Involvement of a Basic Helix-Loop-Helix Gene BHLHE40 in Specification of Chicken Retinal Pigment Epithelium.

The first event of differentiation and morphogenesis in the optic vesicle (OV) is specification of the neural retina (NR) and retinal pigment epithelium (RPE), separating the inner and outer layers of the optic cup, respectively. Here, we focus on a basic helix-loop-helix gene, BHLHE40, which has been shown to be expressed by the developing RPE in mice and zebrafish. Firstly, we examined the expression pattern of BHLHE40 in the developing chicken eye primordia by in situ hybridization. Secondly, BHLHE40 overexpression was performed with in ovo electroporation and its effects on optic cup morphology and expression of NR and RPE marker genes were examined. Thirdly, we examined the expression pattern of BHLHE40 in LHX1-overexpressed optic cup. BHLHE40 expression emerged in a subset of cells of the OV at Hamburger and Hamilton stage 14 and became confined to the outer layer of the OV and the ciliary marginal zone of the retina by stage 17. BHLHE40 overexpression in the prospective NR resulted in ectopic induction of OTX2 and repression of VSX2. Conversely, BHLHE40 was repressed in the second NR after LHX1 overexpression. These results suggest that emergence of BHLHE40 expression in the OV is involved in initial RPE specification and that BHLHE40 plays a role in separation of the early OV domains by maintaining OTX2 expression and antagonizing an NR developmental program.

Toll signalling promotes blastema cell proliferation during cricket leg regeneration via insect macrophages.

Hemimetabolous insects, such as the two-spotted cricket Gryllus bimaculatus, can recover lost tissues, in contrast to the limited regenerative abilities of human tissues. Following cricket leg amputation, the wound surface is covered by the wound epidermis, and plasmatocytes, which are insect macrophages, accumulate in the wound region. Here, we studied the function of Toll-related molecules identified by comparative RNA sequencing during leg regeneration. Of the 11 Toll genes in the Gryllus genome, expression of Toll2-1, Toll2-2 and Toll2-5 was upregulated during regeneration. RNA interference (RNAi) of Toll, Toll2-1, Toll2-2, Toll2-3 or Toll2-4 produced regeneration defects in more than 50% of crickets. RNAi of Toll2-2 led to a decrease in the ratio of S- and M-phase cells, reduced expression of JAK/STAT signalling genes, and reduced accumulation of plasmatocytes in the blastema. Depletion of plasmatocytes in crickets using clodronate also produced regeneration defects, as well as fewer proliferating cells in the regenerating legs. Plasmatocyte depletion also downregulated the expression of Toll and JAK/STAT signalling genes in the regenerating legs. These results suggest that Spz-Toll-related signalling in plasmatocytes promotes leg regeneration through blastema cell proliferation by regulating the Upd-JAK/STAT signalling pathway.

Insights into the genomic evolution of insects from cricket genomes.

Most of our knowledge of insect genomes comes from Holometabolous species, which undergo complete metamorphosis and have genomes typically under 2 Gb with little signs of DNA methylation. In contrast, Hemimetabolous insects undergo the presumed ancestral process of incomplete metamorphosis, and have larger genomes with high levels of DNA methylation. Hemimetabolous species from the Orthopteran order (grasshoppers and crickets) have some of the largest known insect genomes. What drives the evolution of these unusual insect genome sizes, remains unknown. Here we report the sequencing, assembly and annotation of the 1.66-Gb genome of the Mediterranean field cricket Gryllus bimaculatus, and the annotation of the 1.60-Gb genome of the Hawaiian cricket Laupala kohalensis. We compare these two cricket genomes with those of 14 additional insects and find evidence that hemimetabolous genomes expanded due to transposable element activity. Based on the ratio of observed to expected CpG sites, we find higher conservation and stronger purifying selection of methylated genes than non-methylated genes. Finally, our analysis suggests an expansion of the pickpocket class V gene family in crickets, which we speculate might play a role in the evolution of cricket courtship, including their characteristic chirping.

The role of clockwork orange in the circadian clock of the cricket Gryllus bimaculatus.

