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Axonal Outgrowth Within the Abnormal Scaffold of Brain Tracts in a Zebrafish Mutant

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Axonal Outgrowth Within the Abnormal Scaffold of Brain Tracts in a Zebrafish Mutant Axonal Outgrowth within the Abnormal Scaffold of Brain Tracts in a Zebrafish Mutant Chetan K. Patel, Luis C. Rodriguez, and John Y. Kuwada* Department of Biology, University of Michigan, Ann Arbor, Michigan 481 09-1048 SUMMARY The role of specific axonal tracts for the guidance of tion of their pathway the axons they normally follow are growth cones was investigated by examining axonal out- not required for proper navigation. Second, approxi- growth within the abnormal brain tracts of zebrafish cy- mately half of the nuc PC growth cones follow aberrant cfups mutants. Normally, the earliest differentiating neu- longitudinal pathways after the first portion of their rons in the zebrafish brain establish a simple scaffold of pathway. This suggests that for the longitudinal portion axonal tracts. Later-developing axons follow cell-spe- of the pathway, specific growth cone /axon interactions cific pathways within this axonal scaffold. In cyclops em- are important for guiding growth cones. Third, although bryos, this scaffold is perturbed due to the deletion of approximately half of the nuc PC growth cones follow some ventromedial neurons that establish parts of the aberrant longitudinal pathways, the rest follow normal axonal scaffold and the development of an abnormal pathways despite the absence of the axons that they nor- crease in the brain. In these mutant embryos, the growth mally follow. This suggests that cues independent of cones projected by the neurons of the nucleus of the poste- these axons may be capable of guiding nuc PC growth rior commissure (nuc PC) are deprived of the two tracts cones as well. These results suggest that different guid- of axons that they sequentially follow to first extend ven- ance cues or combinations of cues guide specific growth trally, then posteriorly. These growth cones respond to cones along different portions of their pathway. o 1994 the abnormal scaffold in several interesting ways. First, John Wiley & Sons, Inc. nuc PC growth cones initially always extend ventrally as Keywords: growth cone, zebrafish, brain, mutant in wild-type embryos. This suggests that for the first por- INTRODUCTION lished by identifiable clusters of neurons (Fig. 1; Chitnis and Kuwada, 1990; Wilson et al., 1990). Growth cones reach their targets by following cell- Within the diencephalon and mesencephalon, this specific pathways in a variety of embryos (Good- axonal scaffold contains two longitudinal tracts, man and Shatz, 1993). In many cases growth the dorsolateral tract of the postoptic commissure cones accomplish this by interacting with cues in ( TPOC) and the ventromedial medial longitudinal their local environment to select appropriate path- fasciculus (MLF). These tracts are intersected by a ways. Recent experimental manipulations demon- commissure, the posterior commissure (PC), that strated that growth cones utilize specific cues to is located at the border of the forebrain and mid- select pathways in the early brain of zebrafish em- brain. The TPOC is established by neurons of the bryos as well (Chitnis and Kuwada, 199 1; Chitnis nucleus of the TPOC (nuc TPOC) located in the et al., 1992). The brain of early zebrafish embryos anterior diencephalon. These neurons extend contains a simple scaffold of tracts that are estab- axons posteriorly through the ventral diencepha- lon and tegmentum. The MLF is established by the posteriorly directed axons of the neurons of the nu- Received July 19, 1993; accepted November 17, 1993 Journal of Neurobiology, Vol. 25, No. 4, pp. 345-360 (1994) cleus of the MLF (nuc MLF). The nuc MLF is (0 1994 John Wiley & Sons, Inc. located ventromedially in the anterior tegmentum. CCC 0022-3034/94/040345- I6 The PC is established by the dorsally directed 345 346 Putel et ul. Although other trajectories are available at the intersection of tracts in the anterior tegmentum, M nuc PC growth cones always turn posteriorly onto ,<----_ and extend along the TPOC. To see if nuc PC growth cones choose this pathway by selectively bb fasciculating with TPOC axons, the TPOC axons 1 were surgically prevented from entering the mid- MLF brain (Chitnis and Kuwada, 1991; Chitnis et al., '. _-_r 1992). After this manipulation, 39% of the nuc PC 0 GABA positive cells growth cones followed aberrant pathways (usually o GABA & CON1 positive cells e GABA & TH positive cells the MLF). This signifies that cues associated with the TPOC axons normally play an important role Figure 1 A schematic side view of the wild-type zebra- in insuring nearly error-free pathfinding by these fish brain (27 h PF) showing the distribution of neurons growth cones. However, a significant proportion of immunoreactive for GABA, TH, and CON1; and the pathways taken by growth cones through the early scaf- nuc PC growth cones extended on the endfeet of fold of axonal tracts. Nuc PC growth cones extend ven- neuroepithelial cells along an apparently normal, trally along the PC and then caudally along the TPOC; dorsolateral longitudinal