding region and vasa gene 3'-untranslated regions (UTRs); these sequences play a critical role in stabilizing mRNA in zebrafish PGCs. The GFP chimeric RNAs, including vasa 3'-UTR RNAs from rainbow trout, Nibe croaker, and zebrafish, were microinjected into the cytoplasm of fertilized eggs of several Salmonidae species. All the resulting embryos showed specific labeling in PGCs after the somatogenesis stage, which continued to be visible for at least 50 days. To apply this technique to PGC transplantation, PGCs labeled with chimeric RNA were microinjected into the peritoneal cavity of newly hatched salmonid embryos. The GFP labeling was sufficiently long-lived for the initial stage of donor PGC behavior to be followed in the recipient embryos. Importantly, donor PGCs from brown trout and masu salmon were incorporated into xenogeneic genital ridges in recipient rainbow trout. This nontransgenic method for labeling fish PGCs should be extremely useful for applications of PGC transplantation where the resulting progeny are to be released into the environment, such as PGC cryopreservation for fish stocks and surrogate brood stock technology.

[Retinopathy of prematurity in extremely low birth weight infants: a Tokyo multicenter study].

OBJECTIVE: To investigate how the increase in survival rate in extremely low birth weight (a birth weight of 1,000 g or less) infants had affected the incidence of retinopathy of prematurity (ROP) and the frequency of laser treatment. METHODS: We retrospectively reviewed the medical records of 122 surviving premature infants with birthweights less than 1,000 g to determine the severity of ROP observed at 16 neonatal intensive care units in Tokyo between April and October 2002. RESULTS: The survival rate was 85.6%. The mean gestational age was 26.74 weeks and the mean birth weight was 782.25 g. One-hundred-and-five infants (86.1%) developed ROP, fifty (41.0%) received laser treatment, and six (4.9%) had retinal detachment. The median postmenstrual age (gestational age at birth plus chronological age in weeks, PMA) at the onset of ROP was 32.5 weeks, and the first laser treatment was performed at the median PMA of 35.7 weeks. CONCLUSIONS: In these extremely low birth weight infants, there was an increase in the survival rate and in the incidence of severe ROP that progressed to the stage that required treatment.

Biotechnology: surrogate broodstock produces salmonids.

A worldwide decline in the number of wild salmonids calls for strategies to restore endangered populations. Here we show that germ cells can be transplanted between two different salmonid species, with the subsequent production of xenogenic, donor-derived offspring. This pioneering xenotransplantation technology may eventually find applications in facilitating the production of commercially valuable fish, as well as in species conservation.

Molecular cloning, gene expression in albino mutants and gene knockdown studies of tyrosinase mRNA in rainbow trout.

Tyrosinase has a role in melanin synthesis and several defects of the tyrosinase gene lead to albinism. Here, we cloned and characterized rainbow trout tyrosinase cDNAs and carried out the molecular and biochemical characterization of albino mutants. Two types of cDNA were cloned: tyrosinase-1 (Tyr-1) and tyrosinase-2 (Tyr-2). Both contained regions predicted to encode structural features of tyrosinase, and phylogenetic analysis confirmed that Tyr-1 and Tyr-2 were members of the tyrosinase family. Tyr-1 transcripts were first detected in embryos at 5 d post-fertilization (dpf) and Tyr-2 transcripts at 15 dpf. 3,4-dihydroxyphenylalanine assays revealed significantly reduced tyrosinase activities in dominant and recessive albino mutants compared with wild-type embryos. However, reverse-transcription PCR showed no differences in the amounts or lengths of the coding regions of Tyr-1 and Tyr-2 transcripts between wild-type embryos and albino mutants. Antisense morpholino oligonucleotides (AMOs) designed to knockdown tyrosinase gene expression in wild-type embryos led to reduced pigmentation in the retina and skin of embryos at 25 and 35 dpf, respectively. Furthermore, the tyrosinase activities of AMO-treated embryos were significantly reduced. We conclude that both Tyr-1 and Tyr-2 are crucial for melanin synthesis in rainbow trout embryos. Furthermore, we describe a potential application of AMOs in the treatment of hyperpigmentation.

Isolation of highly pure and viable primordial germ cells from rainbow trout by GFP-dependent flow cytometry.

A highly pure and viable primordial germ cell (PGC) population appears to be an essential tool for establishing a cell line that can differentiate into a germ cell lineage and for studying the molecular biology and biochemistry of fish PGCs. Therefore, the aim of the present study was to establish a flow cytometric method for isolating highly pure and viable PGCs. As the material for PGC isolation, we used transgenic rainbow trout possessing the green fluorescent protein (GFP) gene driven by trout vasa-gene regulatory sequences (pvasa-GFP). Four independent transgenic strains were subjected to fluorescence microscopy and GFP-dependent flow cytometric analyses. We found that some of the pvasa-GFP transgenic strains exhibited ectopic background green fluorescence in the somatic cells aside from strong fluorescence in PGCs. Although flow cytometric analysis of genital ridge somatic cells in the four pvasa-GFP transgenic strains revealed a wide range of GFP intensities, we proved that somatic cell contamination of the GFP-positive cell population was markedly reduced if transgenic strains without the ectopic background green fluorescence were used. In addition, the forward light-scattering (FS) property, which is an indication of relative cell size, and the side light-scattering (SS) property, which is determined by cell shape and granularity, were employed to remove non-PGC contaminants from the GFP-positive cell population. By isolating GFP-positive cells with high FS/SS values, we were able to effectively remove cell blebs and the apoptotic fraction. Consequently, the purities and survival rates of isolated PGCs were greatly improved compared with those using GFP intensity as a single indicator. Thus, our flow cytometric method, in combination with the selection of suitable transgenic strains without the ectopic background green fluorescence, is capable of isolating highly pure and viable PGCs from rainbow trout. By using this method in combination with cell-cryopreservation and cell transplantation techniques, the isolated PGCs may also be used for preserving the genetic resources of endangered fish species and domesticated fish strains carrying commercially valuable traits. Mol. Reprod. Dev. 67: 91-100, 2004.

