Microvascular anatomy of olfactory and accessory olfactory (vomeronasal) organs in adult Xenopus laevis: scanning electron microscopy of vascular corrosion casts / Anatomía microvascular de órganos olfatorios y accesorios (vomeronasal) en adultos Xenopus laevis: microscopía electrónica de barrido de moldes de corrosión vascular

Microvascular anatomy and histomorphology of olfactory and vomeronasal organs in adult Xenopus laevis Daudin were studied by scanning electron microscopy of vascular corrosion casts and paraplast embedded stained serial tissue sections. Results show that the arterial supply is bilaterally by terminal arterioles of the medial branch of the nasal artery and by the palatal artery. Arterioles give rise to a capillary meshwork characteristic for respiratory surfaces in principal chambers and in dorsal and caudal areas of middle chambers. Anterior and inferior areas of the middle chambers own a distinctly different capillary network with conspicuous short capillary loops. Loops have a dilated tip and extend in acute angles towards the chamber lumen. The vomeronasal organ (VNO) locates beneath the olfactory organ. It has a medial to lateral extension and attaches with its caudal circumference to the medial nasal glands. Its capillary bed displays rectangular meshes which preferentially orientate along the long axis of the VNO. Locally, capillaries form short hairpin-like or strongly twisted loops with dilated tips which point towards the lumen of the VNO. These capillaries slow-down blood velocity and may lead to an increased exchange of oxygen, nutrients and water-borne odorants in the middle chambers and of pheromones in the VNO. In the latter vascular structures are present which might serve as a vascular pump.

Se estudiaron la anatomía microvascular e histomorfología de los órganos olfatorios y vomeronasales de Xenopus laevis Daudin adultos, mediante microscopía electrónica de barrido de moldes de corrosión vascular y secciones de tejido seriadas, teñidas e incluídas en paraplast. Los resultados muestran que el suministro arterial es bilateral por arteriolas terminales de la rama medial de la arteria nasal y por la arteria palatina. Las arteriolas dan lugar a un lecho capilar característico de las superficies respiratorias en las cámaras principales y en las áreas dorsal y caudal de las cámaras intermedias. Las áreas anterior e inferior de las cámaras centrales poseen una red capilar significativamente diferente con llamativos bucles capilares cortos. Los bucles tienen una punta dilatada y se extienden en ángulos agudos hacia la luz de la cámara. El órgano vomeronasal (VNO) se ubica debajo del órgano olfatorio. Se extiende de medial a lateral y se une con su circunferencia caudal a las glándulas nasales mediales. El lecho capilar muestra mallas rectangulares que se orientan preferentemente a lo largo del eje longitudinal del VNO. Localmente, los capilares forman bucles cortos en forma de horquilla o fuertemente retorcidos con puntas dilatadas que apuntan hacia la luz del VNO. Estos capilares ralentizan la velocidad de la sangre y pueden conducir a un mayor intercambio de oxígeno, nutrientes y odorizantes, a base de agua en las cámaras intermedias y de feromonas, en el VNO. En este último, están presentes estructuras vasculares que podrían servir como una bomba vascular.

Cellular composition and organization of the spinal cord central canal during metamorphosis of the frog Xenopus laevis.

Studying the cellular composition and morphological changes of cells lining the central canal during Xenopus laevis metamorphosis could contribute to understand postnatal development and spinal cord regeneration. Here we report the analysis of central canal cells at different stages during metamorphosis using immunofluorescence for protein markers expression, transmission and scanning electron microscopy and cell proliferation assays. The central canal was regionalized according to expression of glial markers, ultrastructure, and proliferation in dorsal, lateral, and ventral domains with differences between larvae and froglets. In regenerative larvae, all cell types were uniciliated, have a radial morphology, and elongated nuclei with lax chromatin, resembling radial glial cells. Important differences in cells of nonregenerative froglets were observed, although uniciliated cells were found, the most abundant cells had multicilia and revealed extensive changes in the maturation and differentiation state. The majority of dividing cells in larvae corresponded to uniciliated cells at dorsal and lateral domains in a cervical-lumbar gradient, correlating with undifferentiated features. Neurons contacting the lumen of the central canal were detected in both stages and revealed extensive changes in the maturation and differentiation state. However, in froglets a very low proportion of cells incorporate 5-ethynyl-2'-deoxyuridine (EdU), associated with the differentiated profile and with the increase of multiciliated cells. Our work showed progressive changes in the cell types lining the central canal of Xenopus laevis spinal cord which are correlated with the regenerative capacities.