pathway despite the ab- Ep growth cones first extend ventrally to establish the sence of the TPOC axons. This suggests that cues DVDT and then anteriorly along the TPOC. Unless oth- found along a dorsolateral longitudinal strip of erwise stated, anterior is left and dorsal is up in all of the endfeet may also be capable of guiding nuc PC figures. The dashed circle represent the eye, and the dot- growth cones. Furthermore, even growth cones ted lines represent a fold or crease in the brain. Abbrevia- that followed aberrant pathways usually turned tions: T = telencephalon; D = diencephalon; M = mesen- and extended posteriorly through the tegmentum. cephalon; R = rhombencephalon; AC = anterior com- This suggests that directionality cues are not lim- missure; POC = postoptic commissure; PC = posterior ited to the TPOC axons and dorsolateral endfeet. commissure; TT = telencephalic tract: DVDT = dorso- We have investigated these issues further by ex- ventral diencephalic tract: TPOC = tract of the postoptic commissure; MLF = medial longitudinal fasciculus; nuc amining outgrowth by nuc PC growth cones in PC = nucleus of the posterior commissure; Ep = epiphy- mutant cyclops embryos (Hatta et al., 199 1 ). Cy- sial neurons; L = lateral cluster of posterior commissure clops embryos lack the floor plate in the midbrain, neurons; V = ventral cluster of posterior commissure hindbrain, and spinal cord, and have eyes that are neurons. fused on the ventral side due to deletion of the ven- tromedial portions of the brain (Hatta et al., 199 1, 1992a). In the diencephalon the deletion includes axons of two clusters of neurons located in the ven- ventral neurons and longitudinal axonal tracts. tral portion of the anterior tegmentum, the lateral Here we show that these abnormalities prevent the and ventral PC clusters. Within this scaffold the normal formation of the PC and TPOC, the tracts growth cones of the approximately three to four nuc PC growth cones normally follow. Despite the neurons in the nucleus of the PC (nuc PC) (Chitnis absence of the PC, nuc PC growth cones all extend and Kuwada, 1990) and of the two to three epiphy- ventrally to the anterior tegmentum. However, ap- sial (Wilson and Easter, 199 1 ) neurons follow cell- proximately half then followed aberrant pathways specific pathways. The epiphysial neurons are lo- while the rest followed an apparently normal path- cated just lateral to the base of the epiphysis. They way in the absence of the TPOC. Our results con- project growth cones that extend ventrally along firm our earlier findings. Furthermore, the results the diencephalon toward the TPOC to pioneer the suggest that different cues or combination of cues dorsoventral diencephalic tract (DVDT) . Once at guide specific growth cones along different por- the TPOC, epiphysial growth cones turn anteriorly tions of their pathway. to extend along the TPOC. The nuc PC is located in the dorsolateral midbrain just posterior to the PC. They project growth cones that fasciculate METHODS with the PC axons and follow them ventrally into the anterior tegmentum. Once at the anterior teg- Animals mentum, they turn posteriorly onto the TPOC to fasciculate with these axons and extend along them Zebrafish embryos were collected from a laboratory into the hindbrain. breeding colony. Embryos were raised at 28.5"C accord- Aberrant Axonal Outgrowth in Zebrajish 347 ing to the protocol described in Myers et al. ( 1986) and with differential interference contrast (DIC) optics, and were staged from the estimated time of fertilization. Ho- labeled by pressure ejection from a micropipette of a mozygous cyclops embryos were generated by crossing small amount of a 0.2% Dil ( 1,l '-dioctadecyl-3,3,3',3'- heterozygous parents and were identified by their cyclo- tetramethylindocarbocyanine perchlorate) dissolved in pean head phenotype (Hatta et al., 199 I ). N,N-dimethylformamide (Honig and Hume, 1986) into the nuc PC or the base of the epiphysis. Dil was allowed to spread for 10-14 h, and the labeled cells were marked lmmunocytochemistry with the brown dab reaction product by photooxidation (Maranto, 1982; Kuwada et al., 1990). The other axons A guinea pig polyclonal antibody against GABA (Eu- in the embryos were then labeled with the acetylated gene Tech), a mouse monoclonal antibody (MAb) a-tubulin antibody as described above, except a blue- against tyrosine hydroxylase (TH), a MAb against acety- violet dab product was produced by using chloronaphtol lated a-tubulin (Piperno and Fuller, 1985) that labels as the chromagen. Alternatively, other axons were many if not all axons in the zebrafish embryo (Chitnis marked with a blue dab reaction product by a heavy-me- and Kuwada, i990), and a mouse MAb (MAb CON1 ) tal intensification technique ( Adams, I98 1 ) following that recognizes a specific subset of early zebrafish neu- the acetylated a-tubulin antibody. In these embryos the rons (Bernhardt et al., 1990) were used to label embryos Dil-labeled growth cones and axons were brown and using the whole-mount procedure as previously de- could be readily distinguished from the other axons scribed (Kuwada et al., 1990).To detect the GABA anti- which were blue-violet or blue.
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