Characterization of AmphiF-spondin reveals the modular evolution of chordate F-spondin genes

The F-spondin genes are a family of extracellular matrix molecules united by two conserved domains, FS1 and FS2, at the amino terminus plus a variable number of thrombospondin repeats at the carboxy terminus. Currently, characterized members include a single gene in Drosophila and multiple genes in vertebrates. The vertebrate genes are expressed in the midline of the developing embryo, primarily in the floor plate of the neural tube. To investigate the evolution of chordate F-spondin genes, I have used the basal position in chordate phylogeny of the acraniate amphioxus. A single F-spondin-related gene, named AmphiF-spondin, was isolated from amphioxus. Based on molecular phylogenetics, AmphiF-spondin is closely related to a particular subgroup of vertebrate F-spondin genes that encode six thrombospondin repeats. However, unlike these genes, expression of AmphiF-spondin is not confined to the midline but is found through most of the central nervous system. Additionally, AmphiF-spondin has lost three thrombospondin repeats and gained two fibronectin type III repeats, one of which has strong identity to a fibronectin type III repeat from Deleted in Colorectal Cancer (DCC). Taken together, these results suggest a complex evolutionary history for chordate F-spondin genes that includes (1) domain loss, (2) domain gain by tandem duplication and divergence of existing domains, and (3) gain of heterologous domains by exon shuffling.      

Mechanisms of midgut remodeling: juvenile hormone analog methoprene blocks midgut metamorphosis by modulating ecdysone action

In holometabolous insects such as mosquito, Aedes aegypti, midgut undergoes remodeling during metamorphosis. Insect metamorphosis is regulated by several hormones including juvenile hormone (JH) and 20-hydroxyecdysone (20E). The cellular and molecular events that occur during midgut remodeling were investigated by studying nuclear stained whole mounts and cross-sections of midguts and by monitoring the mRNA levels of genes involved in 20E action in methoprene-treated and untreated Ae. aegypti. We used JH analog, methoprene, to mimic JH action. In Ae. aegypti larvae, the programmed cell death (PCD) of larval midgut cells and the proliferation and differentiation of imaginal cells were initiated at about 36h after ecdysis to the 4th instar larval stage (AEFL) and were completed by 12h after ecdysis to the pupal stage (AEPS). In methoprene-treated larvae, the proliferation and differentiation of imaginal cells was initiated at 36h AEFL, but the PCD was initiated only after ecdysis to the pupal stage. However, the terminal events that occur for completion of PCD during pupal stage were blocked. As a result, the pupae developed from methoprene-treated larvae contained two midgut epithelial layers until they died during the pupal stage. Quantitative PCR analyses showed that methoprene affected midgut remodeling by modulating the expression of ecdysone receptor B, ultraspiracle A, broad complex, E93, ftz-f1, dronc and drice, the genes that are shown to play key roles in 20E action and PCD. Thus, JH analog, methoprene acts on Ae. aegypti by interfering with the expression of genes involved in 20E action resulting in a block in midgut remodeling and death during pupal stage.     

Isolation and characterization of genes that are expressed during Ciona intestinalis metamorphosis

In ascidians, the events of metamorphosis transform the non-feeding, mobile tadpole larva into a filter-feeding, fixed juvenile, and the process involves rearrangements of cells, two organs and physiological changes. Differential screening was used to isolate two genes that are not expressed in swimming larvae but are expressed immediately after the initiation of metamorphosis in Ciona intestinalis. One of the genes, Ci-meta1, encodes a polypeptide with a putative secretion signal sequence, 6 epidermal growth factor (EGF)-like repeats and 13 calcium-binding EGF-like repeats. The gene begins to be expressed immediately after the beginning of metamorphosis in the adhesive organ and is likely to be associated with the signal response for metamorphosis. Another gene named Ci-meta2 encodes a protein with a putative secretion signal and three thrombospondin type-1 repeats. Ci-meta2 gene expression begins at the larval stage and is upregulated in the metamorphosing juveniles. Ci-meta2 expression is found in three regions; the adhesive organ which is also associated with settlement, the neck region between the trunk and the tail of the larva which is associated with tail resorption, and dorsal regions of the trunk which correspond to the location of the siphon primordium. This gene may be involved in the dynamic arrangement of cells during ascidian metamorphosis.     

