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Abstract Background It 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. Methods The 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. Results The 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. Conclusions This 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.

In vertebrates, gut coiling proceeds left-right asymmetrically during expansion of the gastrointestinal tract with highly organized muscular structures facilitating peristalsis. In this report, we explored the mechanisms of larval gut coiling morphogenesis relevant to its nascent smooth muscle cells using highly transparent Xenopus early larvae. First, to visualize the dynamics of intestinal smooth muscle cells, whole-mount specimens were immunostained with anti-smooth muscle-specific actin (SM-actin) antibody. We found that the nascent gut of Xenopus early larvae gradually expands the SM-actin-positive region in a stage-dependent manner. Transverse orientation of smooth muscle cells was first established, and next, the cellular longitudinal orientation along the gut axis was followed to make a meshwork of the contractile cells. Finally, anisotropic torsion by the smooth muscle cells was generated in the center of gut coiling, suggesting that twisting force might be involved in the late phase of coiling morphogenesis of the gut. Administration of S-(-)-Blebbistatin to attenuate the actomyosin contraction in vivo resulted in cancellation of coiling of the gut. Development of decapitation embryos, trunk 'torso' explants, and gut-only explants revealed that initial coiling of the gut proceeds without interactions with the other parts of the body including the central nervous system. We newly developed an in vitro model to assess the gut coiling morphogenesis, indicating that coiling pattern of the nascent Xenopus gut is partially gut-autonomous. Using this gut explant culture technique, inhibition of actomyosin contraction was performed by administrating either actin polymerization inhibitor, myosin light chain kinase inhibitor, or calmodulin antagonist. All of these reagents decreased the extent of gut coiling morphogenesis in vitro. Taken together, these results suggest that the contraction force generated by actomyosin-rich intestinal smooth muscle cells during larval stages is essential for the normal coiling morphogenesis of this muscular tubular organ., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

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., (Copyright 2010 S. Karger AG, Basel.)

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.

Signaling by members of TGF-beta superfamily requires the activity of a family of site-specific endopeptidases, known as Subtilisin-like proprotein convertases (SPCs), which cleave these ligands into mature, active forms. To explore the role of SPCs in lateral plate mesoderm (LPM) differentiation in Xenopus, two SPC inhibitors, decanoyl-Arg-Val-Lys-Arg-chloromethylketone (Dec-RVKR-CMK) and hexa-arginine, were injected into the left and right LPM of Xenopus neurulae. Left-side injection caused heart-specific left-right reversal, and this phenotype was rescued by co-injection of mature Nodal protein. In contrast, right-side injection caused left-right reversal of both the heart and gut. Tailbud embryos were less sensitive to SPC inhibitors than neurula embryos. Injection of inhibitors into either side of neurula embryos completely abolished expression of the left-LPM-specific genes, Xnr-1, antivin, and pitx2. SPC1 enzyme (Furin) was injected into the left or right LPM of mid-neurula embryos to determine the effect of enhancing SPC activity. Left-side injection of SPC1 did not cause a significant left-right reversal of the internal organs. However, right-side injection of SPC1 strongly induced the expression of Xnr-1 and pitx2 in the right LPM, and caused 100% left-right reversal of both the heart and gut. These results suggest that moderate level of SPC activity in the right LPM of the neurulae is necessary for proper left-right specification. Taken together, SPC enzymatic activity must be present in both LPMs for expression of the left-handed genes and left-right axis determination of the heart and gut in Xenopus embryos.

