Organization of the sulcus septomedialis and medial cortex

The medial cortex, situated at the dorsomedial border of the cerebral hemisphere, is thought to play an important role in place learning and relational memory37. As for other lizard species, the medial cortex demonstrates a three-layered organization identical to that of the dorsal and lateral cortices: a cell-dense cellular layer nested between cell-sparse inner and outer plexiform layers (Fig. 1A,B). While the outer plexiform layer borders the pial surface, the inner plexiform layer adjoins the cell population contacting the lateral ventricles known as the ventricular zone. The cellular layer demonstrates two morphologically distinct populations of neurons, small type I cells with limited cytoplasm, and larger type II cells with abundant cytoplasm14. On the basis of these cell types, the cellular layer is sometimes subdivided into the medial cortex (type I cells) and dorsomedial cortex (type II cells). For the purpose of this investigation, we focused solely on type I cells of the medial cortex.

Figure 1 Anatomy of the sulcus septomedialis and medial cortex. (A) Transverse section through the telencephalon, stained with hematoxylin and eosin (red line = level of section), yellow box indicates position of (B). (B) Location of the sulcus septomedialis and medial cortex. The ventricular zone of the sulcus septomedialis is separated from the neuron-rich cellular layer of the medial cortex by a cell-sparse inner plexiform layer. The outer plexiform layer separates the cellular layer from the pial surface of the brain. Scale bar: 20 μm. advr = anterior dorsal ventricular ridge, cl = cellular layer, dc = dorsal cortex, dmc = dorsal medial cortex, ipl = inner plexiform layer, lc = lateral cortex, mc = medial cortex, opl = outer plexiform layer, sp = septum, vz = ventricular zone. Full size image

Radial glia are present within the ventricular zone

To identify neural stem/progenitor cells (NSPCs) within the ventricular zone, we first immunostained with SOX2. SOX2 is a hallmark transcription factor of stemness, and is involved in the induction and maintenance of pluripotency38,39. Across species, SOX2 identifies NSPCs within the brain during both embryogenesis and adulthood40. We determined that all ventricular zone cells of the telencephalon demonstrated robust SOX2 expression (Fig. 2A–C). Additional SOX2 labeling was observed among a subset of cells in the inner plexiform layer, but very rarely within the cellular layer. To expand our NSPC panel, we next co-localized SOX2 with the RNA binding protein M u s ash i -1 (MSI-1). MSI-1 has been implicated in asymmetric cell division and (within the brain) mitotically active stem cell populations41,42. Supporting their identification as an NSPC population, we observed strong SOX2/MSI-1 co-localization by all cells of the ventricular zone (Fig. 2D–F). Our data also revealed a population of SOX2+/MSI-1+ cells within the inner plexiform layer. Adjacent to the ventricular zone these double-labeled cells were organized into chains. However, as they approached the cellular layer, they were oriented with their long axes perpendicular to the ventricular surface, consistent with their identification as immature migrating neurons (i.e. neuroblasts). Next, we extended our characterization to include the RNA binding protein HuC /Hu D (HuCD), a marker common to both immature and mature neurons (Fig. 2G–I). HuCD expression was varied across the medial cortex. In the ventricular zone, cells were uniformly SOX2+/HuCD−. Beginning at the interface of the ventricular zone and inner plexiform layer, we first observed HuCD+ cells. Within the inner plexiform layer three distinct cell types were observed: SOX2+/HuCD−, SOX2−/HuCD+, and (less commonly) SOX2+/HuCD+. Notably, SOX2+/HuCD+ cells of the inner plexiform layer were more fusiform and less immunoreactive for HuCD than rounded SOX2−/HuCD+ cells. In contrast, the cellular layer was dominated by SOX2-/HuCD+ cells.