Dopamine is a key factor for the induction of egg diapause of the silkworm, Bombyx mori.

The silkworm, Bombyx mori, enters diapause in the early embryonic stage. Embryonic diapause is induced by incubating eggs of the maternal generation at high temperature (diapause type), whereas incubation at low temperature results in non-diapausing progeny (non-diapause type). Measurement of catecholamine concentrations in haemolymph and brain-subesophageal ganglia (Br-SGs) showed that only dopamine concentrations in both tissues are consistently higher in diapause-type than non-diapause-type larvae and pupae. In particular, the difference in dopamine concentrations in both tissues increases around pupal ecdysis. During the early pupal stage, Dopa decarboxylase activities and mRNA concentrations in Br-SGs were also much higher in diapause-type than non-diapause-type insects. Elevation of dopamine levels induced by feeding Dopa to penultimate-instar and last-instar larvae, and by injecting Dopa or dopamine into pupae 2 days after pupation made the non-diapause-destined insects lay diapause-destined eggs at 59% and approximately 70% frequencies, respectively. Furthermore, injection of Dopa or dopamine elevated mRNA levels of the diapause hormone in the Br-SGs of non-diapause-type pupae 1 day after injection. Incubation of Br-SGs isolated from non-diapause-type day-2 pupae with Dopa or dopamine also stimulated the expression of diapause hormone mRNA. These data indicate that environmental stimuli during embryonic development increase dopamine levels in both hemolymph and Br-SGs from the larval stage to early pupal stage, which results in laying of diapause-destined eggs by female adults through enhanced expression of the diapause hormone gene.     

Synthesis of the same two proteins prior to larval diapause and pupation in the spruce budworm,Choristoneura fumiferana

Spruce budworm larvae produce large quantities of two proteins (Choristoneura fumiferana diapause associated proteins 1 and 2, CfDAP1 and CfDAP2) that are diapause related. These proteins appeared soon after hatching and increased in abundance, reaching maximum levels by four days into the 1st instar, and they remained at high levels until three days after the termination of diapause. These two proteins were purified to homogeneity and their NH2-terminal sequences were obtained. Oligonucleotide primers designed on the basis of these NH2-terminal sequences were used in RT-PCR to isolate the cDNA fragments coding for these proteins. These PCR fragments were then used as probes to isolate the cDNAs that contained the complete coding region. The 2.5kb mRNAs coding for these proteins started to appear 24hr after hatching and large quantities of these mRNAs were detected in 1st instar and 2nd instar larvae until the 2nd instar larvae entered diapause. Low levels of these mRNAs were detected in the 2nd instar larvae that were preparing to enter diapause, in those that were in diapause as well as in those that terminated diapause. Low levels of CfDAP1 mRNA were also detected on days 1 and 2 after ecdysis to the 3rd instar. However, no CfDAP1 and CfDAP2 mRNAs could be detected during the 4th and 5th instar larval stages. The mRNAs reappeared 24hr after the 5th instar larvae molted into the 6th instar and increased to reach maximum levels by 60hr after ecdysis. The mRNA levels remained high until 156hr after ecdysis into the 6th instar (36-48hr before pupal ecdysis), after which they disappeared once again. Immunocytochemical analyses showed that CfDAP1 protein was present in 2nd and 6th instar larval fat body but not in 5th instar larval fat body. Thus, the same two genes were expressed for the first time before C. fumiferana larvae entered diapause and for a 2nd time before pupation.     

