Neuron, Vol. 11, 409-421, September, 1993, Copyright 0 1993 by Cell Press Fasciclin I and II Have Distinct Roles in the Development of Grasshopper Pioneer Neurons Paul Diamond,*+ Aneil Mallavarapu,*+ Jeffrey Schnipper,*+ James Booth,* Lisa Park,* Tim P. O’Connor,* and Daniel G. Jay* *Department of Cellular and Developmental Biology Harvard University Cambridge, Massachusetts 02138 *Department of Cellular Biology University of California Berkeley, California 94720 Summary We have used a new technique, micro-CAL1 (chromo- phore-assisted laser inactivation), to investigate the func- tion of the neural cell adhesion molecules fasciclin I and II in the development of the grasshopper Til neurons. Micro-CAL1 of fasciclin I results in defasciculation of the Til axons similar to that achieved using large scale CALI (Jay and Keshishian, 1990). The initial point of axon sepa- ration corresponds to the site of laser irradiation, and defasciculation always continues distal to this point. Mi- cro-CALI of fasciclin II prevents the initiation of Til axon outgrowth but has no effect on fasciculation. This effect is restricted to a 3 hr interval between cytokinesis and growth cone emergence. Introduction A fundamental understanding of the role of adhesion molecules in neural development is required to un- derstand how neural circuitry is formed (for review see Bixby and Harris, 1991). Molecules that promote adhesion of neurons to each other or to the substra- tum have been reported in many systems (Landmes- ser et al., 1988; Jessell, 1988; Bixby and Harris, 1991; Hynes and Lander, 1992). Whereas many of these mol- ecules have been characterized in vitro, demonstra- tion of their in vivo roles has been moredifficult. Here, we have used a new technique called micro-CAL1 (chromophore-assisted laser inactivation) to studythe roles of two cell adhesion molecules, fasciclin I and II, in the development of the grasshopper Til neurons. The grasshopper is an excellent model system for studying neural development. Though genetic analy- sis of the grasshopper is difficult, it has the advantage of relatively simple neural circuitry that is easy to ac- cess and manipulate (Bate, 1976; Keshishian and Bent- ley, 1983a). In particular, the Til neurons of the grass- hopper limb bud have been extensively studied (Bate, 1976; Keshishian and Bentley, 1983a, 1983b; O’Connor et al., 1990). The Til cells are the first pair of limb bud neurons to differentiate, emerging from a pioneer +These authors contributed equally to this work. mother cell in the distal tip of the limb bud epithelium at the 30% stage of development (Bate, 1976; Keshis- hian, 1980). After cytokinesis, the neurons undergo rapid changes in cytoskeletal and organelle localiza- tion to the proximal pole just before the emergence of the growth cone at this site (Lefcort and Bentley, 1989). They extend axons, which fasciculate, along the epithelium in a stereotypic pathway toward the CNS (Keshishian and Bentley, 1983a). The development of specific neuronal immunostaining and live labeling allows one to observe even subtle changes in the in vivodevelopmentof these identified neurons (Jan and Jan, 1982; O’Connor et al., 1990). Over the last several years, monoclonal antibodies have been used to identify neuron-specific membrane proteins in insects. Some of these proteins localize to subsets of neurons and are expressed in a stage- specific manner during neural development (Bastiani et al., 1987; Pate1 et al., 1987). Of particular interest are fasciclin I and II, which have been cloned and sequenced and are found in both grasshopper and Drosophila (Bastiani et al., 1987; Snow et al., 1988;Zinn et al., 1988; Harrelson and Goodman, 1988). Fasciclin I isa novel 70 kd surfaceglycoprotein (Zinn et al., 1988). In the grasshopper, it is expressed primar- ily on the commissural fascicles of the CNS and also by the Til neurons in the limb bud (Bastiani et al., 1987). Fasciclin II is a 95 kd glycoprotein that is a mem- ber of the immunoglobulin superfamily with a struc- tural similaritywith N-CAM (neural cell adhesion mol- ecule) (Harrelson and Goodman, 1988; Gren- ningloh et al., 1991). Fasciclin II is expressed primarily on the longitudinal fascicles of the CNS and also in the periphery in the grasshopper (Bastiani et al., 1987). Both fasciclin I and II mediate homophilic adhesion in a Drosophila S2 cell adhesion assay (Elkins et al., 1990a; Harrelson, 1991, Sot. Neurosci., abstract). Re- cently, we have used CALI to demonstrate that fas- ciclin I plays a role in the adhesion of the sister axons of the Til neurons during grasshopper limb bud de- velopment (Jay and Keshishian, 1990). CALI provides a means of studying the molecular events underlying neural development bythespecific laser-induced inactivation of proteins bound by mala- chite green-labeled antibodies in vivo (Jay, 1988; Jay and Keshishian, 1990). Specificity results from two properties: using a wavelength of laser light that is absorbed by malachite green, but not by cellular com- ponents; and covalently attaching the dye to an anti- body that binds to the protein of interest. This paper reports the development of micro-CAL1 in which a laser is focused through microscope optics to inacti- vate specific target proteins in single cells during em- bryonic development. Here, we use micro-CAL1 to demonstrate distinct roles for fasciclin I and II in Til neural development. Micro-CAL1 of fasciclin I results in defasciculation of the Til neurons similar to that