Revealing the cells fated to form the cell mass in embryos of temnopleurid sea urchins.

Larvae of temnopleurid sea urchins form a cell mass (CM) instead of an amniotic cavity on the left side at the early developmental stage for formation of the adult rudiment. However, the cell lineage and the mechanisms that form the CM are still unknown. We analyzed the potential to form a CM in partial embryos resulting from microsurgeries, using two temnopleurid species, Mespilia globulus (L.) and Temnopleurus toreumaticus (Leske). CM formation was completed 28-34 h after fertilization at 24°C, corresponding to the period from the late prism to the two-armed pluteus larval stages in both species. In the case of specimens dissected horizontally during the mesenchyme blastula to prism stages, the CM was formed in partial embryos containing enough of the an2 region, a part of the precursor cells that differentiate the ectoderm. The proportion of specimens with a CM was higher in T. toreumaticus than in M. globulus. Additionally, all larvae derived from half embryos obtained from dissection along the animal-vegetal axis at the mesenchyme blastula stage formed the CM. Transplantation of a stained animal or vegetal hemisphere at the 16-cell stage into a nonstained vegetal or animal embryo indicated that the CM derives from the animal half. Exogastrulae vegetalized by lithium chloride treatment did not form the CM. These results indicate that the CM formation is dependent not only on the an2 region but also on signals from the vegetal region after the mesenchyme blastula stage.

Regeneration of the cell mass in larvae of temnopleurid sea urchins.

Mechanisms of cell mass (CM) formation were analyzed by microsurgery in two temnopleurid sea urchins, Mespilia globulus and Temnopleurus toreumaticus. The CM in temnopleurids is formed at the early larval stage from the left ectodermal invagination, and with the hydrocoel derived from the mesoderm, forms an adult rudiment. After serial removal of the CM, it was strongly regenerated until its attachment to the hydrocoel, with the same timing as in control larvae. Embryos that had the tip of the archenteron or the coelomic pouches removed formed a CM in the normal manner. Removal of the CM plus the left somatocoel or the hydrocoel allowed CM regeneration with and without adult rudiment formation. A transplanted CM enlarged autonomously but did not contribute to adult rudiment formation, and larvae formed a new CM. Our observations suggest that the hydrocoel recognizes its distance from the CM to induce the growth of the CM and controls the normal timing of adult rudiment formation.

Effects of Nodal inhibition on development of temnopleurid sea urchins.

Adult rudiment formation in some temnopleurids begins with the formation of a cell mass that is pinched off the left ectoderm in early larval development. The cell mass forms the adult rudiment with the left coelomic pouch of the mesodermal region. However, details of the mechanisms to establish position of the cell mass are still unknown. We analyzed the inhibiting effect of Nodal, a factor for morphogenesis of the oral region and right side, for location of the cell mass, in four temnopleurids. Pulse inhibition, at least 5 min inhibition, during coelomic pouch formation allowed a cell mass to form on both sides, whereas treatments after that period did not. These results indicate that Nodal signaling controls the oral-aboral axis before gastrulation and then affects the position of the cell mass and adult rudiment up to coelomic pouch formation. They also indicate that the position of the adult rudiment under Nodal signaling pathways is conserved in temnopleurids, as adult rudiment formation is dependent on the cell mass.

Specification of Larval Axes of Partial Embryos in the Temnopleurid Temnopleurus toreumaticus and the Strongylocentroid Hemicentrotus pulcherrimus.