The circadian clock generates rhythms of approximately 24 h through periodic expression of the clock genes. In insects, the major clock genes period (per) and timeless (tim) are rhythmically expressed upon their transactivation by CLOCK/CYCLE, with peak levels in the early night. In Drosophila, clockwork orange (cwo) is known to inhibit the transcription of per and tim during the daytime to enhance the amplitude of the rhythm, but its function in other insects is largely unknown. In this study, we investigated the role of cwo in the clock mechanism of the cricket Gryllus bimaculatus. The results of quantitative RT-PCR showed that under a light/dark (LD) cycle, cwo is rhythmically expressed in the optic lobe (lamina-medulla complex) and peaks during the night. When cwo was knocked down via RNA interference (RNAi), some crickets lost their locomotor rhythm, while others maintained a rhythm but exhibited a longer free-running period under constant darkness (DD). In cwoRNAi crickets, all clock genes except for cryptochrome 2 (cry2) showed arrhythmic expression under DD; under LD, some of the clock genes showed higher mRNA levels, and tim showed rhythmic expression with a delayed phase. Based on these results, we propose that cwo plays an important role in the cricket circadian clock.

Fgf10-CRISPR mosaic mutants demonstrate the gene dose-related loss of the accessory lobe and decrease in the number of alveolar type 2 epithelial cells in mouse lung.

CRISPR/Cas9-mediated gene editing often generates founder generation (F0) mice that exhibit somatic mosaicism in the targeted gene(s). It has been known that Fibroblast growth factor 10 (Fgf10)-null mice exhibit limbless and lungless phenotypes, while intermediate limb phenotypes (variable defective limbs) are observed in the Fgf10-CRISPR F0 mice. However, how the lung phenotype in the Fgf10-mosaic mutants is related to the limb phenotype and genotype has not been investigated. In this study, we examined variable lung phenotypes in the Fgf10-targeted F0 mice to determine if the lung phenotype was correlated with percentage of functional Fgf10 genotypes. Firstly, according to a previous report, Fgf10-CRISPR F0 embryos on embryonic day 16.5 (E16.5) were classified into three types: type I, no limb; type II, limb defect; and type III, normal limbs. Cartilage and bone staining showed that limb truncations were observed in the girdle, (type I), stylopodial, or zeugopodial region (type II). Deep sequencing of the Fgf10-mutant genomes revealed that the mean proportion of codons that encode putative functional FGF10 was 8.3 ± 6.2% in type I, 25.3 ± 2.7% in type II, and 54.3 ± 9.5% in type III (mean ± standard error of the mean) mutants at E16.5. Histological studies showed that almost all lung lobes were absent in type I embryos. The accessory lung lobe was often absent in type II embryos with other lobes dysplastic. All lung lobes formed in type III embryos. The number of terminal tubules was significantly lower in type I and II embryos, but unchanged in type III embryos. To identify alveolar type 2 epithelial (AECII) cells, known to be reduced in the Fgf10-heterozygous mutant, immunostaining using anti-surfactant protein C (SPC) antibody was performed: In the E18.5 lungs, the number of AECII was correlated to the percentage of functional Fgf10 genotypes. These data suggest the Fgf10 gene dose-related loss of the accessory lobe and decrease in the number of alveolar type 2 epithelial cells in mouse lung. Since dysfunction of AECII cells has been implicated in the pathogenesis of parenchymal lung diseases, the Fgf10-CRISPR F0 mouse would present an ideal experimental system to explore it.

Functional analysis of Pdp1 and vrille in the circadian system of a cricket.

Circadian rhythms are generated by a circadian clock for which oscillations are based on the rhythmic expression of the so-called clock genes. The present study investigated the role of Gryllus bimaculatus vrille (Gb'vri) and Par domain protein 1 (Gb'Pdp1) in the circadian clock of the cricket Gryllus bimaculatus. Structural analysis of Gb'vri and Gb'Pdp1 cDNAs revealed that they are a member of the bZIP transcription factors. Under light/dark cycles (LD) both genes were rhythmically expressed in the clock tissue, the optic lobes, whereas the rhythm diminished under constant darkness (DD). Gb'vri and Gb'Pdp1 mRNA levels were significantly reduced by RNA interference (RNAi) of Gb'Clk and Gb'cyc, suggesting they are controlled by Gb'CLK/Gb'CYC. RNAi of Gb'vri and Gb'Pdp1 had little effect on locomotor rhythms, although their effects became visible when treated together with Gb'cycRNAi. The average free-running period of Gb'vriRNAi/Gb'cycRNAi crickets was significantly shorter than that of Gb'cycRNAi crickets. A similar period shortening was observed also when treated with Gb'Pdp1RNAi/Gb'cycRNAi. Some Gb'Pdp1RNAi/Gb'cycRNAi crickets showed rhythm splitting into two free-running components with different periods. Gb'vriRNAi and Gb'Pdp1RNAi treatments significantly altered the expression of Gb'Clk, Gb'cyc, and Gb'tim in LD. These results suggest that Gb'vri and Gb'Pdp1 play important roles in cricket circadian clocks.

