Maria del Mar Romero-Alemán

Tenascin‐R and axon growth‐promoting molecules are up‐regulated in the regenerating visual pathway of the lizard (Gallotia galloti)

It is currently unclear whether retinal ganglion cell (RGC) axon regeneration depends on down‐regulation of axon growth‐inhibitory proteins, and to what extent outgrowth‐promoting substrates contribute to RGC axon regeneration in reptiles. We performed an immunohistochemical study of the regulation of the axon growth‐inhibiting extracellular matrix molecules tenascin‐R and chondroitin sulphate proteoglycan (CSPG), the axon outgrowth‐promoting extracellular matrix proteins fibronectin and laminin, and the axonal tenascin‐R receptor protein F3/contactin during RGC axon regeneration in the lizard, Gallotia galloti. Tenascin‐R and CSPG were expressed in an extracellular matrix‐, oligodendrocyte/myelin‐ and neuron‐associated pattern and up‐regulated in the regenerating optic pathway. The expression pattern of tenascin‐R was not indicative of a role in channeling or restriction of re‐growing RGC axons. Up‐regulation of fibronectin, laminin, and F3/contactin occurred in spatiotemporal patterns corresponding to tenascin‐R expression. Moreover, we analyzed the influence of substrates containing tenascin‐R, fibronectin, and laminin on outgrowth of regenerating lizard RGC axons. In vitro regeneration of RGC axons was not inhibited by tenascin‐R, and further improved on mixed substrates containing tenascin‐R together with fibronectin or laminin. These results indicate that RGC axon regeneration in Gallotia galloti does not require down‐regulation of tenascin‐R or CSPG. Presence of tenascin‐R is insufficient to prevent RGC axon growth, and concomitant up‐regulation of axon growth‐promoting molecules like fibronectin and laminin may override the effects of neurite growth inhibitors on RGC axon regeneration. Up‐regulation of contactin in RGCs suggests that tenascin‐R may have an instructive function during axon regeneration in the lizard optic pathway.

Expression of neuronal markers, synaptic proteins, and glutamine synthetase in the control and regenerating lizard visual system

Spontaneous regrowth of retinal ganglion cell (RGC) axons occurs after optic nerve (ON) transection in the lizard Gallotia galloti. To gain more insight into this event we performed an immunohistochemical study on selected neuron and glial markers, which proved useful for analyzing the axonal regrowth process in different regeneration models. In the control lizards, RGCs were beta‐III tubulin‐ (Tuj1) and HuCD‐positive. The vesicular glutamate transporter‐1 (VGLUT1) preferentially stained RGCs and glial somata rather than synaptic layers. In contrast, SV2 and vesicular GABA/glycine transporter (VGAT) labeling was restricted to both plexiform layers. Strikingly, the strong expression of glutamine synthetase (GS) in both Müller glia processes and macroglial somata revealed a high glutamate metabolism along the visual system. Upregulation of Tuj1 and HuCD in the surviving RGCs was observed at all the timepoints studied (1, 3, 6, 9, and 12 months postlesion). The significant rise of Tuj1 in the optic nerve head and optic tract (OTr) by 1 and 6 months postlesion, respectively, suggests an increase of the beta‐III tubulin transport and incorporation into newly formed axons. Persistent Tuj1+ and SV2+ puncta and swellings were abnormally observed in putative degenerating/dystrophic fibers. Unexpectedly, neuron‐like cells of obscure significance were identified in the control and regenerating ON‐OTr. We conclude that: 1) the persistent upregulation of Tuj1 and HuCD favors the long‐lasting axonal regrowth process; 2) the latter succeeded despite the ectopia and dystrophy of some regrowing fibers; and 3) maintenance of the glutamate‐glutamine cycle contributes to the homeostasis and plasticity of the system. J. Comp. Neurol. 518:4067–4087, 2010.

Neuronal differentiation patterns in the optic tectum of the lizard Gallotia galloti.

This study examines in detail the sequences of morphological differentiation and deduces mode of migration into specific layers of all types of neurons present in the optic tectum of the lizard Gallotia galloti. It complements previous similar work on tectal histogenesis in the chick. It was found that the neuronal population diversity in the lizard tectum can be reduced by developmental analysis to three neuroblast classes, called Types I, II and III. These classes correspond closely to those present in the developing avian tectum. Neurons belonging to each developmental class were characterized by their initial polarity, mode of translocation into the mantle layer and pattern of sprouting of primary axonal and dendritic processes. Each class produced along time a subset of the cell types distinguished in the mature tectum. Some aspects of sauropsidian tectal histogenesis are also common of other vertebrates, suggesting that fundamental mechanisms of tectal neuronal differentiation are conserved in tetrapods. Analysis of evolutive differences of tectal structure points to changes affecting the layering and perhaps the population size of specific cell types. Whereas tectal cell-type homology can be easily fundamented on embryological evidence and seems to be consistent with hodological and, to some extent, functional homology, the periventricular, central and superficial strata of the tectum are heterogeneous in cellular composition in different species and therefore represent analogous, rather than homologous entities.