Changes in the activity of dopa quinone imine conversion factor during the development of Bombyx mori

The activity of DOPA quinone imine conversion factor (QICF) in tissues at different developmental stages of the silkworm, Bombyx mori, was determined. QICF activity was detected in all developmental stages from egg to pupa although the activities, other than in fifth instar larvae, were quite low. Activity in whole larvae peaked one day before the onset of larval-pupal development and declined to a low level shortly before ecdysis. In whole pupae, maximal QICF activity was obtained 1 h after pupation. The activity in larval cuticles was elevated on the last day of the fourth instar and again between days 4 and 8 of the fifth instar, decreasing to very low levels before pupal ecdysis. QICF was detectable in pupal cuticles with most of the pupal activity found in homogenates of mid and hind guts. A major part of the total larval QICF activity was found in hemolymph. Activity in hemolymph varied in a different manner from that in cuticles, with markedly raised levels immediately before pupal ecdysis when the cuticular activity had declined. It is postulated that QICF in cuticles plays some role in wound healing and/or sclerotizatio,, while QICF in hemolymph participates in melanization in the humoral immune system.     

Identification and characterization of thrombospondin-4, a new member of the thrombospondin gene family

A new member of the thrombospondin gene family, designated thrombospondin-4, has been identified in the Xenopus laevis genome. The predicted amino acid sequence indicates that the protein is similar to the other members of this gene family in the structure of the type 3 repeats and the COOH-terminal domain. Thrombospondin-4 contains four type 2 repeats and lacks the type 1 repeats that are found in thrombospondin-1 and 2. The amino-terminal domain of thrombospondin-4 has no significant homology with the other members of the thrombospondin gene family or with other proteins in the database. RNAse protection analysis establishes that the initial expression of Xenopus thrombospondin-4 is observed during neurulation. Levels of mRNA expression increase twofold during tailbud stages but decrease by the feeding tadpole stage. The size of the thrombospondin-4 message is 3.3 Kb and 3.4 Kb in the frog and human, respectively. Northern blot analysis of human tissues reveals high levels of thrombospondin-4 expression in heart and skeletal muscle, low levels in brain, lung and pancreas and undetectable levels in the placenta, liver and kidney. These data establish the existence of a new member of the thrombospondin gene family that may participate in the genesis and function of cardiac and skeletal muscle.     

Hormonal regulation of phase polymorphism and storage-protein fluctuation in the common cutworm, Spodoptera litura

Three storage proteins are synthesised by Spodoptera litura last-instar larvae as detected by an antiserum against pupal fat body proteins. The putative pupal storage proteins 1 and 2, appear in the haemolymph of the last-instar larvae 36 h after ecdysis under crowded rearing conditions: they appear 1 day later in isolated conditions. The appearance of these proteins in the haemolymph is prevented by juvenile hormone treatment and enhanced by allatectomy. Injection of 20-hydroxyecdysone into ligatured larvae does not induce appearance of these 2 proteins. Accumulation of protein 3 that reacts with Bombyx mori arylphorin antiserum is not blocked by juvenile hormone and is similar in both phases. It also accumulates to a small extent in the haemolymph during the moult to the final-larval instar and then disappears at ecdysis. One-hundred ng/ml ecdysteroid caused the sequestration of these proteins by the fat body, but a higher concentration of ecdysteroid (200 ng/ml) produced pupal cuticle in the isolated abdomens, suggesting that different ecdysteroid concentrations are necessary for these two events.     

ADAMTS9, a novel member of the ADAM-TS/ metallospondin gene family

ADAM-TS/metallospondin genes encode a new family of proteins with structural homology to the ADAM metalloprotease-disintegrin family. However, unlike other ADAMs, these proteins contain thrombospondin type 1 (TSP1) repeats at the carboxy-terminal end and are secreted proteins instead of being membrane bound. Members of the ADAM-TS family have been implicated in the cleavage of proteoglycans, the control of organ shape during development, and the inhibition of angiogenesis. We have cloned a new member of the ADAM-TS/metallospondin family designated here as ADAMTS9. This protein has a metalloprotease domain, a disintegrin-like domain, one internal TSP1 motif, and three carboxy-terminal TSP1-like submotifs. In contrast to other ADAM-TS family members, ADAMTS9 is expressed in all fetal tissues examined as well as some adult tissues. Using FISH and radiation hybrid analysis, we have localized ADAMTS9 to chromosome 3p14.2-p14.3, an area known to be lost in hereditary renal tumors.     