Many sea urchins, including the strongylocentroid Hemicentrotus pulcherrimus, produce an amniotic cavity on the left for adult rudiment formation at the late larval stage. In contrast, temnopleurids form a cell mass at the early larval stage instead of an amniotic cavity. Although the mechanisms establishing left-right polarity of the amniotic cavity involve cell-cell interactions and signaling pathways, corresponding pathways for the cell mass are unknown. We analyzed the effects of blastomere isolation on the specification of larval axes in the temnopleurid Temnopleurus toreumaticus and compared them to those in H. pulcherrimus. Blastomere isolation at the two- or four-cell stages in T. toreumaticus disturbed the location of the cell mass and adult rudiment in approximately 10-20% of specimens. In contrast, isolation at the two-cell stage in H. pulcherrimus caused the left-right polarity to become random. When blastomeres isolated at the two-cell stage were cultured as pairs, approximately 20% of pairs had atypical polarity in both species. Following isolation at the four-cell stage, 71.4% of quartets produced larvae with atypical polarity in T. toreumaticus. Thus, cell-cell interaction between two daughter blastomeres after the second cleavage may be involved in the mechanism determining left-right polarity. Dye injection into a blastomere and subsequent observations indicated that the location of the boundary of the first cleavage showed similar patterns in both species. These observations suggest that species-specific mechanisms establish the larval axes and blastomeres at the two- and four-cell stages redistribute their cytoplasm, forming gradients that establish left-right polarity.

Morphological diversity of blastula formation and gastrulation in temnopleurid sea urchins.

Embryos of temnopleurid sea urchins exhibit species-specific morphologies. While Temnopleurus toreumaticus has a wrinkled blastula and then invaginates continuously at gastrulation, others have a smooth blastula and their invagination is stepwise. We studied blastula and gastrula formation in four temnopleurids using light and scanning electron microscopy to clarify the mechanisms producing these differences. Unlike T. toreumaticus, blastomeres of mid-blastulae in T. reevesii, T. hardwickii and Mespilia globulus formed pseudopods. Before primary mesenchyme cells ingressed, embryos developed an area of orbicular cells in the vegetal plate. The cells surrounding the orbicular cells extended pseudopods toward the orbicular cell area in three Temnopleurus species. In T. toreumaticus, the extracellular matrix was well-developed and developed a hole-like structure that was not formed in others. Gastrulation of T. reevesii, T. hardwickii and M. globulus was stepwise, suggesting that differences of gastrulation are caused by all or some of the following factors: change of cell shape, rearrangement, pushing up and towing of cells. We conclude that (1) many aspects of early morphogenesis differ even among very closely related sea urchins with indirect development and (2) many of these differences may be caused by the cell shape and structure of blastomeres or by differences in extracellular matrix composition.

Changes of seasonal morph development induced by surgical operations in pupae of the large map butterfly Araschnia burejana Bermer (Lepidoptera: Nymphalidae).

The nymphalid butterfly Araschnia burejana and the papilionid butterfly Papilio xuthus exhibit seasonal diphenism comprising spring-morphs that develop from diapause pupae and summer-morphs that develop from non-diapause pupae. The development of seasonal morphs in A. burejana is regulated by the timing of secretion of ecdysteroids for adult development, whereas that in P. xuthus is regulated by the secretion of summer-morph-producing hormone, which is present in the brains and is under control of the photoperiod. We investigated whether a cerebral factor derived from brains plays a significant role in the regulation of seasonal morph development in A. burejana using surgical operations. Pairs of chilled diapause pupae that had been chilled for more than 3 months at 4°C were joined surgically to each other and then developed into spring-morph or spring-like-morph adults. Chilled diapause pupae that were joined with 1-day-old non-diapause pupae developed into summer-morph or summer-like-morph adults. When the brains of non-diapause pupae were removed surgically 6-8 hr after pupation with and without injection of 20-hydroxyecdysone, a large portion of them developed into spring-morph or spring-like-morph adults, respectively. Furthermore, 90% of non-diapause pupae developed into spring-morph or spring-like-morph adults when the neck was ligated within 5 min after pupation. These results indicated that a cerebral factor showing summer-morph-producing hormone activity, which is secreted from the brain in the early pupal stage, in addition to 20-hydroxyecdysone for adult development, play a significant role in the determination of summer-morph development in non-diapause pupae of A. burejana.

