Tenascin expression in the region between prosomere 2 and the mesencephalon was arranged into two ventro-dorsal columns extending from the basal plate to the dorsal region (D1). One column was located in the superficial stratum, while the other was located in the periventricular stratum (Figure 2K, and dotted arrows Figure 2L). Tenascin was absent in the roof and basal plates (Figures 2F–J). In prosomere 1, especially in the medial region, a third column appeared from the medial layer of the intermediate stratum (solid arrow Figures 2L,M). This third column, in conjunction with the periventricular column, formed a corridor that surrounded the axon fascicles located at the intermediate stratum (dotted double arrow Figure 2M). Tenascin expression abruptly decreased in the dorsal region (D.1) (Figure 2N) and the roof plate, coinciding with SCO-spondin expression (Figure 2O). In the basal region, this protein was present in the floor plate of prosomeres 1 and 2.

HNK-1 expression was almost identical to tenascin expression (Figures 3A,D), with three ventro-dorsal columns extending from the basal plate to D.1 and located in the periventricular, medial intermediate, and superficial strata (solid arrows in Figures 3B,E). The first two columns delimited axon fascicles extending into the dorsal region. Additionally, HNK-1 co-localized with axons in the intermediate stratum of L.1 (dotted arrow in Figures 3B,E), but this co-localization disappeared as axons arrived at the roof plate (asterisk in Figures 3B,E). HNK-1 was also found in the apical region of neuroepithelial cells (arrow in Figure 3C), with the exception of roof plate cells secreting SCO-spondin (Figure 3F).

Osteopontin was highly expressed in the entire prosomere 1 region (Figures 4B–D), in contrast to the cephalic (prosomere 2, Figure 4A) and caudal (mesencephalon, Figure 4E) limits where osteopontin expression drastically decreased. In prosomere 1, osteopontin was found along the intermediate stratum but not in the superficial or periventricular strata (continuous and dotted arrows, respectively, in Figures 4K,L). Osteopontin expression increased along the axon trajectory, and both axons and osteopontin co-localized in the lateral region (arrows in Figures 4M,N). In the dorsal roof plate, osteopontin was found between the axons traversing the medial region. Here, the diencephalic roof plate cells were immunopositive for osteopontin, suggesting possible osteopontin secretion by these cells (arrows in Figure 4P). The horizontal sections along the roof plate presented osteopontin between the axons traversing the midline (Figures 4Q–T).

Laminin expression in the prosomere 1 medial region was recorded from the basal plate to the roof plate, with maximal expressions in the lateral region (asterisk in Figures 5A,B) and roof plate (solid arrow in Figures 5A,D), and minor expression in the dorsal region (dotted arrows in Figure 5A). To more fully analyze expression in the roof plate, horizontal sectioning of this region was conducted (Figures 5C,E,F). Through this, laminin was found located in the ECM surrounding the axons (Figure 5C), co-localizing with SCO-spondin (Figure 5F). Laminin was also highly expressed in the external limiting membrane (dotted arrow in Figure 5D). The expression of laminin was also analyzed in relation with EphA7, a transmembrane protein located in the basal prolongations of the dorsal midline cells that traverse the PC (arrow in Figure 5G). Horizontal sections showed the presence of EphA7 in close contact with the laminin of the extracellular space (Figure 5I).

Fibronectin expression was not detected in the prosomere 1 medial region except in blood vessel walls (solid arrow in Figure 6B), the external limiting membrane (dotted arrow in Figure 6B), and the apical region of neuroepithelial cells (arrow in Figure 6C), with the exception of cells expressing SCO-spondin at the roof plate cells (dotted arrow in Figures 6C–F). However, fibronectin was highly expressed in mesenchymal tissue surrounding the CNS (asterisk in Figure 6A).

Decorin and perlecan expressions were both circumscribed to the external limiting membrane and to blood vessel walls (Figure 7).

The tenascin (TN) gene family includes distinct genes (i.e., TN-C, R, X, and Y), but only TN-C and TN-R have been reported in CNS tissues (Faissner, 1997). Both proteins are characterized by a serial arrangement of a cysteine-rich amino-terminus and varying numbers of fibronectin-type and EGF-type domains (Faissner, 1997). The antibody used in the present study recognize TN-C (Akbareiana et al., 2013).

