Specification of both neural crest cells and Rohon-Beard (RB) sensory neurons involves a complex series of interactions between the neural and non-neural ectoderm. The molecular mechanisms directing this process are not well understood. The zebrafish narrowminded (nrd) mutation is unique, since it is one of two mutations in which defects are observed in both cell populations: it leads to a complete absence of RB neurons and a reduction in neural crest cells and their derivatives. Here, we show that nrd is a mutation in prdm1, a SET/zinc-finger domain transcription factor. A Morpholino-mediated depletion of prdm1 phenocopies the nrd mutation, and conversely overexpression of prdm1 mRNA rescues the nrd RB sensory neuron and neural crest phenotype. prdm1 is expressed at the border of the neural plate within the domain where neural crest cells and RB sensory neurons form. Analysis of prdm1 function by overexpression indicates that prdm1 functions to promote the cell fate specification of both neural crest cells and RB sensory neurons, most likely as a downstream effector of the BMP signaling pathway.

IntroductionDeuterostomes (animals with ‘secondary mouths’) are generally accepted to develop the mouth independently of the blastopore. However, it remains largely unknown whether mouths are homologous among all deuterostome groups. Unlike other bilaterians, in amphioxus the mouth initially opens on the left lateral side. This peculiar morphology has not been fully explained in the evolutionary developmental context. We studied the developmental process of the amphioxus mouth to understand whether amphioxus acquired a new mouth, and if so, how it is related to or differs from mouths in other deuterostomes.ResultsThe left first somite in amphioxus produces a coelomic vesicle between the epidermis and pharynx that plays a crucial role in the mouth opening. The vesicle develops in association with the amphioxus-specific Hatschek nephridium, and first opens into the pharynx and then into the exterior as a mouth. This asymmetrical development of the anterior-most somites depends on the Nodal-Pitx signaling unit, and the perturbation of laterality-determining Nodal signaling led to the disappearance of the vesicle, producing a symmetric pair of anterior-most somites that resulted in larvae lacking orobranchial structures. The vesicle expressed bmp2/4, as seen in ambulacrarian coelomic pore-canals, and the mouth did not open when Bmp2/4 signaling was blocked.ConclusionsWe conclude that the amphioxus mouth, which uniquely involves a mesodermal coelomic vesicle, shares its evolutionary origins with the ambulacrarian coelomic pore-canal. Our observations suggest that there are at least three types of mouths in deuterostomes, and that the new acquisition of chordate mouths was likely related to the dorso-ventral inversion that occurred in the last common ancestor of chordates.

Rohon-Beard sensory neurons, neural crest cells, and sensory placodes can be distinguished at the boundary of the embryonic epidermis (skin) and the neural plate. The inductive signals at the neural plate border region are likely to involve a gradient of bone morphogenic protein (BMP) in conjunction with FGF and Wnts and other signals. However, how these signals are transduced to produce the final cell fate remains to be determined. Recent evidence from Xenopus and chick suggest that Dlx genes are required for the generation of cell fates at the neural plate border (McLarren, K.W., Litsiou, A., Streit, A., 2003. DLX5 positions the neural crest and preplacode region at the border of the neural plate. Dev. Biol. 259, 34-47; Woda, J.M., Pastagia, J., Mercola, M., Artinger, K.B., 2003. Dlx proteins position the neural plate border and determine adjacent cell fates. Development 130, 331-342). In the present study, we extend these findings to zebrafish, where we unequivocally demonstrate that dlx3b and dlx4b function in a dose-dependent manner to specify cell fates such as Rohon-Beard sensory neurons and trigeminal sensory placodes. dlx function was examined by inhibiting: (1) protein levels with antisense morpholino oligonucleotides (MOs), and (2) activity by repressing the ability of dlx-homeodomain to bind to downstream targets (EnR-dlx3bhd mRNA; dlx3b homeodomain fused to Engrailed transcriptional repressor domain). Inhibition of dlx3b and dlx4b protein and activity resulted in the reduction or complete loss of Rohon-Beard (RB) sensory neurons and trigeminal (TG) sensory placodes. These data suggest that dlx3b and dlx4b function in the specification of RB neurons and trigeminal sensory placodes in zebrafish. Further, we have shown that dlx3b and dlx4b function in a non-cell-autonomous manner for RB neuron development; dlx3b and dlx4b act to regulate bmp2b expression at the non-neural ectodermal border. These data suggest that the contribution of dlx3b and dlx4b to neural plate border formation is partially non-cell-autonomous acting via BMP activity.

Insufficient expression of hepatic fibroblast growth factor 21 (FGF21) and stromal cell-derived factor 2 like 1 (Sdf2l1) reportedly leads to insulin resistance and hepatosteatosis in obesity and type 2 diabetes. On the other hand, increased expression of hepatic serotonin receptor 2a (htr2a) in diet-induced obesity contributes to hepatosteatosis. Here we show that increases in circulating FGF21 levels and expression of hepatic FGF21 preceded weight gain, hyperinsulinemia, and hyperglycemia in C57BLJ6 mice fed a high-fat diet. Expression of hepatic htr2a and Sdf2l1 increased in insulin-resistant mice fed a high-fat diet. Intake of whey protein isolate decreased plasma FGF21 levels and expression of hepatic FGF21 in mice fed either a high-fat diet or a chow diet, whereas it only suppressed the overexpression of hepatic Sdf2 and htr2a in insulin-resistant mice fed a high-fat diet. Moreover, intake of whey protein isolate decreased plasma serotonin levels in mice fed either a high-fat diet or a chow diet. Genetic inhibition of tryptophan hydroxylase 1 decreased hepatic FGF21 expression and plasma FGF21 levels in mice. These findings suggest that increased hepatic FGF21 production precedes diet-induced weight gain, hyperinsulinemia, and hyperglycemia, and that intake of whey protein isolate could inhibit hepatic FGF21 production by suppressing peripheral serotonin synthesis.

