Yoshiyasu Ishimaru

Pitx2. a bicoid-type homeobox gene, is involved in a lefty-signaling pathway in determination of left-right asymmetry

Signaling molecules such as Activin, Sonic hedgehog, Nodal, Lefty, and Vg1 have been found to be involved in determination of left-right (L-R) asymmetry in the chick, mouse, or frog. However, a common signaling pathway has not yet been identified in vertebrates. We report that Pitx2, a bicoid-type homeobox gene expressed asymmetrically in the left lateral plate mesoderm, may be involved in determination of L-R asymmetry in both mouse and chick. Since Pitx2 appears to be downstream of lefty-1 in the mouse pathway, we examined whether mouse Lefty proteins could affect the expression of Pitx2 in the chick. Our results indicate that a common pathway from lefty-1 to Pitx2 likely exists for determination of L-R asymmetry in vertebrates.

Mechanism of asymmetric ovarian development in chick embryos

In most animals, the gonads develop symmetrically, but most birds develop only a left ovary. A possible role for estrogen in this asymmetric ovarian development has been proposed in the chick, but the mechanism underlying this process is largely unknown. Here, we identify the molecular mechanism responsible for this ovarian asymmetry. Asymmetric PITX2 expression in the left presumptive gonad leads to the asymmetric expression of the retinoic-acid (RA)-synthesizing enzyme, RALDH2, in the right presumptive gonad. Subsequently, RA suppresses expression of the nuclear receptors Ad4BP/SF-1 and estrogen receptor α in the right ovarian primordium. Ad4BP/SF-1 expressed in the left ovarian primordium asymmetrically upregulates cyclin D1 to stimulate cell proliferation. These data suggest that early asymmetric expression of PITX2 leads to asymmetric ovarian development through up- or downregulation of RALDH2, Ad4BP/SF-1, estrogen receptor α and cyclin D1.

Asymmetric expression of antivin/lefty1 in the early chick embryo

Mammalian lefty and zebrafish antivin, highly related to lefty, are shown to be expressed asymmetrically and involved in the specification of the left body side of early embryos. We isolated a chick homologue of the antivin/lefty1 cDNA and studied its expression pattern during early chick development. We found that antivin/lefty1 is expressed asymmetrically on the left side of the prospective floorplate, notochord and lateral plate mesoderm of the chick embryo.

Analysis of RNA-Seq data reveals involvement of JAK/STAT signalling during leg regeneration in the cricket Gryllus bimaculatus

In the cricket Gryllus bimaculatus, missing distal parts of the amputated leg are regenerated from the blastema, a population of dedifferentiated proliferating cells that forms at the distal tip of the leg stump. To identify molecules involved in blastema formation, comparative transcriptome analysis was performed between regenerating and normal unamputated legs. Components of JAK/STAT signalling were upregulated more than twofold in regenerating legs. To verify their involvement, Gryllus homologues of the interleukin receptor Domeless (Gb’dome), the Janus kinase Hopscotch (Gb’hop) and the transcription factor STAT (Gb’Stat) were cloned, and RNAi was performed against these genes. Gb’domeRNAi, Gb’hopRNAi and Gb’StatRNAi crickets showed defects in leg regeneration. Blastema expression of Gb’cyclinE was decreased in the Gb’StatRNAi cricket compared with that in the control. Hyperproliferation of blastema cells caused by Gb’fatRNAi or Gb’wartsRNAi was suppressed by RNAi against Gb’Stat. The results suggest that JAK/STAT signalling regulates blastema cell proliferation during leg regeneration.