In Xenopus, multiple nodal-related genes are expressed in the organizer region. Among them, only Xenopus nodal related-1 (Xnr-1) is expressed unilaterally in the left lateral plate mesoderm (LPM) at late neurula-early tailbud stage. To elucidate the essential role of Xnr-1 for left-right specification, loss of function experiments using antisense morpholino oligonucleotides (MOs) targeting three different regions of Xnr-1 were performed. Left-side injection of Xnr-1 MO suppresses the left-side specific genes such as Xnr-1, Xenopus antivin (lefty) and Xenopus pitx2 and randomizes cardiac and visceral left-right orientation. In contrast, paraxial bilateral expression of Xnr-1 along the posterior notochord is not affected by the Xnr-1 MO. In embryos injected with the Xnr-1 MO, morphology of dorsal axial structures is normal and dorsal expression of sonic hedgehog and TGF-beta5 is not changed. Right-side injection of Nodal protein, or polyethyleneimine-based gene transfer of Xnr-1 mRNA in the right LPM induces Xnr-1 and pitx2 in the same side and fully (more than 90%) reverses situs of the internal organs. Left-side injection of Nodal protein restores normal left-right orientation in the embryos that were injected with Xnr-1 MO into the left blastomere and would cause randomization of the left-right axis without the Nodal injection. Taken together, unilateral expression of Xnr-1 in the left LPM directs the orientation of the left-right axis by driving the left-specific gene cascade. Knockdown of Xnr-1 function by the MOs suggests that Xnr-1 is indispensable only for the left-right orientation and dispensable for other embryonic axes probably owing to the redundancy in the function of multiple Xnrs.

A variety of TGF-beta-related ligands regulate the left-right asymmetry of vertebrates but the involvement of TGF-betas in left-right specification has not been reported. We assessed whether TGF-beta signaling is involved in the left-right specification of Xenopus post-gastrula embryos by microinjecting Xenopus TGF-beta5 protein into the left or right flank of neurula-tailbud embryos. Injection on the right side of neurulae caused left-right reversal of the internal organs in 93% of the embryos, while injection on the left side caused less than 5% left-right reversal. Expression of Xenopus nodal related-1 (Xnr-1 ), Xenopus antivin and Xenopus Pitx2, which are normally expressed on the left, was unaltered by the left-side injection. In contrast, right-side injection into neurulae induced the expression of these genes predominantly on the right side. Right-side injection into tailbud embryos caused bilateral expression of these handed genes. Time course analysis of asymmetric gene expression revealed that Xnr-1 could be induced by TGF-beta5 at late neurula stage, while antivin and Pitx2 could be induced by TGF-beta5 at the latertail bud stage. Injection of the antisense morpholino oligonucleotide against Xenopus TGF-beta5 into the left dorsal blastomere inhibited the normal left-handed expression of Xnr-1 and Pitx2, and caused the organ reversal in the injected embryos. These results suggest that normal left-right balance of endogenous TGF-beta5 signaling in the neurula embryo may be needed to determine the laterality of the asymmetric genes and to generate the correct left-right axis.

During avian gastrulation, certain cells present in the epiblast layer ingress through the basement membrane sealing the basal surface of themselves. Previously we reported that chick prestreak epiblast cells show two different behavioral phenotypes upon reconstituted basement membrane and laminin gel in vitro. Half of the dissociated epiblast cells invade the gel substratum after one-day of culture, whereas the others attach to the gel but do not invade. It is expected that such heterogeneity in the behavior of the epiblast cells reflects some mechanism that sorts the cells into those that will ingress into the blastocoelic cavity and those that will remain in the epiblast layer. To test this hypothesis, we dissociated chick prestreak epiblast cells into single cells, cultured them on the laminin gel, and then stained them with anti-HNK-1 antibody. This antibody binds to an epitope present on half of the prestreak epiblast cells which are thought to differentiate into presumptive mesoendodermal cells. We found that 80% of the invasive epiblast cells were HNK-1-positive whereas 77% of the non-invasive cells were HNK-1 negative. In the case of invasive cells, the edges of the proteolytic holes made by the invasive cells were often stained. These results suggest that the cells expressing the HNK-1 carbohydrate chain are preferentially invasive, and this induces selective ingression of the carrier cells for mesoendodermal differentiation in vivo.