Regulation of genes for ubiquitination and SUMO-specific protease involved in larval development of the silkworm,Bombyx mori

Protein modifications with highly conserved small proteins, such as ubiquitin (Ub) and small ubiquitin-like modifier (SUMO), regulate various cellular processes; however, the contribution of these protein modifications to larval development in insects has not yet been elucidated. We investigated the regulation of genes for these protein modifications in the posterior silk gland (PSG) during larval development of the silkworm Bombyx mori. We found that several genes encoding enzymes (E1, E2, and E3) for ubiquitination and SUMO-specific protease were upregulated by 20-hydroxyecdysone (20E), and, consistently, increases in ubiquitinated proteins were observed during the fourth molting stage. An injection of 20E into larvae at the fourth feeding stage induced higher expression levels of these E1, E2, and E3 genes and ecdysis approximately one day earlier than in mock-treated larvae. The expression of the fibroin heavy-chain gene (fibH) was simultaneously suppressed approximately one day earlier in 20E-injected larvae. The treatment of cultured PSG with 20E also induced these genes, which could be categorized into at least two types: those induced by a high dose of 20E, or by a pulse of 20E. In contrast to the 20E treatment, the administration of PR-619, an inhibitor of Ub- and SUMO-specific proteases in larvae, delayed ecdysis and prolonged the expression of fibH. These results suggest that the regulation of genes for ubiquitination and SUMO-specific protease is involved in the larval development of B. mori.     

Juvenile hormone acid and ecdysteroid together induce competence for metamorphosis of the Verson's gland in Manduca sexta

In the last larval instar of Lepidoptera, ecdysteroid in the absence of juvenile hormone (JH) is believed to cause the shift from larval to pupal development. In Manduca sexta, tissues such as the Verson's gland and crochet epidermis become pupally committed before the earliest pulse of ecdysteroid that occurs on day 2. What causes the change in commitment in these tissues? First it was necessary to determine at what stage these tissues become competent to express the pupal program. Last instar larvae of different ages were induced to molt prematurely by feeding the ecdysteroid analog RH5992 and Verson's gland proteins were analyzed by SDS-polyacrylamide gel electrophoresis. Glands became competent to make pupal proteins between 24 and 32 h after the last larval ecdysis. Next, hormonal regulation of competence was examined in ligated abdomens of 12h last instar larvae. Treatment with JH II acid or methoprene acid plus a low dose (1/50th of the molt inducing dose) of RH5992 induced competence, whereas RH5992 alone, methoprene acid alone or methoprene plus RH5992 did not. Verson's glands maintained in vitro produced pupal proteins in response to methoprene acid together with RH5992 but not with RH5992 alone. Likewise, crochet epidermis lost the ability to make crochets (metamorphic change) only in isolated abdomens treated with JH II acid or methoprene acid and low doses of RH5992. In conclusion, JH acid in the presence of basal levels of ecdysteroid induces tissue competence for metamorphosis. Metamorphic competence is followed by commitment, induced by a small pulse of ecdysteroid in the absence of JH, and finally by expression caused by a high titer of ecdysteroid. It is proposed that JH acid is an essential metamorphic hormone.     

Identification of molting fluid carboxypeptidase A (MF-CPA) in Bombyx mori

Using microarray analyses, we identified carboxypeptidase A (MF-CPA), which was induced during pupal ecdysis in the wing discs of Bombyx mori. Here, we report the functional characterization of MF-CPA. MF-CPA has amino acid sequence similarities with the proteins in the carboxypeptidase A/B subfamily, from human to nematode. The MF-CPA gene is expressed during the molting periods in the epithelial tissues. MF-CPA is detected in the molting fluid, which fills the space between the old and new cuticle during molting. By Western blot analysis, we show that MF-CPA is secreted as a zymogen and processed in the molting fluid. Recombinant MF-CPA expressed in the insect cells has carboxypeptidase A activity. We propose that MF-CPA degrades the proteins from the old cuticle during the molting periods and contributes to recycling of the amino acids.     