Rohon-Beard (RB) mechanosensory neurons are among the first sensory neurons to develop, and the process by which they adopt their fate is not completely understood. RBs form at the neural plate border (NPB), the junction between neural and epidermal ectoderm, and require the transcription factor prdm1a. Here, we show that prior to RB differentiation, prdm1a overlaps extensively with the epidermal marker dlx3b but shows little overlap with the neuroectodermal markers sox3 and sox19a. Birthdating analysis reveals that the majority of RBs are born during gastrulation in zebrafish, suggesting that it is during this period that RBs become specified. Expression analysis in prdm1a and neurogenin1 mutant and dlx3b/dlx4b morpholino-injected embryos suggests that prdm1a is upstream of dlx3b, dlx4b, and neurogenin1 at the NPB. mRNA for neurogenin1 or dlx3b/dlx4b can rescue the lack of RBs in prdm1a. Based on these data we suggest a preliminary gene regulatory network for RBs development.

The perforated pharynx has generally been regarded as a shared characteristic of chordates. However, there still remains phylogenetic ambiguity between the cilia-driven system in invertebrate chordates and the muscle-driven system in vertebrates. Giant larvae of the genus Asymmetron were reported to develop an orobranchial musculature similar to that of vertebrates more than 100 years ago. This discovery might represent an evolutionary link for the chordate branchial system, but few investigations of the lancelet orobranchial musculature have been completed since. We studied staged larvae of a Japanese population of Branchiostoma japonicum to characterize the developmental property of the orobranchial musculature. The larval mouth and the unpaired primary gills develop well-organized muscles. These muscles function only as obturators of the openings without antagonistic system. As the larval mouth enlarged posteriorly to the level of the ninth myomere, the oral musculature was fortified accordingly without segmental patterning. In contrast, the iterated branchial muscles coincided with the dorsal myomeric pattern before metamorphosis, but the pharynx was remodeled dynamically irrespective of the myomeric pattern during metamorphosis. The orobranchial musculature disappeared completely during metamorphosis, and adult muscles in the oral hood and velum, as well as on the pterygial coeloms developed independently. The lancelet orobranchial musculature is apparently a larval adaptation to prevent harmful intake. However, vestigial muscles appeared transiently with the secondary gill formation suggest a bilateral ancestral state of muscular gills, and a segmental pattern of developing branchial muscles without neural crest and placodal contributions is suggestive of a precursor of vertebrate branchiomeric pattern.

Cancer stem cells are an important target for effective therapy, since they show tumorigenicity, chemoresistance, and radioresistance. We isolated cancer stem cells from glioma cell lines and tissues and examined the expression of cancer testis antigen (CTA) genes as potential target molecules for cancer vaccine therapy. CTA genes were highly and frequently expressed in cancer stem cells compared with differentiated cells. In addition, histone acetylation levels in the promoter regions of CTA genes were high in cancer stem cells and low in differentiated cells, while DNA methylation analysis of the promoter regions revealed hypomethylation in cancer stem cells. This epigenetic difference between cells leads to heterogeneous expression of CTA genes in the tumor mass, which consists of cells at various levels of differentiation. Moreover, the expression level of HLA class I antigens was not affected by the differentiation status, suggesting that CTA genes may present as surface antigens in cancer stem cells. Taken together, these findings suggest that CTA genes may be attractive candidates for targeted vaccine therapy against cancer stem cells in glioma patients.

Lancelets (amphioxus) exhibit a remarkable asymmetric development in the anterior body region, which is reflected in the peripheral nervous system even at adulthood. Not all of the anterior nerves are involved, but the left third to fifth nerves are clearly asymmetric. To trace the developmental process responsible for asymmetric innervation, the peripheral nerves in the anterior region were studied in pre- and mid-metamorphic larvae, 1-cm-long juveniles, and in adults by using whole-mount immunostaining. The mouth changes in size and location during larval life before moving ventrally and, in conjunction with this change, nerves in the oral region are also modified. The left second nerve initially innervates the oral region, but this connection is secondarily lost. As the mouth expands and shifts posteriorly, the left fifth to ninth nerves join the left third and fourth in the innervation of the oral region. The left third to sixth nerves anastomose with the oral nerve ring, which encircles the mouth on the left side. In the juveniles and adults, there are two nerve plexuses that run parallel to the margin of the oral hood. The innermost of these, the "inner oral-hood nerve plexus", is asymmetrically connected with the left third to fifth nerves on both sides. The other, the "outer oral-hood nerve plexus", is ipsilaterally connected with the third to seventh nerves on both sides. The velar nerve ring is also innervated asymmetrically by the left fourth and fifth nerves. From these observations, we suggest that the oral nerve ring is the precursor of both the inner oral-hood nerve plexus and the velar nerve ring, and that the asymmetric innervation retained in adult lancelets is related to the early anastomosis of the left nerves with the oral nerve ring. We also show that, contrary to the persistent asymmetric innervation, the axonal patterns of the anterior peripheral nervous system in developing lancelets can change.