TN-C has been related to various aspects of CNS development, including the regulation of proliferation and differentiation processes, participation in axon guidance, and the modulation of responsiveness to other cell matrix components, such as FGF2 (Gotz et al., 1997; Besser et al., 2012). Furthermore, the importance of TN during commissure development was studied in the optic tract. Specifically, TN was found bordering the developing optic projection, thus preventing optic axons from exiting the optic tract in a medio-caudal direction. TN inhibition in this context results in axon misrouting and increased axonal branches (Becker et al., 2003). The TN expression pattern found in the present study suggests a similar function during PC development. In particular, tenascin was located at the border of dorsally extending axonal fascicles, and this positioning could generate a repulsion barrier that would prevent axons from leaving the fascicle.

The HNK-1 carbohydrate epitope is comprised of a unique trisaccharide, HSO3-3GlcAb1-3Galb1-4GlcNAc. This epitope is only carried by certain molecule types, such as cell adhesion molecules (e.g., NCAM, L1; Ong et al., 2002) or some ECM proteins, especially tenascin (Yagi et al., 2010). This glycoepitope is predominantly expressed in the nervous system, being used as axonal marker in some anamniotes (Ware et al., 2015), and contributes to regulating neural functions (Senn et al., 2002; Yamamoto et al., 2002; Kizuka et al., 2006; Morita et al., 2009).

The observed localization of HNK-1 during PC development suggests that it was carried by tenascin, particularly as both molecules presented similar expression patterns consisting of three ventro-dorsal columns. Additionally, HNK-1 was present in L.1 and D.2 located axons, but expression decreased as the axons reached the dorsal region and was absent in the roof plate region.

These results suggest the participation of HNK-1 and tenascin in guiding axons from the ventral to the dorsal region of the prosomere 1 through the generation of an axonal corridor limited by repulsive tenascin and HNK-1 walls.

Laminins are heterotrimeric glycoproteins consisting of α, β, and γ subunits that assemble into a characteristic cruciform structure. In turn, this structure is a major ECM component during the development and in the mature CNS. Currently, 11 laminin chains have been identified, including α1–5, β1–3, and γ1–3. These chains combine to form up to 15 different laminin isoforms (for a review see Colognato and Yurchenco, 2000). Laminins play important roles in vitro in migration, differentiation, and axonal growth for a variety of neuronal subtypes (Liesi et al., 1989; Adams et al., 2005; Turney and Bridgman, 2005).

The antibody used in the present study recognize laminin 1. In culture, when laminin 1 is used as substratum, it promotes robust axon outgrowth for a variety of CNS and peripheral nervous system neurons. Analyses of laminin mutants in Caenorhabditis elegans, Drosophila, Danio rerio, and Mus musculus provide strong evidence that this protein is required for correct axon guidance (Garcia-Alonso et al., 1996; Karlstrom et al., 1996; Forrester and Garriga, 1997; Huang et al., 2003; Paulus and Halloran, 2006; Chen et al., 2009). Indeed, bashful/Laminin-α1 zebrafish mutants present defects in most CNS axon pathways, including the PC (Paulus and Halloran, 2006). Additionally, axon tracts in bashful mutants display defects in fasciculation and extension, as well as increased branching. Interestingly, and in contrast to CNS axons, most peripheral axons appear normal in bashful mutants. In mice, the conditional knockout of laminin γ1 subunit in cortical neurons results in profound defects in neuronal migration and morphogenesis (Chen et al., 2009).

A number of in vitro studies demonstrate that laminin1 not only acts as a simple permissive substratum but, if provided locally, can also direct growing axons (Adams et al., 2005; Turney and Bridgman, 2005). Laminin isoforms primarily stimulate axon extension through adhesion and by signaling downstream of integrin receptors (Kuhn et al., 1995). Nevertheless, other receptors, such as syndecans or dystroglycan, may be involved in certain neurons (Hamill et al., 2009). Regarding the PC, integrin β1 has been found in PC axons (Caprile et al., 2009) and could therefore be a possible receptor for the laminin found in the present analyses.