Figure 2 SOX2, Musashi-1 and HuC/D expression in the ventricular zone and cellular layer. (A–C) Ventricular zone cells ubiquitously express the NSPC marker SOX2. A subset of SOX2+ cells are also observed within the inner plexiform (hatched ellipses and inset), but not cellular layers. (D–F) Ventricular zone cells co-express NSPC marker Musashi-1. SOX2+/MSI-1+ cells are also present within the inner plexiform layer, often appearing in chains or with their long axes perpendicular to the ventricular lumen (hatched ellipses and inset). The shape, position, and immunoreactivity of these cells are consistent with their identification as neuroblasts. (G–I) Double immunofluorescence for SOX2 and neuronal marker HuC/D revealed that SOX2+/HuCD− (hatched ellipse), SOX2+HuCD+ (inset), and SOX2-HuCD+ cells reside within the inner plexiform layer. In contrast, the cellular layer is dominated by SOX2−/HuCD+ cells. All scale bars: 15 μm. cl = cellular layer, ipl = inner plexiform layer, opl = outer plexiform layer, vz = ventricular zone, white boxes = high magnification insets. Full size image

Amongst adult reptiles, NSPCs of the ventricular zone are most commonly identified as radial glia14,17. To determine if radial glia were present in leopard geckos, we first used a classic marker of this cell type, the intermediate filament glial f ibrillary a cidic p rotein (GFAP). Virtually all cells of the ventricular zone were GFAP+ (Fig. 3A). Moreover, these cells demonstrated the characteristic morphology of radial glia, contacting both the ventricular lumen and, via a lengthy radial (basal) process, the pial surface or the perivascular membranes surrounding blood vessels. Radial processes passed directly through the cellular layer often bifurcating in the outer plexiform layer before contacting the pial surface. We corroborated our GFAP data using a second radial glia marker, the intermediate filament Vimentin, which is most widely recognized as a neurodevelopmental marker. Cell bodies in the ventricular zone and radial processes were strongly and ubiquitously Vimentin+/GFAP+ (Fig. 3B,C). Although Vimentin immunostaining additionally revealed the endothelium of blood vessels, clear contact between Vimentin+/GFAP+ end feet and Vimentin+ vessels could not be discerned. It is also worth noting we did not observe any stellate-shaped GFAP+ or Vimentin+ astrocytes within the cortex.

Figure 3 Ventricular zone cells express radial glial markers glial fibrillary acidic protein (GFAP) and Vimentin. (A) Cells of the ventricular zone are ubiquitously GFAP+, and extend lengthy basal processes that span the cortex to terminate at the pial surface of the brain. (B) Ventricular zone cells and processes additionally express developmental radial glial marker Vimentin. Vimentin staining also reveals endothelial cells of blood vessels (white arrows). (C) GFAP+/Vimentin+ processes can be unambiguously traced from the ventricular zone through the inner plexiform layer and cellular layer (white arrows). Scale bar A,B: 20 μm, C: 10 μm. cl = cellular layer, ipl = inner plexiform layer, opl = outer plexiform layer, p = pial surface, vz = ventricular zone. Full size image

Another characteristic feature of radial glia is that they are constitutively active. To identify proliferative activity in the gecko cerebral cortex, we used the M-phase marker p hosphorylated h istone- H3 (pHH3), and the S-phase marker p roliferating c ell n uclear a ntigen (PCNA). pHH3+ cells were restricted exclusively to the ventricular zone (Fig. 4A). Similarly, PCNA immunostained cells were most abundant in the ventricular zone, although several positive cells were also located in the inner plexiform layer in close proximity to the ventricular zone (Fig. 4B). In stark contrast, pHH3+ or PCNA+ cells were never observed within the cellular layer of the medial cortex.

