Ryuji Toyoizumi

Adrenergic neurotransmitters and calcium ionophore‐induced situs inversus viscerum in Xenopus laevis embryos

Xenopus laevis embryos at the blastula–early tail bud stage were exposed to norepinephrine or octopamine dissolved in culture saline until they reached the larval stage. The left–right asymmetry of the heart and gut was then examined. We found that these adrenergic neurotransmitters induced situs inversus in the heart and/or gut in up to 35% of tested neurula embryos. Norepinephrine‐induced situs inversus was blocked by the α‐1 adrenergic antagonist prazosin. Furthermore, A23187, a calcium ionophore, also increased the incidence of situs inversus up to 54% when late‐neurula embryos were exposed to the solution. A23187 treatment initiated before neural groove formation was less effective. The incidence of situs inversus induced by these reagents decreased towards the control level (2.2%, 25 untreated embryos out of 1127 embryos in total) in embryos past the stage of neural tube closure. In the present experiments we obtained 22 gut‐only situs inversus embryos having an inverted gut and a normal heart. In contrast, such embryos were not observed among the 1127 untreated embryos. An adrenergic signal mediated by an increase in intracellular free calcium may be involved in the asymmetrical visceral morphogenesis of Xenopus embryos.

Visualisation of cerebrospinal fluid flow patterns in albino Xenopus larvae in vivo.

BackgroundIt has long been known that cerebrospinal fluid (CSF), its composition and flow, play an important part in normal brain development, and ependymal cell ciliary beating as a possible driver of CSF flow has previously been studied in mammalian fetuses in vitro. Lower vertebrate animals are potential models for analysis of CSF flow during development because they are oviparous. Albino Xenopus laevis larvae are nearly transparent and have a straight, translucent brain that facilitates the observation of fluid flow within the ventricles. The aim of these experiments was to study CSF flow and circulation in vivo in the developing brain of living embryos, larvae and tadpoles of Xenopus laevis using a microinjection technique.MethodsThe development of Xenopus larval brain ventricles and the patterns of CSF flow were visualised after injection of quantum dot nanocrystals and polystyrene beads (3.1 or 5.8 μm in diameter) into the fourth cerebral ventricle at embryonic/larval stages 30-53.ResultsThe fluorescent nanocrystals showed the normal development of the cerebral ventricles from embryonic/larval stages 38 to 53. The polystyrene beads injected into stage 47-49 larvae revealed three CSF flow patterns, left-handed, right-handed and non-biased, in movement of the beads into the third ventricle from the cerebral aqueduct (aqueduct of Sylvius). In the lateral ventricles, anterior to the third ventricle, CSF flow moved anteriorly along the outer wall of the ventricle to the inner wall and then posteriorly, creating a semicircle. In the cerebral aqueduct, connecting the third and fourth cerebral ventricles, CSF flow moved rostrally in the dorsal region and caudally in the ventral region. Also in the fourth ventricle, clear dorso-ventral differences in fluid flow pattern were observed.ConclusionsThis is the first visualisation of the orchestrated CSF flow pattern in developing vertebrates using a live animal imaging approach. CSF flow in Xenopus albino larvae showed a largely consistent pattern, with the exception of individual differences in left-right asymmetrical flow in the third ventricle.

Optic chiasm in the species of order Clupeiformes, family Clupeidae: Optic chiasm of Spratelloides gracilis shows an opposite laterality to that of Etrumeus teres

In most teleost fishes, the optic nerves decussate completely as they project to the mesencephalic region. Examination of the decussation pattern of 25 species from 11 different orders in Pisces revealed that each species shows a specific chiasmic type. In 11 species out of the 25, laterality of the chiasmic pattern was not determined; in half of the individuals examined, the left optic nerve ran dorsally to the right optic nerve, while in the other half, the right optic nerve was dorsal. In eight other species the optic nerves from both eyes branched into several bundles at the chiasmic point, and intercalated to form a complicated decussation pattern. In the present study we report our findings that Spratelloides gracilis, of the order Clupeiformes, family Clupeidae, shows a particular laterality of decussation: the left optic nerve ran dorsally to the right (n=200/202). In contrast, Etrumeus teres, of the same order and family, had a strong preference of the opposite (complementary) chiasmic pattern to that of S. gracilis (n=59/59), revealing that these two species display opposite left–right optic chiasm patterning. As far as we investigated, other species of Clupeiformes have not shown left–right preference in the decussation pattern. We conclude that the opposite laterality of the optic chiasms of these two closely related species, S. gracilis and E. teres, enables investigation of species-specific laterality in fishes of symmetric shapes.