Development of an embryonic skeletogenic mesenchyme lineage in a sea cucumber reveals the trajectory of change for the evolution of novel structures in echinoderms.

BACKGROUND: The mechanisms by which the conserved genetic "toolkit" for development generates phenotypic disparity across metazoans is poorly understood. Echinoderm larvae provide a great resource for understanding how developmental novelty arises. The sea urchin pluteus larva is dramatically different from basal echinoderm larval types, which include the auricularia-type larva of its sister taxon, the sea cucumbers, and the sea star bipinnaria larva. In particular, the pluteus has a mesodermally-derived larval skeleton that is not present in sea star larvae or any outgroup taxa. To understand the evolutionary origin of this structure, we examined the molecular development of mesoderm in the sea cucumber, Parastichopus parvimensis. RESULTS: By comparing gene expression in sea urchins, sea cucumbers and sea stars, we partially reconstructed the mesodermal regulatory state of the echinoderm ancestor. Surprisingly, we also identified expression of the transcription factor alx1 in a cryptic skeletogenic mesenchyme lineage in P. parvimensis. Orthologs of alx1 are expressed exclusively within the sea urchin skeletogenic mesenchyme, but are not expressed in the mesenchyme of the sea star, which suggests that alx1+ mesenchyme is a synapomorphy of at least sea urchins and sea cucumbers. Perturbation of Alx1 demonstrates that this protein is necessary for the formation of the sea cucumber spicule. Overexpression of the sea star alx1 ortholog in sea urchins is sufficient to induce additional skeleton, indicating that the Alx1 protein has not evolved a new function during the evolution of the larval skeleton. CONCLUSIONS: The proposed echinoderm ancestral mesoderm state is highly conserved between the morphologically similar, but evolutionarily distant, auricularia and bipinnaria larvae. However, the auricularia, but not bipinnaria, also develops a simple skelotogenic cell lineage. Our data indicate that the first step in acquiring these novel cell fates was to re-specify the ancestral mesoderm into molecularly distinct territories. These new territories likely consisted of only a few cells with few regulatory differences from the ancestral state, thereby leaving the remaining mesoderm to retain its original function. The new territories were then free to take on a new fate. Partitioning of existing gene networks was a necessary pre-requisite to establish novelty in this system.

Diapause pupal color diphenism induced by temperature and humidity conditions in Byasa alcinous (Lepidoptera: Papilionidae).

We investigated whether diapause pupae of Byasa alcinous exhibit pupal color diphenism (or polyphenism) similar to the diapause pupal color polyphenism shown by Papilio xuthus. All diapause pupae of B. alcinous observed in the field during winter showed pupal coloration of a dark-brown type. When larvae were reared and allowed to reach pupation under short-day conditions at 18°C under a 60±5% relative humidity, diapause pupae exhibited pupal color types of brown (33%), light-brown (25%), yellowish-brown (21%), diapause light-yellow (14%) and diapause yellow (7%). When mature larvae reared at 18°C were transferred and allowed to reach pupation at 10°C and 25°C under a 60±5% relative humidity after a gut purge, the developmental ratio of brown and light-brown, yellowish-brown, and diapause light-yellow and diapause yellow types was 91.2, 8.8 and 0.0% at 10°C, and 12.2, 48.8 and 39.0% at 25°C, respectively. On the other hand, when mature larvae reared at 18°C were transferred and allowed to reach pupation at 10°C, 18°C and 25°C under an over 90% relative humidity after a gut purge, the developmental ratio of brown and light-brown, yellowish-brown, and diapause light-yellow and diapause yellow types was 79.8, 16.9 and 3.3% at 10°C, 14.5, 26.9 and 58.6% at 18°C, and 8.3, 21.2 and 70.5% at 25°C, respectively. These results indicate that diapause pupae of brown types are induced by lower temperature and humidity conditions, whereas yellow types are induced by higher temperature and humidity conditions. The findings of this study show that diapause pupae of B. alcinous exhibit pupal color diphenism comprising brown and diapause yellow types, and suggest that temperature and humidity experienced after a gut purge are the main factors that affect the diapause pupal coloration of B. alcinous as environmental cues.

