This study provides three lines of evidence that ephrin-A2, a glycosylphosphatidylinositol (GPI)-linked guidance molecule, is transiently expressed within cortical neurons of SI in early postnatal rats. Specifically, immunohistochemical localization demonstrated that the ephrin-A2 protein was expressed during the first two postnatal weeks in neurons within the supragranular and infragranular layers as well as in the subplate, but not within layer IV or its anlage in the cortical plate. Ephrin-A2 mRNA was also expressed in the same strata, indicating that the cortical neurons mainly in layers II/III and V, as well as in the subplate, are the source of the protein expression. No spatial gradients parallel to the cortical surface were noted for ephrin-A2 protein or mRNA. Ephrin-A2 mRNA, extracted from PMBSF samples and analyzed by RTPCR, increased during the first four postnatal days and then sharply declined, as much as 20-fold by P18. Thus, the specific temporal and laminar expression patterns of ephrin-A2 are consistent with the suggestion that this molecule may play a transient role in cortical development during the period of TCA arbor formation.
Comparison With Previous Studies
In situ hybridization for cortical ephrin-A2 has previously been performed in postnatal mice (Cang et al.,2005; Peuckert et al.,2008), and our results show both similarities and differences. The similarities are that ephrin-A2 expression in mouse visual cortex and in SI up as a band in the supragranular layers, with little hybridization signal seen in layer IV, other than at its superficial border. Also, SI in mouse showed no obvious tangential gradients of ephrin-A2 expression (Peuckert et al.,2008), although this differed from visual cortex in the same species (Cang et al.,2005). The features present in rat, but lacking in mouse sensory cortex, are the bands found in the infragranular layers and in the subplate. Interestingly, in ephrin-A5 knockout mice on C57BL6 background, there was up-regulation of ephrin-A2 in layers II/III and uppermost layer IV and, in addition, expression in the subplate, although none appeared in layers V/VI (Peuckert et al.,2008). Analysis of protein expression was not undertaken in these two studies.
Although many developmental features of barrel cortex are similar in rats and mice, the two species are known to differ slightly in laminar organization and timing. In mice, for example, acetylcholinesterase (AChE) activity associated with TCA ingrowth and arborization in SI forms a continuous band in upper layer V preceding its segmented appearance in layer IV as the TCA arbors develop. In contrast, rat cortex reveals little AChE activity in layer V until well after its appearance in the layer IV barrels (Sendemir et al.,1996). The molecular factors that differentially regulate AChE expression by TCAs at the layer IV/V boundary in SI of rats and mice are not presently known, but these findings provide an example in which molecular involvement at this border may differ across rodent species.
Ephrin-A2 as a Laminar Guidance Cue?
In rat and mouse SI, TCAs form terminal arbors in cortical layer IV without undergoing a phase of initial overgrowth and back-pruning (Killackey et al.,1995). Molnár and Blakemore (1991) have previously hypothesized that a repulsive signal within the cortex could be necessary for confining TCA termination to layer IV during cortical development. In vitro, coculture models involving TCAs from VB forming projections into cortical explants suggested that the molecular signals guiding this process were associated with neuronal membranes or the extracellular matrix (Yamamoto et al.,1992). The ephrin-A family of guidance molecules were viewed as ideal candidates for TCA arbor confinement to layer IV, that is, a “stop signal,” by Yamamoto and coworkers who showed that TCAs would continue their growth past layer IV and into supragranular layers following cleavage of GPI-anchored proteins (Yamamoto et al.,2000). Other findings from cocultures of embryonic thalamus with postnatal visual cortex suggested further that the molecular stop signal for TCA termination did not become well established until approximately P3 (Molnár and Blakemore,1999). Thus, a GPI-anchored molecule, such as an ephrin-A protein family member that is characterized by expression beginning about the time of TCA penetration into the cortical plate and then temporarily upregulated during the period of peak TCA arbor elaboration, would be a suitable candidate for an interlaminar stop signal.
Previous studies of ephrin-A family contributions to TCA pathfinding and connectivity in SI have largely focused on perinatal expression of ephrin-A5 (Castellani et al.,1998; Gao et al.,1998; Mackarehtschian et al.,1999; Vanderhaeghen et al.,2000; Yabuta et al.,2000; Mann et al.,2002; Dufour et al.,2003). At P3, ephrin-A5 mRNA has a weak medio-lateral gradient in barrels within layer IV (Vanderhaeghen et al.,2000; Dufour et al.,2003) and, while still present at later ages (P6 and P8), shows no marked density gradation (Castellani et al.,1998; Bolz et al.,2004). Moreover, ephrin-A5 is not expressed in supragranular or infragranular cortical layers (Yabuta et al.,2000). Thus, expression timing and laminar distribution of ephrinA5 are not well suited to constrain the termination of TCAs within layer IV. Consistent with this suggestion, results from ephrin-A5 deletion mutants show tangential distortions of the PMBSF (Prakash et al.,2000; Vanderhaeghen et al.,2000; Uziel et al.,2002; Dufour et al.,2003), but reveal no deficits in laminar targeting precision (Yabuta et al.,2000; Uziel et al.,2008). Taken together, the results from these studies indicate that ephrin-A5 may be involved in directing TCA growth relevant to topography, but is insufficient for restricting axonal growth to the correct layers.
Our results suggest that during normal development, transient ephrin-A2 expression at upper and lower layer IV borders may constrain the laminar distribution of TCAs until P12–P14 when arbor formation is well advanced. Within a different context, a similar role for ephrin-A2 has been proposed in the hippocampus. Several days following axotomy of the perforant pathway in adult mouse, ephrin-A2 undergoes long term upregulation in a band spanning the denervated terminal zone and may thus serve to limit local axonal sprouting while stimulating synaptogensis (Wang et al.,2005).
Several EphA receptors (EphA3, EphA4, and EphA7) capable of binding with ephrin-A2 (Gale et al.,1996) are expressed in VB neurons during the late embryonic and early postnatal period (Mackarehtschian et al.,1999; Vanderhaeghen et al.,2000; Sestan et al.,2001; Dufour et al.,2003; Bolz et al.,2004), a time when VB TCAs reach the cortex, make connections in layer IV, and refine those connections (Catalano et al,1996). We suggest that ephrin-A2 expression in cortical neurons acting in concert with EphA receptors on TCA terminals may serve a distinct role in regulating TCA arbor development in the appropriate lamina of SI after their topographic location has been specified by additional molecules.