Morphometry of cellular protrusions of mesodermal cells and fibrous extracellular matrix in the primitive streak stage chick embryo

SummaryChick mesodermal cells, having become invaginated and beginning to locomote prior to the formation of the mesodermal cell layer at an early primitive streak stage, extend many filopodia and flatten themselves against the basal surface of the epiblast. Morphometry on scanning electron micrographs of chick mesodermal cells revealed two statistically significant tendencies. Each cell took an extended form and protruded filopodia, preferably along its major axis, suggesting that the force extending the cell body was generated by both ends rich in filopodia. The cells also tended to protrude filopodia most frequently in a direction away from Hensens node. The orientation of the fibrous extracellular matrix (fECM), running on the basal surface of the epiblast, was assessed quantitatively, and it was proved statistically that the orientation of the fECM was radial around the primitive streak: With an immunogold staining technique, fECM, to which the filopodia of the mesodermal cells attached frequently and closely, was confirmed to be rich in fibronectin (FN). These results lead us to conclude that the mesodermal cells in chick gastrula were guided to locomote towards the periphery of the area pellucida by FN-rich fECM laid on the basal surface of the epiblast, and that this movement was due to an in vivo locomotive mechanism using filopodia.

Invasion by Matrix Metalloproteinase-Expressing Cells Is Important for Primitive Streak Formation in Early Chick Blastoderm

Epiblast cells in the early chick embryo differentiate to form all three germ layers through ingression of cells at the primitive streak across the basement membrane that underlies the epiblast. We tested the idea that degradation of the extracellular matrix components by matrix metalloproteinase(s) (MMPs) is involved in this process. Epiblast cells and primitive streak cells were dissociated into single cells and seeded onto a reconstituted basement membrane gel in vitro. Following overnight culture, approximately half the cells made holes in the substratum by dissolving the gel matrix. This invasive phenomenon was reproduced in vitro even when the cells were cultured upside down using a hanging culture system. We detected gelatinase activity in the culture supernatants from both prestreak epiblast cells and primitive streak cells. Pro-MMP-2 was detected in the culture media of the prestreak/streak cells as a 72-kDa band by gelatin zymography. In RT-PCR experiments, mRNAs for MMP-2, membrane-type (MT)3-MMP and MMP-11(stromelysin-3) were expressed in the epiblast cells before and during primitive streak formation. Injection of GM 6001 or other MMP inhibitors into the subgerminal cavity of the embryo inhibited the formation of the primitive streak and/or the primitive groove in more than 82% of the injected embryos. On the other hand, injection of a negative control compound instead of GM 6001 did not cause substantial inhibition. These results suggest that MMPs are involved in the enzymatic degradation of the basement membrane underlying the epiblast and are thus important for the ingression of mesendodermal cells along the primitive streak.

Invasion and migration of a single chick pre-streak stage epiblast cell in vitro: Its implication to the primitive streak formation

To investigate the contribution of the epiblast cell behavior to the primitive streak formation, we examined the motility of a single epiblast cell from pre‐streak stage embryo in vitro. On the substratum that was evenly coated with laminin gel, epiblast cells attached well to the gel and one or a few very long and broad cellular processes protruded from their spherical cell bodies; however, they hardly locomoted on it. Unexpectedly, after overnight culture, half of the single cells dissolved the laminin gel beneath them to make well‐like holes, and invaded in the holes. On the substratum lined parallel with the fibrous laminin gels supplemented with fibronectin, they locomoted actively in accordance with the alignment. That is, they were subjected to contact guidance. In locomotion they looked like snails, extending one or a few long and broad processes in a forward direction from the spherical cell bodies. However, on the substratum lined with laminin or fibronectin only, they did not locomote actively. Individual chick pre‐streak epiblast cells had already been committed to invade, and their migratory nature existed in each cell, even though they were isolated from the epithelial sheet. The implication of these findings on the cellular basis of primitive streak formation will be discussed.

Left–Right Specification in the Embryonic and Larval Development of Amphibians

Biomolecules in living organisms, such as amino acids and double-stranded DNA, show left–right asymmetry. Even unicellular organisms, such as ciliates, have species-specific left- or right-handedness. Such molecular chiral asymmetry and cellular left–right asymmetry have likely provided a basis for the evolution of genetically determined left–right asymmetry of organ situs and the morphology of the heart, visceral organs, and central nervous system in eumetazoans. To study left–right asymmetry of the body plan, we believe that Xenopus laevis is a valuable model organism. The early Xenopus larva forms a transparent epidermis in the ventral abdominal region; thus organ development and morphology can be easily observed without dissection.