Novel morphological traits in the early developmental stages of Temnopleurus toreumaticus.

We have re-observed in detail the development of the sea urchin species Temnopleurus toreumaticus, which is considered to be a typical indirect-developing species with a feeding larval stage. In this re-observation, we discovered two new morphological traits in the early embryonic stages of T. toreumaticus. The first trait is that, immediately after fertilization, the egg enters a stage in which wrinkles form on its surface as a result of actin polymerization. The second new trait is that the blastulae form wrinkles; in sea urchins, this has previously been known only in direct-developing species that have a nonfeeding larval stage and form wrinkles during the blastula stage, before hatching. These phenomena indicate that after fertilization, the egg of T. toreumaticus undergoes a surface transformation that is unprecedented in echinoderms, and that an indirect-developing sea urchin can form a wrinkled blastula.

Hormonal control of pupal coloration in the painted lady butterfly Vanessa cardui.

Pupae of the painted lady butterfly Vanessa cardui exhibit pupal color polyphenism consisting of white, dark and intermediate types. We investigated environmental factors affecting pupal coloration and the physiological mechanisms underlying the control of pupal color polyphenism in this species. Over 80% of larvae reared at 16 degrees C developed into pupae of dark types, whereas over 82% of larvae at 32 degrees C developed into pupae of white types irrespective of long/short-day photoperiod conditions. When mature larvae reared at 32 degrees C were ligatured between thoracic and abdominal parts at three different pharate pupal stages, all of the head-thoracic parts developed into white pupae regardless of pupal stage, but all abdominal parts ligatured at the early pharate pupal stage only developed into dark pupae. These results indicate that temperature during larval stages is an important element affecting pupal coloration as an environmental cue in V. cardui, and that a factor(s) inducing white pupae is released from head-thoracic parts under conditions of high temperature. Additionally, when ligatured abdomens destined to develop into dark pupae were treated with crude extracts prepared from the central nervous system, all of the ligatured abdomens developed into white pupae at a level dependent on dose and pupal stage. These results suggest that the factor inducing white pupae is a key molecule controlling pupal color polyphenism in V. cardui.

Gene regulatory networks and developmental plasticity in the early sea urchin embryo: alternative deployment of the skeletogenic gene regulatory network.

Cell fates in the sea urchin embryo are remarkably labile, despite the fact that maternal polarity and zygotic programs of differential gene expression pattern the embryo from the earliest stages. Recent work has focused on transcriptional gene regulatory networks (GRNs) deployed in specific embryonic territories during early development. The micromere-primary mesenchyme cell (PMC) GRN drives the development of the embryonic skeleton. Although normally deployed only by presumptive PMCs, every lineage of the early embryo has the potential to activate this pathway. Here, we focus on one striking example of regulative activation of the skeletogenic GRN; the transfating of non-skeletogenic mesoderm (NSM) cells to a PMC fate during gastrulation. We show that transfating is accompanied by the de novo expression of terminal, biomineralization-related genes in the PMC GRN, as well as genes encoding two upstream transcription factors, Lvalx1 and Lvtbr. We report that Lvalx1, a key component of the skeletogenic GRN in the PMC lineage, plays an essential role in the regulative pathway both in NSM cells and in animal blastomeres. MAPK signaling is required for the expression of Lvalx1 and downstream skeletogenic genes in NSM cells, mirroring its role in the PMC lineage. We also demonstrate that Lvalx1 regulates the signal from PMCs that normally suppresses NSM transfating. Significantly, misexpression of Lvalx1 in macromeres (the progenitors of NSM cells) is sufficient to activate the skeletogenic GRN. We suggest that NSM cells normally deploy a basal mesodermal pathway and require only an Lvalx1-mediated sub-program to express a PMC fate. Finally, we provide evidence that, in contrast to the normal pathway, activation of the skeletogenic GRN in NSM cells is independent of Lvpmar1. Our studies reveal that, although most features of the micromere-PMC GRN are recapitulated in transfating NSM cells, different inputs activate this GRN during normal and regulative development.