Dkk3/REIC, an N-glycosylated Protein, Is a Physiological Endoplasmic Reticulum Stress Inducer in the Mouse Adrenal Gland.

Dickkopf 3 (Dkk3) is a secreted protein belonging to the Dkk family and encoded by the orthologous gene of REIC. Dkk3/REIC is expressed by mouse and human adrenal glands, but the understanding of its roles in this organ is still limited. To determine the functions of Dkk3 in the mouse adrenal gland, we first identified that the mouse Dkk3 protein is N-glycosylated in the adrenal gland as well as in the brain. We performed proteome analysis on adrenal glands from Dkk3-null mice, in which exons 5 and 6 of the Dkk3 gene are deleted. Twodimensional polyacrylamide gel electrophoresis of adrenal proteins from wild-type and Dkk3-null mice revealed 5 protein spots whose intensities were altered between the 2 genotypes. Mass spectrometry analysis of these spots identified binding immunoglobulin protein (BiP), an endoplasmic reticulum (ER) chaperone. To determine whether mouse Dkk3 is involved in the unfolded protein response (UPR), we carried out a reporter assay using ER-stress responsive elements. Forced expression of Dkk3 resulted in the induction of distinct levels of reporter expression, showing the UPR initiated by the ER membrane proteins of activating transcription factor 6 (ATF6) and inositol-requring enzyme 1 (IRE1). Thus, it is possible that Dkk3 is a physiological ER stressor in the mouse adrenal gland.

Dickkopf3 (Dkk3) is required for maintaining the integrity of secretory vesicles in the mouse adrenal medulla.

REIC (reduced expression in immortalized cells) has been identified as a gene whose expression was reduced in immortalized cultured cells. The REIC gene is identical to Dickkopf-3 (Dkk3), which encodes a secreted glycoprotein belonging to the Dkk family. Previously, we showed that Dkk3 protein is present in the mouse adrenal medulla. However, its role in this tissue has not been elucidated. To explore it, we performed electron microscopic (EM) studies and RNA-sequencing (RNA-seq) analysis on Dkk3-null adrenal glands. EM studies showed that the number of dense core secretory vesicles were significantly reduced and empty vesicles were increased in the medulla endocrine cells. Quantitative PCR (qPCR) analysis showed relative expression levels of chromogranin A (Chga) and neuropeptide Y (Npy) were slightly but significantly reduced in the Dkk3-null adrenal glands. From the result of RNA-seq analysis as a parallel study, we selected three of the downregulated genes, uncoupled protein-1 (Ucp1), growth arrest and DNA-damage-inducible 45 gamma (Gadd45g), and Junb with regard to the estimated expression levels. In situ hybridization confirmed that these genes were regionally expressed in the adrenal gland. However, expression levels of these three genes were not consistent as revealed by qPCR. Thus, Dkk3 maintains the integrity of secreting vesicles in mouse adrenal medulla by regulating the expression of Chga and Npy.

Localization of the ultraviolet-sensor Opn5m and its effect on myopia-related gene expression in the late-embryonic chick eye.

