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

<p>Abstract</p> <p>Background</p> <p>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 <it>in vitro</it>. Lower vertebrate animals are potential models for analysis of CSF flow during development because they are oviparous. Albino <it>Xenopus laevis </it>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 <it>in vivo </it>in the developing brain of living embryos, larvae and tadpoles of <it>Xenopus laevis </it>using a microinjection technique.</p> <p>Methods</p> <p>The development of <it>Xenopus </it>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.</p> <p>Results</p> <p>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.</p> <p>Conclusions</p> <p>This is the first visualisation of the orchestrated CSF flow pattern in developing vertebrates using a live animal imaging approach. CSF flow in <it>Xenopus </it>albino larvae showed a largely consistent pattern, with the exception of individual differences in left-right asymmetrical flow in the third ventricle.</p

The potentiation of Nodal signaling in the right lateral plate mesoderm inverts the left-right specification of the internal organs.

In Xenopus, multiple nodal-related genes are expressed during early embryogenesis. Among them, only Xenopus nodal related-1(Xnr-1) is expressed unilaterally in the left lateral plate mesoderm(LPM) at the late neurula-early tailbud stage. Early studies report that ectopic administration of Xnr-1 in the right hemisphere at the cleavage stage alters the left-right specification of the heart and visceral organs, or else makes a secondary axis. However, because Xnr-1 and other Xnrs function already at the blastula-gastrula stage, it is very difficult to evaluate the correct timing of the effects of excessively administered Xnr-1 from such a method. To elucidate the essential role of Xnr-1 within the left LPM, ectopic potentiation of Nodal signaling in the right lateral plate mesoderm was performed. Right-side injection of Nodal protein changed the laterality of Xnr-1 and Xenopuspitx2, but lefty, and fully (more than 90%) reversed the situs of the internal organs. Polyethyleneimine-based gene transfer of Xnr-1 mRNA in the right LPM also changed the laterality of pitx2 and fully (more than 90%) reversed the situs of the internal organs. Taken together, the potentiation of Xnr-1 signaling in the right LPM induces pitx2 in the right side and fully inverts the left-right axis of the heart and visceral organs, suggesting that the right LPM can transduce Nodal signaling, and only the absence of the Xnr-1 ligand silences the Nodal signaling in the right LPM. Normal left-right balance of Xnr-1 signaling is needed for the normal left-right specification of the internal organs