Effects of juvenile hormone on some phase characteristics in the common cutworm, Spodoptera litura

Phase characters of the common cutworm, Spodoptera litura, were influenced by different rearing densities from the 4th-larval instar. Primarily the final feeding period of isolated larvae was 1 day longer than that of crowded larvae causing an increase in pupal weight. Applications of juvenile hormone I, II, or methoprene to crowded larvae caused an increased feeding period similar to that of isolated larvae when the juvenile hormones were applied within 1 day after the last-larval ecdysis. Allatectomy of isolated Spodoptera during the moult to the final-larval instar decreased the duration of the final feeding period to that of intact crowded larvae. These results suggested that one of the characters of phase variation, pupal weight, is influenced by the differences in the regulation and activity of the corpora allata during the last-larval instar. Other characteristics of phase variation such as behaviour (feigned death) and colour were not affected by alteration in juvenile hormone levels after the last larva ecdysis.     

ADAM-TS5, ADAM-TS6, and ADAM-TS7, novel members of a new family of zinc metalloproteases. General features and genomic distribution of the ADAM-TS family.

We report the primary structure of three novel, putative zinc metalloproteases designated ADAM-TS5, ADAM-TS6, and ADAM-TS7. All have a similar domain organization, comprising a preproregion, a reprolysin-type catalytic domain, a disintegrin-like domain, a thrombospondin type-1 (TS) module, a cysteine-rich domain, a spacer domain without cysteine residues, and a COOH-terminal TS module. These genes are differentially regulated during mouse embryogenesis and in adult tissues, with Adamts5 highly expressed in the peri-implantation period in embryo and trophoblast. These proteins are similar to four other cognate gene products, defining a distinct family of human reprolysin-like metalloproteases, the ADAM-TS family. The other members of the family are ADAM-TS1, an inflammation-induced gene, the procollagen I/II amino-propeptide processing enzyme (PCINP, ADAM-TS2), and proteins predicted by the KIAA0366 and KIAA0688 genes (ADAM-TS3 and ADAM-TS4). Individual ADAM-TS members differ in the number of COOH-terminal TS modules, and some have unique COOH-terminal domains. The ADAM-TS genes are dispersed in human and mouse genomes.     

ADAMTSL-3/punctin-2, a novel glycoprotein in extracellular matrix related to the ADAMTS family of metalloproteases.

The complete primary structure of ADAMTSL-3/punctin-2, a novel member of the family designated ADAMTSL (a disintegrin-like and metalloprotease domain with thrombospondin type I motifs-like), was determined by cDNA cloning from a human placenta library. The predicted open reading frame encodes a protein of 1690 amino acids that has considerable similarity to ADAMTSL-1/punctin-1. These multi-domain proteins lack both a protease domain and a disintegrin-like domain but are remarkably similar in their domain organization to the ADAMTS proteases, hence the name ADAMTS-like. Punctin-2 contains thrombospondin type 1 repeats (TSRs), a cysteine-rich domain and a cysteine-free spacer domain in the precise order in which they occur in the ADAMTS proteases. However, the number and organization of the TSRs in punctin-2 is unique with respect to the ADAMTS proteases. Punctin-2 contains 13 TSRs arranged in two arrays separated by a region containing three immunoglobulin-like repeats. Northern blot analysis of RNA from human adult tissues demonstrated that ADAMTSL3 is widely expressed, with highest expression in liver, kidney, heart and skeletal muscle, whereas it is expressed at low levels in mouse embryos. We characterized two punctin-2 polyclonal antisera. Using these and a monoclonal antibody to a C-terminal myc tag, we show that in transfected COS-7 cells, punctin-2 is expressed as a 210-kDa glycoprotein that is located in the extracellular matrix. The domain structure of punctin-2 and its matrix localization suggest that it might play a role in cell-matrix interactions or in assembly of specific extracellular matrices.