Electron microscopy of the amphibian model systems Xenopus laevis and Ambystoma mexicanum.

In this chapter we provide a set of different protocols for the ultrastructural analysis of amphibian (Xenopus, axolotl) tissues, mostly of embryonic origin. For Xenopus these methods include: (1) embedding gastrulae and tailbud embryos into Spurr's resin for TEM, (2) post-embedding labeling of methacrylate (K4M) and cryosections through adult and embryonic epithelia for correlative LM and TEM, and (3) pre-embedding labeling of embryonic tissues with silver-enhanced nanogold. For the axolotl (Ambystoma mexicanum) we present the following methods: (1) SEM of migrating neural crest (NC) cells; (2) SEM and TEM of extracellular matrix (ECM) material; (3) Cryo-SEM of extracellular matrix (ECM) material after cryoimmobilization; and (4) TEM analysis of hyaluronan using high-pressure freezing and HABP labeling. These methods provide exemplary approaches for a variety of questions in the field of amphibian development and regeneration, and focus on cell biological issues that can only be answered with fine structural imaging methods, such as electron microscopy.

Early requirement of Hyaluronan for tail regeneration in Xenopus tadpoles.

Tail regeneration in Xenopus tadpoles is a favorable model system to understand the molecular and cellular basis of tissue regeneration. Although turnover of the extracellular matrix (ECM) is a key event during tissue injury and repair, no functional studies to evaluate its role in appendage regeneration have been performed. Studying the role of Hyaluronan (HA), an ECM component, is particularly attractive because it can activate intracellular signaling cascades after tissue injury. Here we studied the function of HA and components of the HA pathway in Xenopus tadpole tail regeneration. We found that transcripts for components of this pathway, including Hyaluronan synthase2 (HAS2), Hyaluronidase2 and its receptors CD44 and RHAMM, were transiently upregulated in the regenerative bud after tail amputation. Concomitantly, an increase in HA levels was observed. Functional experiments using 4-methylumbelliferone, a specific HAS inhibitor that blocked the increase in HA levels after tail amputation, and transgenesis demonstrated that the HA pathway is required during the early phases of tail regeneration. Proper levels of HA are required to sustain proliferation of mesenchymal cells in the regenerative bud. Pharmacological and genetic inhibition of GSK3beta was sufficient to rescue proliferation and tail regeneration when HA synthesis was blocked, suggesting that GSK3beta is downstream of the HA pathway. We have demonstrated that HA is an early component of the regenerative pathway and is required for cell proliferation during the early phases of Xenopus tail regeneration. In addition, a crosstalk between HA and GSK3beta signaling during tail regeneration was demonstrated.

Dissecting CNBP, a zinc-finger protein required for neural crest development, in its structural and functional domains.

Cellular nucleic-acid-binding protein (CNBP) plays an essential role in forebrain and craniofacial development by controlling cell proliferation and survival to mediate neural crest expansion. CNBP binds to single-stranded nucleic acids and displays nucleic acid chaperone activity in vitro. The CNBP family shows a conserved modular organization of seven Zn knuckles and an arginine-glycine-glycine (RGG) box between the first and second Zn knuckles. The participation of these structural motifs in CNBP biochemical activities has still not been addressed. Here, we describe the generation of CNBP mutants that dissect the protein into regions with structurally and functionally distinct properties. Mutagenesis approaches were followed to generate: (i) an amino acid replacement that disrupted the fifth Zn knuckle; (ii) N-terminal deletions that removed the first Zn knuckle and the RGG box, or the RGG box alone; and (iii) a C-terminal deletion that eliminated the three last Zn knuckles. Mutant proteins were overexpressed in Escherichia coli, purified, and used to analyze their biochemical features in vitro, or overexpressed in Xenopus laevis embryos to study their function in vivo during neural crest cell development. We found that the Zn knuckles are required, but not individually essential, for CNBP biochemical activities, whereas the RGG box is essential for RNA-protein binding and nucleic acid chaperone activity. Removal of the RGG box allowed CNBP to preserve a weak single-stranded-DNA-binding capability. A mutant mimicking the natural N-terminal proteolytic CNBP form behaved as the RGG-deleted mutant. By gain-of-function and loss-of-function experiments in Xenopus embryos, we confirmed the participation of CNBP in neural crest development, and we demonstrated that the CNBP mutants lacking the N-terminal region or the RGG box alone may act as dominant negatives in vivo. Based on these data, we speculate about the existence of a specific proteolytic mechanism for the regulation of CNBP biochemical activities during neural crest development.