Micromere-derived signal regulates larval left-right polarity during sea urchin development.

The micromeres (Mics) lineage functions as a morphogenetic signaling center in early embryos of sea urchins. The Mics lineage releases signals that regulate the specification of cell fates along the animal-vegetal and oral-aboral axes. We tested whether the Mics lineage might also be responsible for differentiation of the left-right (LR) axis by observing of the placement of the adult rudiment, which normally forms only on the left side of the larvae, after removal of the Mics lineage. When all of the Mics lineage were removed from embryos of the regular sea urchin Hemicentrotus pulcherrimus between the 16- and 64-cell stages, the LR placement of the rudiment became randomized. However, the immediate retransplantation of the Mics rescued the normal LR placement of the rudiment, indicating that the Mics lineage releases a signal that specifies LR polarity. Additionally, we investigated whether the specification of LR polarity of whole embryos in the indirect-developing sea urchin H. pulcherrimus is affected by LiCl exposure, which disturbs the establishment of LR asymmetry in a direct-developing sea urchin. Larvae derived from normal animal caps combined with LiCl-exposed Mics descendants were defective in normal LR placement of the rudiment, suggesting that LiCl interferes with the Mics-derived signal. In contrast, embryos of two sand dollar species (Scaphechinus mirabilis and Astriclypeus manni) were resistant to alteration of LR placement of the rudiment by either removal of the Mics lineage or LiCl exposure. These results indicate that the Mics lineage is involved in specification of LR polarity in the regular sea urchin H. pulcherrimus, and suggest that LiCl impairs the normal LR patterning by affecting Mics-derived signaling.

Properties of Orange-Pupa-Inducing Factor (OPIF) in the swallowtail butterfly, Papilio xuthus L.

Diapause pupae of the swallowtail butterfly Papilio xuthus L. exhibit diapause-green, orange and brownish-orange color polymorphism. Development of orange pupae involves a neuroendocrine factor inducing orange pupa (Orange-Pupa-Inducing Factor, OPIF), which is secreted from the head-thoracic region during late pharate pupal stages, in particular from the ganglia of short-day animals located posteriorly from the second thoracic ganglion2 (TG2). This report describes certain properties of OPIF using bioassays involving ligated abdomens of short-day pharate pupae. Localization of OPIF in the central nervous system of short-day larvae indicated that it was present predominantly in TG2, thoracic ganglion3 (TG3) and abdominal ganglion1 (AG1) complexes. OPIF activity in TG(2,3)-AG1 complexes was over two times higher than in the more posteriorely located ganglia. The developmental profile of OPIF in last instar short-day larvae revealed that OPIF activity in larval ganglia posterior to TG2 became gradually higher as larval growth proceeded, suggesting that OPIF might be accumulated in TG(2,3)-AG(1-7) complexes as larvae prepare for pupal molting. Furthermore, ligated abdomens of short-day larvae developed into pupae of an orange type when a 2% NaCl extract containing OPIF prepared from TG(2,3)-AG(1-7) complexes of long-day larvae was injected into ligated abdomens of short-day pharate pupae, indicating that OPIF is also present in long-day larvae. Additionally, a biochemical investigation using gel filtration chromatography showed that the molecular weight of OPIF was about 10 kDa.

Environmental factors affecting black/white coloration of the silken girdle in the swallowtail butterfly, Atrophaneura alcinous (Lepidoptera: Papilionidae).