Fish eggs as bioreactors: the production of bioactive luteinizing hormone in transgenic trout embryos.

We demonstrated the production of goldfish luteinizing hormone (gfLH) by the use of 4-day-old rainbow trout embryos as novel bioreactors. This expression system has several advantages: target proteins can be rapidly expressed at low cost, and recombinant proteins can be synthesized at low temperatures and can undergo complex post-translational modifications (PTMs). An expression vector containing gfLH cDNA was microinjected into fertilized trout eggs. After 4 days of incubation at 10 degrees C, transgenic embryos were harvested and glycosylated recombinant gfLH was recovered, which stimulated testosterone production in testicular fragments from the goldfish. This is the first report on the successful production of bioactive recombinant gonadotropin originated from cyprinid. Further, these results demonstrate that trout-embryo bioreactors are a potentially powerful tool for the production of functional recombinant proteins.

Specific gene silencing using small interfering RNAs in fish embryos.

Recently, small interfering RNAs (siRNAs) have been used for gene knockdown in mammalian cultured cells, but their utility in fish has remained unexplored. Here we demonstrate a siRNA-mediated gene silencing technique in rainbow trout embryos. We found that siRNAs effectively suppressed the transient expression of episomally located foreign GFP genes at an early developmental stage and inhibited the expression of GFP genes in stable transgenic trout embryos. Similar gene silencing was observed with an siRNA against the endogenous tyrosinase A gene. siRNAs interfered with the expression of maternally inherited mRNA. siRNAs did not affect non-relevant gene expression and siRNAs with a 4 base mismatch did not affect target gene expression. siRNA gene silencing is therefore highly sequence-specific. Our findings are the first evidence that siRNA-mediated gene silencing is effective in fish. This technique could be a powerful tool for studying gene function during embryonic development in aquacultural fish species, zebrafish, and medaka.

Generation of live fry from intraperitoneally transplanted primordial germ cells in rainbow trout.

Germ cell transplantation has tremendous applications in transgenic animal production, assisted reproductive technology, and germline stem cell research. Here, we report for the first time the production of individuals from intraperitoneally transplanted primordial germ cells (PGCs) in animals. To trace the behavior of exogenous PGCs in recipients, PGCs visualized by a green fluorescent protein gene were used as donors. The PGCs prepared from the genital ridges of hatching embryos were transplanted into recipients at various developmental stages. The PGCs injected into the peritoneal cavities of hatching embryos had the ability to migrate toward, and to colonize, the genital ridges of recipient embryos. Furthermore, donor-derived PGCs proliferated and differentiated into mature eggs and sperm in the allogenic gonads; the resulting gametes produced live fry, showing the donor-derived phenotype, through fertilization. Combined with in vitro culture, genetic modification, and cryopreservation of PGCs, this technique provides new approaches for fish bioengineering.

Differentiation of chondrocytes and scleroblasts during dorsal fin skeletogenesis in flounder larvae.

In teleosts, the embryonic fin fold consists of a peridermis, an underlying epidermis and a small number of mesenchymal cells. Beginning from such a simple structure, the fin skeletons, including the proximal and distal radials and lepidotrichia (finrays), develop in the dorsal fin fold at the larval stage. Their process of skeletogenesis and embryonic origin are unclear. Using flounder larvae, we report the differentiation process for chondrocytes and scleroblasts prior to fin skeletogenesis and the effects of retinoic acid (RA) on it. In early larvae, the mesenchymal cells grow between the epidermis and spinal cord to form a line of periodical condensations, which are proximal radial primordia, to produce chondrocytes. The prescleroblasts, which ossify the proximal radial cartilages, differentiate in the mesenchymal cells remaining between the cartilages. Then, mesenchymal condensations occur between the distal ends of the proximal radials, forming distal radial primordia, to produce chondrocytes. Simultaneously, condensations occur between the distal radial primordia and peridermis, which are lepidotrichia primordia, to produce prescleroblasts. Exogenous RA specifically inhibits the mesenchymal condensation prior to the proximal radial formation together with the down-regulation of sonic hedgehog (shh) and patched (pta) expression, resulting in the loss of proximal radials. Thus, it was indicated that differentiation of the precursor cells of radials and lepidotrichia begins in the proximal part of the fin fold and that the initial mesenchymal condensation prior to the proximal radial formation is highly susceptible to the effects of RA. Lepidotrichia formation does not occur where proximal radials are absent, indicating that lepidotrichia differentiation requires interaction with the radial cartilages. To examine the suggestion that neural crest cells contribute to the medial fin skeletons, we localized the HNK-1 positive cells in flounder embryos and slug and msxb-positive cells in pufferfish, Fugu rubripes, embryos. That the positive cells commonly arrive at the proximal part of the fin fold does not contradict the suggestion, but their final destiny as radial chondrocytes or lepidotrichia scleroblasts, should be further investigated.