In recent years, genes that show left-right (L-R) asymmetric expression patterns have been identified one after another in vertebrate gastrula-neurula embryos. However, we still have little information about when the irreversible L-R specification is established in vertebrate embryos. In this report, we show that almost 100% of the embryos develop to be L-R-inverted larvae after microinjection of activin molecules into the right lateral hypodermic space of Xenopus neurula embryos. After right-side injection of 10-250 pg activin protein, both early neurulae just after gastrulation movement (stage 13-14) and late neurulae just before neural tube closure (stage 17-18) showed almost 100% reversal of the heart and gut L-R axes. At higher doses of activin, more than 90% of the L-R-inverted embryos showed L-R reversal of both heart and gut. The survival ratio of the right-injected 4-day embryos was 90% on average. In the left-injected embryos, the occurrence of L-R inversion was less than 2% as observed in normal untreated siblings (1.7%). When the same amount of activin (1-50 pg) was microinjected into both sides of neurula embryos, the incidence of L-R inversion was reduced to 58%. The injection of activin along the dorsal midline in the trunk region also randomized the visceral L-R axis. Injection of activin into the right side changed normal left-handed expression of Xnr-1 to right-handed or bilateral expression. In contrast, left-handed expression of Pitx2 was switched to the right side by right activin injection. This is the first report of a method that achieves complete inversion of the visceral L-R axis by treatment of embryos at the neurula stage. Activin not only acts on the neurulae to cancel the original L-R specification up to the late neurula stage, but also rebuilds a new L-R axis whose left side coincides with the injection side. It is suggested that the left and right halves of neurulae have equal potential for L-R differentiation., (Copyright 2000 Academic Press.)

In early chick blastodermal morphogenesis, the hypoblast layer is organized beneath the epiblast and induces an axial structure. However, the origin of hypoblast cells and the mechanism of hypoblast layer formation are poorly understood. We hypothesized that the hypoblast layer is formed by an invasive process across the basement membrane of the juxtaposing epiblast, and tested the idea in vitro. Primary and secondary hypoblast cells from embryos at various pre-streak stages were dissociated into single cells and cultured on reconstituted basement membrane gel, laminin gel or fibronectin gel in the culture medium with or without serum for 24-48 h. As a result, we found that after 24h of serum-supplemented culture, up to 35% of the hypoblast cells dissolved the gel and made holes on it. Similarly, up to 36% of the hypoblast cells showed invasiveness after 48 h in the serum-free culture. Furthermore, it was observed that Koller's sickle cells, which are regarded to be the progenitors of secondary hypoblast cells, penetrated those gels on which they were seeded. The posterior epiblast cells covering Koller's sickle were also invasive. These results suggest that the presumptive primary hypoblast cells that are known to mingle with epiblast cells invade through the basement membrane to form the hypoblast layer. Furthermore, the present results imply that invasion through the basement membrane may be involved in the formation of Koller's sickle, the anlage of secondary hypoblast.

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 alpha-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.

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.

Chick 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 Hensen's 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.

In order to investigate the mechanism of the formation of the mesodermal layer during chick gastrulation, we observed the behavior of fragments of mesodermal cells explanted and cultured on substrata coated with parallel lines of fibronectin (FN). We also examined the distribution of F-actin, alpha-actinin, and vinculin in explanted fragments by immunocytochemical methods noting particularly their distribution with respect to FN lines. Explants of mesodermal cells flattened on FN-coated substrata and then became elliptical with the major axis of the ellipse oriented along the FN lines and migrated along them. The peripheral cells of explants extended filopodia and lamellipodia which attached preferentially to FN lines and then contracted, pulling other mesodermal cells in explants along passively. Vinculin and alpha-actinin in peripheral anchoring filopodia and lamellipodia co-localized with the terminations of F-actin bundles and with FN lines, suggesting that the peripheral cells were the moving force for explant translocation. We propose based on these results that in vivo, peripheral cells of invaginated cell mass are guided by the known FN-rich fibrous extracellular matrix on the basal surface of epiblast to move outwards; the rest linked to the peripheral cells are pulled away from the primitive streak to spread in tandem to form the mesodermal layer.