Recent studies show that exposure to ultraviolet (UV) light suppresses ocular elongation, which causes myopia development. However, the specific mechanisms of this process have not been elucidated. A UV-sensor, Opsin 5 (Opn5) mRNA was shown to be present in extraretinal tissues. To test the possibility that UV-signals mediated by Opn5 would have a direct effect on the outer connective tissues of the eye, we first examined the expression patterns of a mammalian type Opn5 (Opn5m) in the late-embryonic chicken eye. Quantitative PCR showed Opn5m mRNA expression in the cornea and sclera. The anti-Opn5m antibody stained a small subset of cells in the corneal stroma and fibrous sclera. We next assessed the effect of UV-A (375 nm) irradiation on the chicken fibroblast cell line DF-1 overexpressing chicken Opn5m. UV-A irradiation for 30 min significantly increased the expression of Early growth response 1 (Egr1), known as an immediate early responsive gene, and of Matrix metalloproteinase 2 (Mmp2) in the presence of retinal chromophore 11-cis-retinal. In contrast, expression of Transforming growth factor beta 2 and Tissue inhibitor of metalloproteinase 2 was not significantly altered. These results indicate that UV-A absorption by Opn5m can upregulate the expression levels of Egr1 and Mmp2 in non-neuronal, fibroblasts. Taken together with the presence of Opn5m in the cornea and sclera, it is suggested that UV-A signaling mediated by Opn5 in the extraretinal ocular tissues could influence directly the outer connective tissues of the chicken late-embryonic eye.

Congenital eye anomalies: More mosaic than thought?

The eye is a sensory organ that primarily captures light and provides the sense of sight, as well as delivering non-visual light information involving biological rhythms and neurophysiological activities to the brain. Since the early 1990s, rapid advances in molecular biology have enabled the identification of developmental genes, genes responsible for human congenital diseases, and relevant genes of mutant animals with various anomalies. In this review, we first look at the development of the eye, and we highlight seminal reports regarding archetypal gene defects underlying three developmental ocular disorders in humans: (1) holoprosencephaly (HPE), with cyclopia being exhibited in the most severe cases; (2) microphthalmia, anophthalmia, and coloboma (MAC) phenotypes; and (3) anterior segment dysgenesis (ASDG), known as Peters anomaly and its related disorders. The recently developed methods, such as next-generation sequencing and genome editing techniques, have aided the discovery of gene mutations in congenital eye diseases and gene functions in normal eye development. Finally, we discuss Pax6-genome edited mosaic eyes and propose that somatic mosaicism in developmental gene mutations should be considered a causal factor for variable phenotypes, sporadic cases, and de novo mutations in human developmental disorders.

A novel photic entrainment mechanism for the circadian clock in an insect: involvement of c-fos and cryptochromes.

BACKGROUND: Entrainment to the environmental light cycle is an essential property of the circadian clock. Although the compound eye is known to be the major photoreceptor necessary for entrainment in many insects, the molecular mechanisms of photic entrainment remain to be explored. RESULTS: We found  that cryptochromes (crys) and c-fos mediate photic entrainment of the circadian clock in a hemimetabolous insect, the cricket Gryllus bimaculatus. We examined the effects of RNA interference (RNAi)-mediated knockdown of the cry genes, Gb'cry1 and Gb'cry2, on photic entrainment, and light-induced resetting of the circadian locomotor rhythm. Gb'cry2 RNAi accelerated entrainment for delay shifts, while Gb'cry1/ Gb'cry2 double RNAi resulted in significant lengthening of transient cycles in both advance and delay shifts, and even in entrainment failure in some crickets. Double RNAi also strongly suppressed light induced resetting. The Gb'cry-mediated phase shift or resetting of the rhythm was preceded by light-induced Gb'c-fosB expression. We also found that Gb'c-fosB, Gb'cry2 and Gb'period (Gb'per) were likely co-expressed in some optic lobe neurons. CONCLUSION: Based on these results, we propose a novel model for photic entrainment of the insect circadian clock, which relies on the light information perceived by the compound eye.

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.

timeless2 plays an important role in reproduction and circadian rhythms in the cricket Gryllus bimaculatus.