Neuronal and glial differentiation during lizard (Gallotia galloti) visual system ontogeny

We studied the histogenesis of the lizard visual system (E30 to adulthood) by using a selection of immunohistochemical markers that had proved relevant for other vertebrates. By E30, the Pax6+ pseudostratified retinal epithelium shows few newborn retinal ganglion cells (RGCs) in the centrodorsal region expressing neuron‐ and synaptic‐specific markers such as betaIII‐tubulin (Tuj1), synaptic vesicle protein‐2 (SV2), and vesicular glutamate transporter‐1 (VGLUT1). Concurrently, pioneer RGC axons run among the Pax2+ astroglia in the optic nerve and reach the superficial optic tectum. Between E30 and E35, the optic chiasm and optic tract remain acellular, but the latter contains radial processes with subpial endfeet expressing vimentin (Vim). From E35, neuron‐ and synaptic‐specific stainings spread in the retina and optic tectum, whereas retinal Pax6, and Tuj1/SV2 in RGC axons decrease. Müller glia and abundant optic nerve glia express a variety of glia‐specific markers until adulthood. Subpopulations of optic nerve glia are also VGLUT1+ and cluster differentiation‐44 (CD44)‐positive but cytokeratin‐negative, unlike the case in other regeneration‐competent species. Specifically, coexpression of CD44/Vim and glutamine synthetase (GS)/VGLUT1 reflects glial specialization, insofar as most CD44+ glia are GS−. In the adult optic tract and tectum, radial glia and free astroglia coexist. The latter show different immunocharacterization (Pax2−/CD44−/Vim−) compared with that in the optic nerve. We conclude that upregulation of Tuj1 and SV2 is required for axonal outgrowth and search for appropriate targets, whereas Pax2+ optic nerve astroglia and Vim+ radial glia may aid in early axonal guidance. Spontaneous axonal regrowth seems to succeed despite the heterogeneous mammalian‐like glial environment in the lizard optic nerve. J. Comp. Neurol. 520:2163–2184, 2012.

Expression of BDNF and NT-3 During the Ontogeny and Regeneration of the Lacertidian (Gallotia galloti) Visual System

Retinal ganglion cell (RGC) axons regrow spontaneously after optic nerve (ON) transection in G. galloti. Because brain‐derived neurotrophic factor (BDNF) is considered the major neurotrophin participating in vertebrate visual system development and promotes RGC survival, we investigated its distribution using dual‐labeling immunohistochemistry for neuronal and glial markers. We examined the developing and regenerating lizard visual system at 1, 3, 6, 9, and 12 months postlesion to comparatively evaluate BDNF expression patterns. BDNF was detected from midembryonic stages (E35) in both retinal plexiform layers, and in radial glial processes in the tectum. Moreover, RGC axon staining was detected at late prenatal stages (E39), showing a transient punctate staining which progressed in a temporo‐spatial pattern that was similar to myelination. Strong expression in RGC axons was maintained in adults. However, transient downregulation of BDNF staining occurred on the experimental side one month after ON transection followed by a gradual recovery with extensive punctate/swelling distribution and persistent upregulation at 12 months. Conversely, quantitative PCR analysis for 1 and 12 months regenerate lizards showed downregulation of the ratio of BDNF mRNA expression at 12 months and nonsignificant changes of NT‐3 transcripts. In summary, we demonstrate that BDNF and NT‐3 are abundantly expressed during lizard visual system ontogeny and regeneration suggesting their participation inthe development, maintenance and plasticity of the system.

Development of astroglia heterogeneously expressing Pax2, vimentin and GFAP during the ontogeny of the optic pathway of the lizard (Gallotia galloti): an immunohistochemical and ultrastructural study.

The successful regrowth of retinal ganglion cell (RGC) axons after optic nerve (ON) axotomy in Gallotia galloti indicates a permissive role of the glial environment. We have characterised the astroglial lineage of the lizard optic pathway throughout its ontogeny (embryonic stage 30 [E30] to adults) by using electron microscopy and immunohistochemistry to detect the proliferation marker PCNA (proliferating cell nuclear antigen), the transcription factor Pax2 and the gliofilament proteins vimentin (Vim) and GFAP (glial fibrillary acidic protein). PCNA+ cells were abundant until E39, with GFAP+/PCNA+ astrocytes being observed between E37 and hatching. Proliferation diminished markedly afterwards, being undetectable in the adult optic pathway. Müller glia of the central retina expressed Pax2 from E37 and their endfeet accumulated Vim from E33 and GFAP from E37 onwards. Astrocytes were absent in the avascular lizard retina, whereas abundant Pax2+ astrocytes were observed in the ON from E30. A major subpopulation of these astrocytes coexpressed Vim from E35 and also GFAP from E37 onwards; thus the majority of mature astrocytes coexpressed Pax2/Vim/GFAP. The astrocytes were ultrastructurally identified by their gliofilaments, microtubules, dense bodies, desmosomes and glycogen granules, which preferentially accumulated in cell processes. Astrocytes in the adult ON coexpressed both gliofilaments and presented desmosomes indicating a reinforcement of the ON structure; this is physiologically necessary for local adaptation to mechanical forces linked to eye movement. We suggest that astrocytes forming this structural scaffold facilitate the regrowth of RGCs after ON transection.