Roles of OA1 octopamine receptor and Dop1 dopamine receptor in mediating appetitive and aversive reinforcement revealed by RNAi studies

Revealing reinforcing mechanisms in associative learning is important for elucidation of brain mechanisms of behavior. In mammals, dopamine neurons are thought to mediate both appetitive and aversive reinforcement signals. Studies using transgenic fruit-flies suggested that dopamine neurons mediate both appetitive and aversive reinforcements, through the Dop1 dopamine receptor, but our studies using octopamine and dopamine receptor antagonists and using Dop1 knockout crickets suggested that octopamine neurons mediate appetitive reinforcement and dopamine neurons mediate aversive reinforcement in associative learning in crickets. To fully resolve this issue, we examined the effects of silencing of expression of genes that code the OA1 octopamine receptor and Dop1 and Dop2 dopamine receptors by RNAi in crickets. OA1-silenced crickets exhibited impairment in appetitive learning with water but not in aversive learning with sodium chloride solution, while Dop1-silenced crickets exhibited impairment in aversive learning but not in appetitive learning. Dop2-silenced crickets showed normal scores in both appetitive learning and aversive learning. The results indicate that octopamine neurons mediate appetitive reinforcement via OA1 and that dopamine neurons mediate aversive reinforcement via Dop1 in crickets, providing decisive evidence that neurotransmitters and receptors that mediate appetitive reinforcement indeed differ among different species of insects.

Short germ insects utilize both the ancestral and derived mode of Polycomb group-mediated epigenetic silencing of Hox genes.

In insect species that undergo long germ segmentation, such as Drosophila, all segments are specified simultaneously at the early blastoderm stage. As embryogenesis progresses, the expression boundaries of Hox genes are established by repression of gap genes, which is subsequently replaced by Polycomb group (PcG) silencing. At present, however, it is not known whether patterning occurs this way in a more ancestral (short germ) mode of embryogenesis, where segments are added gradually during posterior elongation. In this study, two members of the PcG family, Enhancer of zeste (E(z)) and Suppressor of zeste 12 (Su(z)12), were analyzed in the short germ cricket, Gryllus bimaculatus. Results suggest that although stepwise negative regulation by gap and PcG genes is present in anterior members of the Hox cluster, it does not account for regulation of two posterior Hox genes, abdominal-A (abd-A) and Abdominal-B (Abd-B). Instead, abd-A and Abd-B are predominantly regulated by PcG genes, which is the mode present in vertebrates. These findings suggest that an intriguing transition of the PcG-mediated silencing of Hox genes may have occurred during animal evolution. The ancestral bilaterian state may have resembled the current vertebrate mode of regulation, where PcG-mediated silencing of Hox genes occurs before their expression is initiated and is responsible for the establishment of individual expression domains. Then, during insect evolution, the repression by transcription factors may have been acquired in anterior Hox genes of short germ insects, while PcG silencing was maintained in posterior Hox genes.

Mesonephric Wnt Signaling Associate with a Formation of an Adreno‐Gonadal Primordium in Chick Embryos

The adrenal cortex as well as the reproductive tract, kidney, and gonad is derived from the intermediate mesoderm. In addition, the mesonephros essential for the early adrenal development also originates from the intermediate mesoderm. A contribution of mesonephros to adrenal formation through expression of growth factors has been largely unknown. Moreover, Ad4 binding protein/steroidogenic factor 1[Ad4BP/SF-1(NR5A1)], and Dmrt-1(doublesex- and mab-3-related transcription factor) are expressed in adrenal and gonadal development chick embryos. What growth factor(s) regulate the expression of Ad4BP/SF-1 and Dmrt-1, has not been determined. Here we show with chick embryos that Wnt4 is expressed in only certain parts of the mesonephric tubules, while the expression was also detected in the mesenchyme at stage 21. Moreover, Ad4BP/SF-1 and Dmrt-1 were expressed in the developing adreno-gonadal and gonadal primordial at the same stage adjacent to the expressions of Wnt4. To examine the possibility that Ad4BP/ SF-1 and Dmrt-1 were influenced by Wnt4, we performed with misexpression. Our studies showed that Wnt4 expanded marker gene expression for the adreno-gonadal primordium, whereas Wnt4 does not stimulated gonad formation. We conclude that Wnt signaling is involved in the formation of the adreno-gonadal primordium.