The timeless2 (tim2) gene is an insect orthologue of the mammalian clock gene Timeless (mTim). Although its functional role has been extensively studied in mammals, little is known regarding its role in insects. In the present study, we obtained tim2 cDNA (Gb'tim2) from the cricket Gryllus bimaculatus and characterized its functional role in embryonic development, egg production, and circadian rhythms. Gb'tim2 gave rise to a 1432 amino acid protein, and showed approximately 65% homology to that of Drosophila melanogaster. When treated with parental Gb'tim2RNAi, less than 2% of the treated eggs hatched. On the other hand, control eggs treated with DsRed2RNAi demonstrated a hatching rate of 70%. In most of the Gb'tim2RNAi treated embryos, development was arrested in early stages. Egg production in ovaries of adult virgin females treated with Gb'tim2RNAi was significantly reduced. In addition, while Gb'tim2RNAi crickets exhibited clear locomotor rhythm synchronized with light cycles, their light-on peak was weaker than that of control crickets. Under constant darkness, the activity rhythm of Gb'tim2RNAi crickets was often split into two components running with different periods. Molecular analysis revealed that Gb'tim2RNAi treatment downregulated mRNA levels of Gb'per and Gb'Clk, and enhanced Gb'cyc expression rhythm; no distinct effect was found on Gb'tim expression levels. The change in Gb'per, Gb'Clk and Gb'cyc levels may underlie the altered behavioral rhythms in Gb'tim2RNAi crickets. Both Gb'ClkRNAi and Gb'cycRNAi downregulated Gb'tim2 expression, which suggested that transcription of Gb'tim2 is mediated by Gb'CLK and Gb'CYC through E-box. These results suggested that Gb'tim2 may be involved in both reproduction and circadian rhythm regulation in crickets.

cryptochrome genes form an oscillatory loop independent of the per/tim loop in the circadian clockwork of the cricket Gryllus bimaculatus.

BACKGROUND: Animals exhibit circadian rhythms with a period of approximately 24 h in various physiological functions, including locomotor activity. This rhythm is controlled by an endogenous oscillatory mechanism, or circadian clock, which consists of cyclically expressed clock genes and their product proteins. cryptochrome (cry) genes are thought to be involved in the clock mechanism, and their functions have been examined extensively in holometabolous insects, but in hemimetabolous insects their role is less well understood. RESULTS: In the present study, the role of cry genes was investigated using RNAi technology in a hemimetabolous insect, the cricket Gryllus bimaculatus. Using a molecular cloning approach, we obtained cDNAs for two cry genes: Drosophila-type cry1 (Gb'cry1) and mammalian-type cry2 (Gb'cry2). Gb'cry2 has six splicing variants, most of which showed rhythmic mRNA expression. Gb'cry1RNAi treatment had only a limited effect at the behavioral and molecular levels, while Gb'cry2RNAi had a significant effect on behavioral rhythms and molecular oscillatory machinery, alone or in combination with Gb'cry1RNAi. In Gb'cry1/Gb'cry2 double-RNAi crickets, most clock genes showed arrhythmic expression, except for timeless, which retained clear rhythmic expression. Molecular analysis revealed that some combination of Gb'cry1 and Gb'cry2 variants suppressed CLK/CYC transcriptional activity in cultured cells. CONCLUSION: Based on these results, we propose a new model of the cricket's circadian clock, including a molecular oscillatory loop for Gb'cry2, which can operate independent of the Gb'per/Gb'tim loop.

Relationship between somatic mosaicism of Pax6 mutation and variable developmental eye abnormalities-an analysis of CRISPR genome-edited mouse embryos.

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) system is a rapid gene-targeting technology that does not require embryonic stem cells. To demonstrate dosage effects of the Pax6 gene on eye formation, we generated Pax6-deficient mice with the CRISPR/Cas system. Eyes of founder embryos at embryonic day (E) 16.5 were examined and categorized according to macroscopic phenotype as class 1 (small eye with distinct pigmentation), class 2 (pigmentation without eye globes), or class 3 (no pigmentation and no eyes). Histologically, class 1 eyes were abnormally small in size with lens still attached to the cornea at E16.5. Class 2 eyes had no lens and distorted convoluted retinas. Class 3 eyes had only rudimentary optic vesicle-like tissues or histological anophthalmia. Genotyping of neck tissue cells from the founder embryos revealed somatic mosaicism and allelic complexity for Pax6. Relationships between eye phenotype and genotype were developed. The present results demonstrated that development of the lens from the surface ectoderm requires a higher gene dose of Pax6 than development of the retina from the optic vesicle. We further anticipate that mice with somatic mosaicism in a targeted gene generated by CRISPR/Cas-mediated genome editing will give some insights for understanding the complexity in human congenital diseases that occur in mosaic form.

