Genetic Analysis on the Role of Integrin during Axon Guidance in Drosophila

βPS integrin protein subunit in the embryonic CNS at hour 18. Fillet-dissected embryos were processed for βPS immunocytochemistry (see Materials and Methods). Panelsshow four abdominal CNS segments. A, In a wild-type embryo (FM7c/Y), the βPS protein subunit is detected in the major axon tracts that include the pair of longitudinal connectives (LC) as well as the anterior nerve tract (ANt) and the posterior nerve tract (PNt). The latter two axon tracts contain motoneuron axons. In addition, surfaces of many neuronal cell bodies, including those of the pair of RP3 motoneurons at this focus (arrowheads), accumulate low levels of the βPS protein subunit. B, βPS null embryo (mys^xb87/Y), which has been dissected and immunoprocessed in the same minipool as the wild-type control, shows only the nonspecific background staining and serves as a negative control. Scale bar, 20 μm.

CNS axon defects in loss-of-function mutants at hour 18. Whole embryos were processed for mAb 1D4 immunocytochemistry (see Materials and Methods). Panels show five abdominal CNS segments in fillet-dissected embryos. A, In wild type (Canton S), each of the bilaterally paired longitudinal connectives (LC) contains three discrete axon fascicles that show little crossover among themselves.B, C, Axon fascicles are disorganized in the CNS. The two most common defects are “gaps” in the longitudinal fascicles (B, arrows) and “wiggles” within the axon fascicles (C, circle). With the level of analysis, it is difficult to resolve whether the gaps represent axons stalling or axons changing the fascicles. See Figure 4for summary. Scale bar, 20 μm.

Summary of axon defects in loss-of-function mutants. Data are based on the hour 18 embryos after immunoprocessing with mAb 1D4 and fillet dissection. CNS axon fascicles were analyzed only in the longitudinal connectives (LC). Various classes of defects, such as the gaps and wiggles shown in Figure 3,B and C, are included as axon “errors.” In the periphery, all five groups of motoneuron axons (ISN, SNa, SNb,SNc, and SNd) were examined. In each group, axon defects are collectively summarized as errors. Examples of various peripheral nervous system (PNS) axon defects are shown below (see Fig. 5G–L). Both the error rates and sample sizes (the numbers of abdominal hemisegments scored; numbersin parentheses below each bar) are indicated in the charts. The data for the αPS1 mutants are based on 10 mew^M8 and eightmew⁴⁹⁸ embryos, and the penetrance of the axon errors either in the CNS or PNS in each allele is ∼90%. Similarly, the data for the αPS2 mutants are based on ninemew^M8 and eightmew⁴⁹⁸ embryos, and the penetrance of the axon errors either in the CNS or PNS ranges between 78 and 100%. There is no obvious correlation between the CNS and PNS segments in which axon defects occur. Taken together, these observations support the idea that the loss of integrin leads to widespread and stochastic axon guidance errors throughout the nervous system. A,B, Wild type (Canton S) shows very low axon error rates (0–5%) in the CNS (A) as well as in the periphery (PNS) (B). The data are based on eight embryos. C, D, αPS1 null mutants (mew^M6/Y andmew⁴⁹⁸/Y) both show low-to-medium rates (0–24%) of axon errors in the CNS (C) and PNS (D).E, F, The αPS2 null mutant allele (if^K27E/Y) exhibits medium-to-high rates (8–69%) of axon errors in the CNS (E) and PNS (F). The hypomorphic allele (if^B2/Y) shows similar defects at slightly reduced rates (1–37%).

PNS axon defects in loss-of-function mutants at hour 18. Embryos were processed for mAb 1D4 immunocytochemistry and fillet-dissected. B–L show parts of the motoneuron axon pathways in a right PNS hemisegment. Boxes in theschematics above and below B–Japproximately correspond to the areas shown in thepanels. A, Each hemisegment contains 30 uniquely identified muscles and is innervated by five groups (fascicles) of motoneuron axons: SNd,SNc, SNb, SNa, andISN. The muscles that are targeted by each motoneuron group are labeled with identification numbers (13 of 30). B–F, In wild type (Canton S),SNd targets the ventral- (proximal) most muscles including 15 and 16 (B). SNctargets the next ventral-most muscles including 26 (C). SNb branches into several subfascicles and innervates ventrolateral muscles including 6, 7, 12, and 13 (D). The SNa motoneuron group extends toward the lateral muscles and bifurcates into two subfascicles; one subfascicle reaches transverse muscles 21, 22, and others, whereas another turns posteriorly to innervate 5 and 8 (E). ISN extends farthest and targets the dorsal- (distal) most muscles such as 1, 2, and 3 (F). G–L, In the null mutant embryos, the PNS axon fascicles exhibit various defects. The types of axon pathfinding defects seen for the αPS1 and αPS2 null mutants are very similar. SNd and SNc are sometimes missing (G, H,arrows). Axons that would normally reach this muscle region may be either stalling or bypassing. SNbfrequently extends beyond the normal stopping point and invades into the neighboring segment (J, circle) or fails to form sub-branches at muscles 6 and 7 (I,arrow). SNa occasionally misses its posteriorly directed sub-branch and fails to innervate muscles 5 and 8 (K, arrow). ISN sometimes fails to obey segmental boundaries and merges with the ISN from adjacent segments (L, circle). See Figure4 for data summary. Scale bar, 20 μm.

A, B, The axon defects in αPS2 null mutants (if^K27E/Y) can be partially rescued by supplying wild-type αPS2 gene to the nervous system in the mutant background (striped bars;if^K27E/Y; UAS-αPS2^wt/elav’-GAL4^III; see Materials and Methods). In this neuron rescue experiment, the rates of axon defects in both the CNS (A) and PNS (B) revert toward those of wild type, yielding error rates significantly lower than those in αPS2 null mutants (asterisks; p < 0.001 by Chi²t test). (See Fig. 7 for examples.) The parental lines used for the neuron rescue experiment were either wild-type–like (UAS-αPS2^wt andelav’-GAL4^III) or indistinguishable from the αPS2 null mutants (if^K27E/Y; UAS-αPS2^wt) in their axon fascicle organizations.

Neuron rescue experiment. Axon fascicle organizations in the αPS2 null mutant embryos (hour 18) after neuron rescue (see Materials and Methods) were visualized using mAb 1D4.A, Four abdominal CNS segments in a fillet-dissected embryo are shown. The longitudinal connectives (LC) revert to virtually wild type and exhibit three discrete fascicles (compare with Fig. 3A). B–F, In the PNS, axon fascicle organizations are very similar to that in wild type (compare with Fig. 5B–F). BothSNd (B) and SNc(C) innervations are present at high frequencies.SNb axons show much-reduced error rates (D) compared with those in the αPS2 null mutants (compare with Fig. 5I, J).SNa increases its rate of forming the posteriorly directed sub-branch that reaches muscles 5 and 8 (E). ISN obeys the segment boundaries and looks very similar to wild type (F). See Figure 6, A andB, for data summary. Scale bar, 20 μm.