Bone morphogenetic protein signaling in distal patterning and intercalation during leg regeneration of the cricket, Gryllus bimaculatus

The cricket, Gryllus bimaculatus, is a classic model of leg regeneration following amputation. We previously demonstrated that Gryllus decapentaplegic (Gb’dpp) is expressed during leg regeneration, although it remains unclear whether it is essential for this process. In this study, double‐stranded RNA targeting the Smad mathers‐against‐dpp homolog, Gb’mad, was used to examine the role of bone morphogenetic protein (BMP) signaling in the leg regeneration process of Gryllus bimaculatus. RNA interference (RNAi)‐mediated knockdown of Gb’mad led to a loss of tarsus regeneration at the most distal region of regenerating leg segments. Moreover, we confirmed that the phenotype obtained by knockdown of Dpp type I receptor, Thick veins (Gb’tkv), closely resembled that observed for Gb’mad RNAi crickets, thereby suggesting that the BMP signaling pathway is indispensable for the initial stages of tarsus formation. Interestingly, knockdown of Gb’mad and Gb’tkv resulted in significant elongation of regenerating tibia along the proximodistal axis compared with normal legs. Moreover, our findings indicate that during the regeneration of tibia, the BMP signaling pathway interacts with Dachsous/Fat (Gb’Ds/Gb’Ft) signaling and dachshund (Gb’dac) to re‐establish positional information and regulate determination of leg size. Based on these observations, we discuss possible roles for Gb’mad in the distal patterning and intercalation processes during leg regeneration in Gryllus bimaculatus.

Molecular mechanisms of limb regeneration: insights from regenerating legs of the cricket Gryllus bimaculatus

This review summarizes recent advances in leg regeneration research, focusing on the cricket Gryllus bimaculatus. Recent studies have revealed molecular mechanisms on blastema formation, establishment of positional information, and epigenetic regulation during leg regeneration. Especially, these studies have provided molecular bases in classical conceptual models such as the polar coordinate model, the intercalation model, the boundary model, the steepness model, etc., which were proposed to interpret regeneration processes of the cockroach legs. When a leg is amputated, a blastema is formed through the activation of the Janus-kinase (Jak)/Signal-Transduction-and-Activator-of-Transcription (STAT) pathway. Subsequently, the Hedgehog/Wingless/Decapentaplegic/Epidermal-growth-factor pathways instruct distalization in the blastema, designated as the molecular boundary model. Downstream targets of this pathway are transcription factors Distal-less (Dll) and dachshund (dac), functioning as key regulators of proximodistal pattern formation. Dll and dac specify the distal and proximal regions in the blastema, respectively, through the regulation of tarsal patterning genes. The expression of leg patterning genes during regeneration may be epigenetically controlled by histone H3K27 methylation via Enhancer-of-zeste and Ubiquitously-transcribed-tetratricopeptide-repeat-gene-X-chromosome. For the molecular mechanism of intercalation of the missing structures between the amputated position and the most distal one, Dachsous/Fat (Ds/Ft) steepness model has been proposed, in which the Ds/Ft pathway maintains positional information and determines leg size through dac expression. This model was theoretically verified to interpret the experimental results obtained with cricket legs. Availability of whole-genome sequence information, regeneration-dependent RNA interference, and genome editing technique will have the cricket be an ideal model system to reveal gene functions in leg regeneration.

Growth factors from mesonephros implicated in gonadal and adrenal differentiation.

The mesonephros is a tissue formed transiently as a basement structure of early stages of the gonad and adrenal cortex, and thus it has been believed that the mesonephros is strictly related to differentiation of these two tissues. In this regard, it is interesting to note which growth factors are expressed in the mesonephros or intermediate mesoderm, from which the mesonephros is derived. In addition to studies with mouse fetuses, we detected expression of FGFs and Wnt in the developing mesonephros and intermediated mesoderm in chick embryos. In order to clarify the functions of these growth factors, overexpression and chemical inhibition studies were performed with the chick. In consequence, it was found that FGF signal is essential for the gonad development probably by stimulation of cell proliferation and determination of cell-fate. These studies also showed that another unknown signal is required for the adrenal development.