Expression analysis of Dickkopf-related protein 3 (Dkk3) suggests its pleiotropic roles for a secretory glycoprotein in adult mouse.

Dickkopf-related protein 3 (Dkk3) is the third member of the Dkk gene family and identical to the gene, whose expression was reduced in immortalized cells. Therefore, its another name is reduced expression in immortalized cells. Since the intratumoral introduction of Dkk3 inhibits tumor growth in mouse models of cancers, Dkk3 is likely a tumor suppressor gene. However, the functions of Dkk3 in vivo remain unclear. As the first step to decipher the physiological roles of this gene, we examined the expression pattern of Dkk3 in various tissues from adult mice. In situ hybridization showed that Dkk3 mRNA was detected in the brain, retina, heart, gastrointestinal tract, adrenal glands, thymus, prostate glands, seminal vesicles, testes, and ovaries in a regionally specific manner. Furthermore, we raised anti-mouse Dkk3 antibody and performed immunohistochemistry. Cytoplasmic localization of Dkk3 protein was observed in the cells of the adrenal medulla, while Dkk3 immunoreactivity was observed in the lumen of the stomach and intestine, implying that the Dkk3 protein may be secreted into the lumen of the gastrointestinal tract. These results suggest that Dkk3 has pleiotropic roles for a secretory glycoprotein that acts primarily in the gastrointestinal tract, thymus, endocrine and reproductive organs of the mouse.

TGF-ß signaling in insects regulates metamorphosis via juvenile hormone biosynthesis.

Although butterflies undergo a dramatic morphological transformation from larva to adult via a pupal stage (holometamorphosis), crickets undergo a metamorphosis from nymph to adult without formation of a pupa (hemimetamorphosis). Despite these differences, both processes are regulated by common mechanisms that involve 20-hydroxyecdysone (20E) and juvenile hormone (JH). JH regulates many aspects of insect physiology, such as development, reproduction, diapause, and metamorphosis. Consequently, strict regulation of JH levels is crucial throughout an insect's life cycle. However, it remains unclear how JH synthesis is regulated. Here, we report that in the corpora allata of the cricket, Gryllus bimaculatus, Myoglianin (Gb'Myo), a homolog of Drosophila Myoglianin/vertebrate GDF8/11, is involved in the down-regulation of JH production by suppressing the expression of a gene encoding JH acid O-methyltransferase, Gb'jhamt In contrast, JH production is up-regulated by Decapentaplegic (Gb'Dpp) and Glass-bottom boat/60A (Gb'Gbb) signaling that occurs as part of the transcriptional activation of Gb'jhamt Gb'Myo defines the nature of each developmental transition by regulating JH titer and the interactions between JH and 20E. When Gb'myo expression is suppressed, the activation of Gb'jhamt expression and secretion of 20E induce molting, thereby leading to the next instar before the last nymphal instar. Conversely, high Gb'myo expression induces metamorphosis during the last nymphal instar through the cessation of JH synthesis. Gb'myo also regulates final insect size. Because Myo/GDF8/11 and Dpp/bone morphogenetic protein (BMP)2/4-Gbb/BMP5-8 are conserved in both invertebrates and vertebrates, the present findings provide common regulatory mechanisms for endocrine control of animal development.

Autopsy Case of Bilateral Optic Nerve Aplasia with Microphthalmia: Neural Retina Formation Is Required for the Coordinated Development of Ocular Tissues.

Human congenital anomalies provide information that contributes to the understanding of developmental mechanisms. Here we report bilateral optic nerve aplasia (ONA) with microphthalmia in the autopsy of the cadaver of a 70-year-old Japanese female. The gross anatomical inspection of the brain showed a cotton thread-like cord in the presumed location of the optic nerve tract or chiasm. Histologically, no neural retina, optic nerve bundle or retinal central vessels were formed in the eye globe, and the retinal pigment cells formed rosettes. The cornea, iris, and lens were also histologically abnormal. Immunohistochemically, no retinal cells expressed beta III tubulin, and Pax6- immunoreactive cells were present in the ciliary non-pigmented epithelial cells. This case of ONA could be attributed to the agenesis of retinal projection neurons as a sequel to the disruption of neural retina development. The neural retina formation would coordinate the proper development of ocular tissues.