Figure 4 Ventricular zone cells express proliferation markers phosphorylated histone -H3 (pHH3) and proliferating cell nuclear antigen (PCNA). (A) Within the sulcus septomedialis, pHH3+ cells are restricted to the ventricular zone. Expression was confirmed using immunohistochemistry (inset). (B) Likewise, PCNA+ cells are most abundant in the ventricular zone, although occasionally observed in close contact to the ventricular zone within the inner plexiform layer (white arrow). Neither pHH3+ nor PCNA+ cells were ever observed within the cellular layer. Asterisk indicates an artifact. Scale bar A: 10 μm. cl = cellular layer, ipl = inner plexiform layer, opl = outer plexiform layer, vz = ventricular zone. Full size image

A supportive microenvironment

Next, we sought to characterize the microenvironment associated with the ventricular zone, with a focus on vasculature. Previous studies in mammals have revealed a relationship between blood vessels and vascular-derived factors, and neurogenesis43,44,45,46,47. We reasoned that a comparable vascular niche should also exist in neurogenic regions of the gecko brain. We co-localized GFAP, a marker of radial glia, with t omato l ectin (TL), a carbohydrate binding protein common to microglia and blood vessels. Matching our previous findings with Vimentin, we observed TL+ blood vessels throughout the telencephalon (Fig. 5A). Blood vessels were conspicuously arranged so that those at the pial surface were oriented radially, whereas closer to the ventricular lumen they were cut in transverse. GFAP+ end-feet appeared to surround the lumen of all TL+ blood vessels (Fig. 5B,C).

Figure 5 Microenvironment and associated vasculature of the ventricular zone. (A) T omato l ectin (TL) expressing blood vessels are cut in transverse in close proximity to the ventricular zone, and in longitudinal directed toward the pial surface. All TL+ vessels appear to be surrounded by GFAP+ radial glia endfeet (B,C). (D,E) The ventricular zone expresses both fibroblast growth factor-2 (FGF2) and vascular endothelial growth factor (VEGF). Subsets of FGF2+ and VEGF+ cells are also identified within the inner plexiform layer (hatched ellipses), and are abundant within the cellular layer (white arrows in E). (F) Investigating VEGF receptors, we found populations of VEGFR1+ cells within the ventricular zone, inner plexiform layer (hatched ellipses) and cellular layer. All cells were VEGFR2-. Scale bar A:15 μm, D: 10 μm. cl = cellular layer, ipl = inner plexiform layer, opl = outer plexiform layer, p = pial surface, vz = ventricular zone. Full size image

We then explored the expression pattern of two growth factors involved in angiogenesis: basic fibroblast growth factor (FGF2) and vascular endothelial growth factor A (VEGF). Along with their pro-angiogenic roles, both FGF2 and VEGF are known to maintain NSPC pools, and to be potent mitogenic and neurogenic factors in vivo and in vitro43,48,49,50. Although the telencephalon is enriched in both FGF2 and VEGF, expression is most pronounced within the ventricular zone of the sulcus septomedialis (Fig. 5D,E). Both growth factors were also expressed by a subset of cells within the inner plexiform layer, and most cells within the adjacent cellular layer. In the mammalian dentate gyrus of the hippocampus, VEGF signalling by NSPCs occurs primarily via VEGFR248,51. However, in leopard geckos, co-localization of VEGFR1 and VEGFR2 show the ventricular zone to house a population of VEGR1+ cells but to be uniformly VEGFR2- (Fig. 5F). A subset of cells within the inner plexiform layer, and the majority of cells within the cellular layer are also VEGFR1+/VEGFR2−.

Cells generated in the ventricular zone become neurons in the cellular layer

To track the fate of newly generated cells, we conducted an acute 5-bromo-2′-deoxyuridine (BrdU) pulse-chase experiment. BrdU is a thymidine analogue that is incorporated into cells during DNA synthesis52. Subsequently, cells that have incorporated the BrdU label can be visualized using immunofluorescence. We administered BrdU (intraperitoneal injection; 50 mg/kg) twice daily for a 2-day pulse period. Geckos were collected at three chase time points: day 0 (immediately following the pulse), day 10 post-pulse, and day 30 post-pulse (Fig. 6A). At day 0, BrdU+ cells were restricted to the ventricular zone, indicating that proliferation was localized to this population during the pulse period (Fig. 6B). At day 10, BrdU+ cells were identified within both the ventricular zone and the inner plexiform layer, demonstrating that cells born in the ventricular zone had begun to migrate (Fig. 6C). At day 30, BrdU+ cells were located within each of the ventricular zone, inner plexiform layer, and their presumptive final destination, the cellular layer of the medial cortex (Fig. 6D).

