Growth-associated protein-43 is required for commissural axon guidance in the developing vertebrate nervous system

Abstract

Growth-associated protein-43 (GAP-43) is a major growth cone protein whose phosphorylation by PKC in response to extracellular guidance cues can regulate F-actin behavior. Here we show that 100% of homozygote GAP-43 (-/-) mice failed to form the anterior commissure (AC), hippocampal commissure (HC), and corpus callosum (CC) in vivo. Instead, although midline fusion was normal, selective fasciculation between commissural axons was inhibited, and TAG-1-labeled axons tangled bilaterally into Probst's bundles. Moreover, their growth cones had significantly smaller lamellas and reduced levels of F-actin in vitro. Likewise, 100% of GAP-43 (+/-) mice with one disrupted allele also showed defects in HC and CC, whereas the AC was unaffected. Individual GAP-43 (+/-) mice could be assigned to two groups based on the amount that PKC phosphorylation of GAP-43 was reduced in neocortical neurons. In mice with approximately 1% phosphorylation, the HC and CC were absent, whereas in mice with approximately 10% phosphorylation, the HC and CC were smaller. Both results suggest that PKC-mediated signaling in commissural axons may be defective. However, although Probst's bundles formed consistently at the location of the glial wedge, both GAP-43 (-/-) and GAP-43 (+/+) cortical axons were still repulsed by Slit-2 in vitro, precluding failure of this deflective signal from the glial wedge as the source of the phenotype. Nonetheless, the data show that a functional threshold of GAP-43 is required for commissure formation and suggests that failure to regulate F-actin in commissural growth cones may be related to inhibited PKC phosphorylation of GAP-43.

Fig. 1.

a, b, Nissl-stained parasagittal sections at P0. a, GAP-43 (+/+) showing corpus callosum (cc), hippocampal commissure (hc), and anterior commissure (ac). b, GAP-43 (−/−) showing no commissures. c, d, Nissl-stained horizontal sections at P0. c, GAP-43 (+/+) showing CC and HC. d, GAP-43 (−/−) showing that midline commissural axons tangle into Probst's bundles (pb). Asterisks indicate inappropriate fasciculation of axons in the corpus striatum.e, f, Coronal sections at P0 labeled with the anti-TAG1 mAb 4D7, followed by TRITC-labeled secondary antibody.e, GAP-43 (+/+) showing CC and HC. f, GAP-43 (−/−) showing no midline crossing (Probst's bundles themselves are located more anteriorly than this section illustrates). No callosal or hippocampal commissures ever formed in GAP-43 (−/−) mice (n = 12). Scale bars, 500 μm.

Fig. 2.

a, b, Nissl-stained coronal sections at P0. a, GAP-43 (+/+) showing both anterior (ab) and posterior (pb) branches of the anterior commissure. b, GAP-43 (−/−) showing only posterior branches. Neither branch crossed the midline.c, f, Coronal sections at P0.c, GAP-43 (+/+) visualized by differential interference contrast showing the anterior and posterior branches.d, Same section after TAG-1 immunohistochemistry to show that only the anterior branch is labeled with TAG-1. e, GAP-43 (+/+) after TAG-1 immunohistochemistry showing TAG-1-labeled (anterior branch) axons crossing the midline. f, GAP-43 (−/−) after TAG-1 immunohistochemistry showing TAG-1-labeled (anterior branch) axons formed Probst's bundles (arrows). Scale bar: a, b, 500 μm; c, d, 100 μm;e, f, 250 μm.

Fig. 3.

Growth cones from dissociated cortical cultures from GAP-43 (+/+) (a–d) or GAP-43 (−/−) (e–h) mice double labeled with TAG-1 (a–h) and rhodamine phalloidin (a′–h′) after 24 hr in vitro. Arrows indicate the growth cone used for measuring both lamellal area and pixel intensity of rhodamine phalloidin labeling with IP Lab (Scanalytics Inc.).Lines bisecting neurites indicate limit of growth cones used for measurement. Scale bar, 100 μm.

Fig. 4.

a, Coronal confocal section of E17.5 GAP-43 (+/+) cortex labeled with GFAP mAb, followed by TRITC showing bilateral localization of the glial wedge (gw; arrows), which is highly immunoreactive with GFAP. b, c, Similar sections of GAP-43 (−/−) cortex double labeled with GFAP, followed by TRITC and RC1 followed by FITC. Note that the glial wedge (gw; arrows) is detected by RC1 immunoreactivity but is very poorly immunoreactive with GFAP. The Probst's bundles (pb) are consistently localized in the vicinity of the glial wedge and are immunoreactive with GFAP. Scale bar, 125 μm. d, Quantitation of the responses of each explant culture to Slit-2-expressing HEK 293 cells suspended in a collagen gel capsule. A response was defined as attractive (A) when >70% of the total neurites grew in the hemisphere adjacent to the cell–gel capsule, whereas a response was defined as repulsive (R) when >70% of the total neurites grew in the hemisphere distant from the cell–gel capsule. A response was defined as neutral (N) when neurites in neither hemisphere were >70% of total. Black barsrepresent GAP-43 (+/+) cultures (n = 14), andwhite bars represent GAP-43 (−/−) cultures (n = 12). Dashed bars represent GAP-43 (+/+) cultures (n = 4) cocultured with HEK 293 cells expressing vector alone as control.

Fig. 5.

Parasaggital sections of GAP-43 (+/+) (a, b) and GAP-43 (−/−) (c, d) mice that have been labeled in frontal cortex with a crystal of DiI (green) and in occipital cortex with a crystal of DiA (red).a, c, Sections at the midline showing appropriate topographic organization of GAP-43 (+/+) callosal axons (a) but no GAP-43 (−/−) axons crossing the midline (c). b, d, Sections taken at the distance from the midline in which Probst's bundles are found in the (−/−) mice (d). Again, topographic organization of label is seen in the GAP-43 (+/+) section (b) but abnormal mixing and projection of axons in the GAP-43 (−/−) section (d). Scale bar, 500 μm.

Fig. 6.

a–c, Nissl-stained parasagittal sections close to the midline of GAP-43 (+/−) forebrains at P0 showing corpus callosum (cc), hippocampal commissure (hc), and anterior commissure (ac). The severity of phenotypes in the (+/−) ranges from reduced size of CC and HC (a, b) to complete absence of both commissures (c). Note that the anterior commissure is normal in all cases. Scale bar, 250 μm.d, The area of the CC–HC at midsagittal levels was measured at P0, P7, and P21 in GAP-43 (+/+) (▪) and those GAP-43 (+/−) (■) mice with commissures. Results presented as mean ± SEM; n ≥ 3 in each case. There was a significant reduction in size of the HC and CC at both P7 and P21 (p < 0.05).

Fig. 7.

a–c, Parasaggital sections at P0, from GAP-43 (+/+) (a) and (+/−) (b, c) mice labeled with the anti-phospho-GAP-43-specific mAb 2G12, followed by FITC mice and arranged according to severity of callosal phenotype (CC).Arrows indicate position of the CC. There is less PKC phosphorylated in both cortex and CC in GAP-43 (+/−) compared with (+/+). On the other hand, phosphorylation in the AC appears normal. Scale bar, 500 μm. d, Reduction in callosal area of GAP-43 (+/−) mice correlates with the amount of phosphorylated GAP-43 in anterior cortex. White bars indicate area of AC,hatched bars indicate area of CC, and black bars indicate the amount of PKC-phosphorylated GAP-43 in anterior cortex quantitated from Western blots by¹²⁵I-labeled secondary antibody, followed by PhosphorImager analysis. Note that the quantitative results are presented on a log scale.