Nogo‐A does not inhibit retinal axon regeneration in the lizard, Gallotia galloti

The myelin‐associated protein Nogo‐A contributes to the failure of axon regeneration in the mammalian central nervous system (CNS). Inhibition of axon growth by Nogo‐A is mediated by the Nogo‐66 receptor (NgR). Nonmammalian vertebrates, however, are capable of spontaneous CNS axon regeneration, and we have shown that retinal ganglion cell (RGC) axons regenerate in the lizard Gallotia galloti. Using immunohistochemistry, we observed spatiotemporal regulation of Nogo‐A and NgR in cell bodies and axons of RGCs during ontogeny. In the adult lizard, expression of Nogo‐A was associated with myelinated axon tracts and upregulated in oligodendrocytes during RGC axon regeneration. NgR became upregulated in RGCs following optic nerve injury. In in vitro studies, Nogo‐A‐Fc failed to inhibit growth of lizard RGC axons. The inhibitor of protein kinase A (pkA) activity KT5720 blocked growth of lizard RGC axons on substrates of Nogo‐A‐Fc, but not laminin. On patterned substrates of Nogo‐A‐Fc, KT5720 caused restriction of axon growth to areas devoid of Nogo‐A‐Fc. Levels of cyclic adenosine monophosphate (cAMP) were elevated over sustained periods in lizard RGCs following optic nerve lesion. We conclude that Nogo‐A and NgR are expressed in a mammalian‐like pattern and are upregulated following optic nerve injury, but the presence of Nogo‐A does not inhibit RGC axon regeneration in the lizard visual pathway. The results of outgrowth assays suggest that outgrowth‐promoting substrates and activation of the cAMP/pkA signaling pathway play a key role in spontaneous lizard retinal axon regeneration in the presence of Nogo‐A. Restriction of axon growth by patterned Nogo‐A‐Fc substrates suggests that Nogo‐A may contribute to axon guidance in the lizard visual system. J. Comp. Neurol. 525:936–954, 2017.

Regrowth of transected retinal ganglion cell axons despite persistent astrogliosis in the lizard (Gallotia galloti)

We analysed the astroglia response that is concurrent with spontaneous axonal regrowth after optic nerve (ON) transection in the lizard Gallotia galloti. At different post‐lesional time points (0.5, 1, 3, 6, 9 and 12 months) we used conventional electron microscopy and specific markers for astrocytes [glial fibrillary acidic protein (GFAP), vimentin (Vim), sex‐determining region Y‐box‐9 (Sox9), paired box‐2 (Pax2)¸ cluster differentiation‐44 (CD44)] and for proliferating cells (PCNA). The experimental retina showed a limited glial response since the increase of gliofilaments was not significant when compared with controls, and proliferating cells were undetectable. Conversely, PCNA+ cells populated the regenerating ON, optic tract (OTr) and ventricular wall of both the hypothalamus and optic tectum (OT). Subpopulations of these PCNA+ cells were identified as GFAP+ and Vim+ reactive astrocytes and radial glia. Reactive astrocytes up‐regulated Vim at 1 month post‐lesion, and both Vim and GFAP at 12 months post‐lesion in the ON‐OTr, indicating long‐term astrogliosis. They also expressed Pax2, Sox9 and CD44 in the ON, and Sox9 in the OTr. Concomitantly, persistent tissue cavities and disorganised regrowing fibre bundles reaching the OT were observed. Our ultrastructural data confirm abundant gliofilaments in reactive astrocytes joined by desmosomes. Remarkably, they also accumulated myelin debris and lipid droplets until late stages, indicating their participation in myelin removal. These data suggest that persistent mammalian‐like astrogliosis in the adult lizard ON contributes to a permissive structural scaffold for long‐term axonal regeneration and provides a useful model to study the molecular mechanisms involved in these beneficial neuron–glia interactions.

Glial Cells Ontogeny in the Telencephalon and Mesencephalon of the Lizard Gallotia galloti

In contrast to the extensive literature on mammalian gliogenesis, relatively little attention has been paid to lower vertebrates and particularly to reptiles. Along this chapter we have documented the distribution of macroglial cells and the modifications of their precursors during ontogeny in the central nervous system (CNS) of the lizard Gallotia galloti by using histological, immunohistological methods, and electron microscopy. Our observations showed that most glial cell phenotypes in Gallotia galloti and the sequence of their morphological differentiation and maturation do not greatly differ from those of mammals. However there are some differences that should be pointed. The first striking feature is the different glial distribution between telencephalon and mesencephalon as demonstrated by double-labelling immunocytochemistry. Another remarkable feature is the large proportion of radial glial cells present in the adult brain. Finally, another particularity of special interest, is the presence of immature cells in some ventricular sulci throughout lizard lifetime. Probably, the abundance of immature cells in mesencephalic and telencephalic sulci of adult nuclei could be related to the regenerative capacity of reptilian CNS.