The silken girdles of pupae of the swallowtail butterfly Atrophaneura alcinous show black and white color diphenism. Field observations revealed that all pupae observed on non-food plants and the leaves and stems of the larval food plant Aristolochia debilis were classified as a silken girdle of a black type, while a large portion of pupae pupating on the twigs and trunks of cherry trees in close proximity to A. debilis were classified as a silken girdle of a black type. Additionally, all pupae observed on the surfaces of artificial objects in areas where there are no surrounding plants or trees were classified as a silken girdle of a white type. We demonstrated the effect of day length and the texture, light, plant odor and humidity of pupation sites on the coloration of the silken girdle in A. alcinous. Regardless of long-day or short-day day length conditions, light conditions of constant light or dark, or the presence of a plant odor of A. debilis as environmental cues, all larvae placed at over 80% relative humidity (R.H.) developed into pupae with a silken girdle of a black type. However, all larvae developed into pupae with a silken girdle of a white type when R.H. was below 75%. Furthermore, when pupae with a silken girdle of a white type were transferred to conditions of 90% R.H. within 24 hr of pupation, the white color of the silken girdle changed into a black type within 24 hr of the transfer. The present data suggest that the induction of a black coloration of the silken girdle in A. alcinous requires a R.H. of approximately 80% or more as an environmental factor.

LiCl inhibits the establishment of left-right asymmetry in larvae of the direct-developing echinoid Peronella japonica.

The effect of LiCl on the establishment of left-right (LR) asymmetry in larvae of the direct-developing echinoid Peronella japonica was investigated with special attention to the location of the amniotic opening and ciliary band pattern. The larvae of echinoids are LR symmetric, but shortly before metamorphosis the larval LR symmetry is lost as a result of the formation of an amniotic cavity (vestibule), part of the adult rudiment, on the left side of the body. P. japonica has been considered to be the only exception among the echinoids, because the amniotic cavity forms at the midline of the larval body. In the present study we discovered the following two different LR asymmetric traits in larvae of P. japonica: the opening of the amniotic cavity initially forms at the midline of the larval body but shifts to the left dorsal side, and a looped ciliary band that initially forms with LR symmetry becomes LR asymmetric as a result of the formation of a bulge on left dorsal side. The establishment of LR asymmetry in both the location of the amniotic opening and the change in the shape of the ciliary band was influenced by exposing embryos to LiCl. Quantitative analysis of the shift in amniotic opening showed that exposure of embryos to LiCl causes repression of leftward shifting of the amniotic opening in earlier stage larvae, and leftward or rightward shifting in later stage larvae. These findings suggest that LiCl is an effective means of impairing the establishment of LR asymmetry in sea urchin embryos.

Hormonal control of the orange coloration of diapause pupae in the swallowtail butterfly, Papilio xuthus L. (Lepidoptera: Papilionidae).

Diapause pupae of Papilio xuthus show color polymorphism, represented by diapause-green, orange, and brownish-orange types that are each associated with specific pupation sites. We investigated the role of the site of pupation on the induction of the development of orange types (or brownish-orange types), and the endocrine mechanism underlying the control of color polymorphism in short-day pupae. All short-day larvae of the wandering stage developed into orange or brownish-orange type pupae when they were placed in rough-surfaced containers after gut-purge. Utilizing a pharate pupal ligation between the thorax and abdomen, the endocrine mechanism underlying the control of color polymorphism was shown to involve a head-thorax factor (Orange-Pupa-Inducing Factor: OPIF) that induced orange types in short-day pupae. OPIF was bioassayed using the ligated abdomens of short-day pharate pupae. OPIF was extractable with 2% NaCl solution from 5th-instar larval ganglia complexes following the mesothoracic complex (TG(2,3)-AG(1-7)), but it could not be extracted with either acetone or 80% ethanol solution. OPIF may not exist in the brains of day-0 pupae or in brain-subesophageal ganglion and prothoracic ganglion complexes of 5th-instar larvae. The short-day pharate pupae responded to OPIF in a dose-dependent manner.