Although, laminin alone has potent effects on neuronal morphogenesis, this protein could also function by modulating responses to axon guidance cues, such as netrins (Hopker et al., 1999) and different Eph/ephrin family members. In this context, while EphB displays no effect on retinal ganglion cell axons when growing on the cell adhesion molecule L1, when cultured on a combined laminin-L1 substratum, growth cones pause but remain dynamic in response to EphB (Suh et al., 2004). Laminin-mediated modulation can be important for PC development since Eph family members have been reported at the midline, where axons must decide to cross or not cross the midline (Stanic et al., 2014). At this point, PC axons (positive for NCAM, similar to L1) become defasciculated, and the presence of laminin may be important to pause the growth cones, thereby allowing the axons to detect Eph members expressed at the midline. The presence of laminin can also switch or nullify inhibitory axon guidance cues. For example, ephrin-A5 is an important repulsive factor toward retinal ganglion cell axons. This repulsive effect is maintained in vitro by retinal neurons using fibronectin as a substrate. However, when cultured on laminin, retinal ganglion cells are attracted toward a gradient of soluble ephrin-A5 (Weinl et al., 2003).

Considering the modulatory effects of laminin on Eph/ephrin family members, detailed analyses were performed to establish the presence of this protein in the roof plate, where EphA7 (Figures 5G–I; Stanic et al., 2014) and other Eph/ephrin family members have been found (results not published). In the roof plate, laminin was located in the ECM co-localizing with SCO-spondin and in close contact with PC axons and basal prolongations of the roof plate cells, positives for EphA7. The PC axons expressed integrin β1, a potential receptor for laminin and SCO-spondin, thus suggesting that laminin might modulate the effects of both SCO-spondin and the EphA7.

At postnatal stage, osteopontin is principally associated with peripheral axon regeneration and it is upregulated during CNS injury. However, osteopontin appears to have different roles depending on axon type. For example, this protein increases motor axon outgrowth (Wright et al., 2014) but inhibits sensory axon outgrowth (Kury et al., 2005). Furthermore, postnatal hippocampal neurons cultured in an osteopontin substrate show more primary neurites than neurons grown in a laminin substrate (Plantman, 2012). Osteopontin expression and function during CNS development has not been previously described, but in vitro experiments with embryonic retinal ganglion cell neurons show that using this protein as a substrate promotes axonal growth, and the additional presence of laminin produces an additive effect (Ries et al., 2007).

Osteopontin contains an Arg-Gly-Asp cell-binding sequence specific to integrins. In fact, it has been reported the binding to αvβ3, αvβ1, αvβ5, and α9β1, and α4β1 integrin (Liaw et al., 1995; Smith et al., 1996; Bayless et al., 1998). PC axons express the β1 integrin subunit, although the alpha subunit has not been described, suggesting a possible axon-osteopontin interaction through these receptors.

Fibronectin has been broadly used in vitro as a substrate for embryonic neurons due to its permissive effect on axonal growth. However, fibronectin expression in embryonic mice is restricted to the pial surface and blood vessel walls, with expression absent in the CNS (Milner and Campbell, 2002; Lathia et al., 2007). The presently obtained results revealed the same expression pattern in chick embryos, with expression restricted to the external membrane and blood vessels. Therefore, fibronectin does not appear to participate in the guidance of commissural axons.

Perlecan is a major heparan sulfate proteoglycan constituent of basement membranes. Additionally, perlecan surrounds axonal fascicle bundles from olfactory sensory neurons in embryonic mice (Shay et al., 2008). Perlecan can also promote neurite extension in vitro (Nakamura et al., 2015). In chick embryos, the expression pattern of this protein has been studied until HH17, where, as in the present report, perlecan becomes circumscribed to the external limiting membrane (Soulintzi and Zagris, 2007) and does not appear to participate in axon guidance.

Previous studies have been unable to detect decorin expression in mouse brain embryos, with the exception of the meninges and a region of the fourth ventricle floor plate (Scholzen et al., 1994). The present work found a similar expression pattern in chick embryos, with expression restricted to the external limiting membrane.

In conclusion, the present results support that posterior commissure axons navigate a complex extracellular matrix. While some molecules in this matrix generate a barrier surrounding the axons, others are expressed in the roof plate. At the midline, axons must decide whether or not to cross to the contralateral side in a medium containing laminin, osteopontin, SCO-spondin, and members of the Eph/ephrin family. Understanding the interrelation and modulation of these molecules in axonal guidance will provide a clearer image of how each axon navigates in a highly specific manner to find its synaptic counterpart. This knowledge will also aid in the creation of new regenerative therapies for CNS injuries, a field in which extracellular matrix technology is particularly promising (Ren et al., 2015).