Figure 6 Cells generated by the ventricular zone become neurons in the cellular layer. (A) Experimental design for the 5-bromo-2′-deoxyuridine (BrdU) cell tracking experiment. BrdU was injected intraperitoneally twice daily for two days (pulse). Geckos were collected at days 0, 10, and 30 following the pulse. BrdU+ cells (hatched ellipses) were restricted to the ventricular zone at day 0 (B), had migrated into the inner plexiform layer by day 10 (C), and were observed in the cellular layer by day 30 (D). At day 0 (E), BrdU+ cells were of the ventricular zone co-localized with SOX2. By day 10 (F), BrdU+ cells within the inner plexiform layer were closely associated with GFAP+ radial processes. At day 30 (G), BrdU+ cells in the cellular layer co-localized with mature neuronal marker NeuN. Scale bar: 15 μm. cl = cellular layer, ipl = inner plexiform layer, vz = ventricular zone. Full size image

To explore the identity of these BrdU+ populations we conducted a series of co-localizations using SOX2, GFAP, and the mature neuronal marker NeuN. At each chase time point (day 0, 10, and 30) all BrdU+ cells in the ventricular zone co-localized with SOX2 (Fig. 6E, Fig. S1). At day 10, BrdU+ cells within the inner plexiform layer were fusiform in shape, SOX2- and NeuN-, and demonstrated a close association with GFAP+ radial processes (Fig. 6F). Specifically, the long axis of BrdU+ cells was arranged exactly parallel to that of the GFAP+ processes. By day 30, all BrdU+ cells present within the cellular layer of the medial cortex co-expressed NeuN (but neither GFAP nor SOX2) (Fig. 6G).

Finally, we sought to determine if there was evidence for long-term survival of newly generated cells. Continuous cell proliferation within the ventricular zone raises the question of whether the newly generated cells are retained, and possibly incorporated into the existing neurocircuitry, or if these cells routinely undergo apoptosis and are eliminated. To identify long-term surviving cells, we used a long duration BrdU pulse-chase experiment. Geckos were administered BrdU (intraperitoneal injection twice daily; 50 mg/kg) for a 7-day pulse period, and collected at day 0 (immediately following the pulse) and day 140 post-pulse (Fig. 7A). As previously demonstrated, at day 0 BrdU+ cells were abundant within the ventricular zone, and all co-expressed SOX2 (Fig. 7B,C). In addition, likely due to the extended pulse period, a subset of BrdU+ cells were identified within the inner plexiform layer suggesting that they had begun migration. Within the inner plexiform layer, some BrdU+ cells were additionally SOX2+ (typically those in closer proximity to the ventricular zone) (Fig. 7D). By day 140, BrdU+ cells were identified within each of the ventricular zone, inner plexiform layer and cellular layer of the medial cortex (Fig. 7E). Moreover, all BrdU+ cells in both the inner plexiform layer and cellular layer co-expressed NeuN (Fig. 7F,G).

Figure 7 Newly generated neuronal cells persist long-term. (A) Experimental design for the long-duration 5-bromo-2′-deoxyuridine (BrdU) cell tracking experiment. BrdU was injected intraperitoneally twice daily for seven days (the pulse). Experimental geckos were collected immediately following, and at 140 days post-pulse. (B) At day 0, BrdU+ cells were located in the ventricular zone and the inner plexiform layer. All BrdU+ cells of the ventricular zone co-localized with SOX2 (C). A subset of BrdU+ cells in the inner plexiform layer also co-expresses SOX2 (D). (E) At day 140, BrdU+ cells were located in the ventricular zone, inner plexiform layer and cellular layer. All BrdU+ cells in the inner plexiform (F) and cellular layers (G) were NeuN+. BrdU+ cells in the ventricular zone were NeuN-. Scale bar: 15 μm. cl = cellular layer, ipl = inner plexiform layer, vz = ventricular zone. Full size image

Cell proliferation in the medial cortex is not altered in response to tail loss

A hallmark of postnatal neurogenesis is its sensitivity to physiological and pathological stimuli, including central nervous system injuries53,54. Curiously, leopard geckos (and many other lizard species) spontaneously rupture their tail spinal cord during tail loss (caudal autotomy), a naturally evolved predation avoidance behaviour29,30,31,32,33. We took advantage of this reflexive ability to determine if tail spinal cord injury had an acute influence on cell proliferation in the medial cortex. We compared BrdU uptake following a 2-day pulse between post-autotomy geckos (n = 3) and original-tailed controls (n = 3) (Fig. 8A). All geckos were sacrificed 12 hours after the last BrdU administration. Focusing on the ventricular zone of the sulcus septomedialis, we quantified the number of BrdU+ cells and the total number of DAPI+ cells for both groups of geckos. For these analyses the medial cortex was serially sectioned in the transverse plane, and the histological series was divided into four equal subareas allowing for observation of any rostrocaudal differences in BrdU uptake (Fig. 8B). The data set included three sections (between 30 μm and 60 μm apart) per subarea, for a total of 12 sections per gecko. The ventricular zone of the sulcus septomedialis was imaged bilaterally (one field of view per hemisphere).

Figure 8 5-bromo-2′-deoxyuridine (BrdU) incorporation is not altered by tail autotomy. (A) Experimental design. Geckos with original (intact) or autotomized tails were injected with BrdU (pulse). All geckos were collected immediately following the pulse. (B) Brains were serially sectioned through the cerebral hemispheres and divided into four subareas. From each subarea, three sections were selected and immunostained for BrdU. (C) A BrdU to DAPI ratio was established for each section. (D) No significant difference in BrdU incorporation was observed between original and post-autotomy geckos (p = 0.42). (E) Considering original-tailed and autotomized geckos together, a rostrocaudal difference in BrdU uptake was observed. Subarea 1 took up significantly more BrdU than all other subareas (*p = 0.0002, **p < 0.0001). (F) For each subarea, no significant differences were observed between the two groups. Scale bar: 15 μm. CI = confidence interval, sub = subarea. Full size image

Consistent with our previous findings, in all sections examined for both experimental groups, BrdU+ cells were restricted to the ventricular zone (Fig. 8C). In the original-tailed group, 3.33% were BrdU+ (with a 95% confidence interval (CI) [lower limit = 0.95%, upper limit = 7.60%]), whereas in the post-autotomy group, 5.26% of cells were BrdU+ (with a 95% CI [lower limit = 0.99%, upper limit = 9.54%]) (Fig. 8D). Comparing the groups, we observed no significant difference in BrdU incorporation (p = 0.42), indicating that cell proliferation within the medial cortex is resilient to the effects of spinal cord rupture following tail loss.

Curiously, there did appear to be rostrocaudal differences in BrdU incorporation. Considering both groups simultaneously, BrdU uptake was significantly higher in the rostral-most subarea (subarea 1) than in all other subareas (compared to: subarea 2, p = 0.002; subarea 3 and 4, p < 0.001) (Fig. 8E). To examine if there was any effect of tail-loss specific to different subareas we conducted post-hoc Tukey pairwise comparisons. As the most constitutively active region of the sulcus septomedialis, it stands to reason that the rostal-most subarea may be the most affected following spinal cord rupture. We determined that 8.13% of cells were BrdU+ in the post-autotomy group (with a 95% CI [lower limit = 4.89%, upper limit = 11.37%]), and 4.58% of cells were BrdU+ in the original-tailed group (with a 95% CI [lower limit = 1.34%, upper limit = 7.81%]) (Fig. 8F). Consistent with the results achieved by surveying the entirety of the medial cortex, differences in BrdU incorporation were not statistically significant (p = 0.12).