Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount <em>In Situ</em> Hybridization in Larval Sea Lamprey

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several ...

Temple University School of Medicine

The large, conserved semaphorin gene family encodes axon guidance molecules in both invertebrates and vertebrates. The primitive vertebrate lamprey diverged near the time of vertebrate origins and is useful for understanding the gene duplication events that led to the increased complexity of the vertebrate genome. We characterized the sequence and expression pattern of semaphorins and their receptors genes in the sea lamprey, Petromyzon marinus. We uncovered two members of the semaphorin family in sea lamprey. The first encodes a diffusible class 3 type semaphorin protein that is most similar to the human and mouse Sema3F (71% amino acid identity). The second encodes a transmembrane class 4 type semaphorin that is most similar to mouse Sema4D and human Sema4G, with 38% amino acid identity within the Sema domain. We also identified in lamprey two members of the semaphorin receptor family, lamprey Plexin A1 and Plexin A2. Phylogenetic analysis indicates that lamprey Sema3 and Sema4 represent precursor genes existing prior to the origin of the vertebrate Sema3A-G and Sema4A-G subfamilies. Therefore, the gene duplication event that gave rise to those subfamilies must have occurred after the divergence of jawed vertebrates from jawless fish. These semaphorins and plexins are expressed in unique and dynamic patterns in lamprey spinal cord and brain during development.

Semaphorins and their receptors in lamprey CNS: Cloning, phylogenetic analysis, and developmental changes during metamorphosis.

To explore the role of axon guidance molecules during regeneration in the lamprey spinal cord, we examined the expression of mRNAs for semaphorin 3 (Sema3), semaphorin 4 (Sema4), and netrin during regeneration by in situ hybridization. Control lampreys contained netrin-expressing neurons along the length of the spinal cord. After spinal transection, netrin expression was downregulated in neurons close (500 mum to 10 mm) to the transection at 2 and 4 weeks. A high level of Sema4 expression was found in the neurons of the gray matter and occasionally in the dorsal and the edge cells. Fourteen days after spinal cord transection Sema4 mRNA expression was absent from dorsal and edge cells but was still present in neurons of the gray matter. At 30 days the expression had declined to some extent in neurons and was absent in dorsal and edge cells. In control animals, Sema3 was expressed in neurons of the gray matter and in dorsal and edge cells. Two weeks after transection, Sema3 expression was upregulated near the lesion, but absent in dorsal cells. By 4 weeks a few neurons expressed Sema3 at 20 mm caudal to the transection but no expression was detected 1 mm from the transection. Isolectin I-B(4) labeling for microglia/macrophages showed that the number of Sema3-expressing microglia/macrophages increased dramatically at the injury site over time. The downregulation of netrin and upregulation of Sema3 near the transection suggests a possible role of netrin and semaphorins in restricting axonal regeneration in the injured spinal cord.

Differential expression of class 3 and 4 semaphorins and netrin in the lamprey spinal cord during regeneration.

The sea lamprey recovers normal-appearing locomotion after spinal cord transection and its spinal axons regenerate selectively in their correct paths. However, among identified reticulospinal neurons some are consistently bad regenerators and only about 50% of severed reticulospinal axons regenerate through the site of injury. We previously suggested (Shifman, M. I., and Selzer, M. E., 2000a. Expression of netrin receptor UNC-5 in lamprey brain; modulation by spinal cord transection. Neurorehabilitation and Neural Repair 14, 49-58; Shifman, M. I., and Selzer, M. E., 2000b. In situ hybridization in wholemounted lamprey spinal cord: localization of netrin mRNA expression. Journal of Neuroscience Methods 104, 19-25) that selective chemorepulsion might explain why some neurons are bad regenerators and others not. To explore the role of additional chemorepulsive axonal guidance molecules during regeneration, we examined the expression of the repulsive guidance molecule (RGM) and its receptor neogenin by in situ hybridization and quantitative PCR. RGM mRNA was expressed in the spinal cord, primarily in neurons of the lateral gray matter and in dorsal cells. Following spinal cord transection, RGM message was downregulated in neurons close (within 10 mm) to the transection at 2 and 4 weeks, although it was upregulated in reactive microglia at 2 weeks post-transection. Neogenin mRNA expression was unchanged in the brainstem after spinal cord transection, and among the identified reticulospinal neurons, was detected only in "bad regenerators", neurons that are known to regenerate well never expressed neogenin. The downregulation of RGM expression in neurons near the transection may increase the probability that regenerating axons will regenerate through the site of injury and entered caudal spinal cord.

Expression of the repulsive guidance molecule RGM and its receptor neogenin after spinal cord injury in sea lamprey.

UNC5 receptors mediate repulsive signaling of netrin on neurons. Although only one UNC5 receptor has been identified in invertebrates, four members of the UNC5 family have been identified in gnathostomes. Lampreys, together with mixynes, belong to the oldest branch of extant vertebrates, and their phylogenetic position near to the vertebrate root makes them an interesting model for understanding molecular evolution. Here, we cloned three sea lamprey UNC5 (UNC5L) receptors, and phylogenetic analyses indicated that the first two duplications of the ancestral UNC5 gene occurred before the separation of jawless and jawed vertebrates. UNC5 receptors play important roles during early development, but expression studies have also suggested that UNC5 receptors play roles in the mature nervous system. Here, we report the expression of the different UNC5L receptor transcripts in identified reticulospinal neurons of mature larval or adult sea lampreys detected by in situ hybridization in wholemounted brain preparations. In addition, an extensive expression of the UNC5 receptors was also observed in most brain regions of the adult lamprey. An increase in the types of identifiable reticulospinal neurons expressing the UNC5L receptors was observed in adults compared with larvae. Expression of UNC5 receptors at late developmental stages appears to be a shared characteristic of lampreys and mammals. In larvae, expression of UNC5L receptors was observed in reticulospinal neurons that when axotomized are known to be "bad regenerators." Results in lampreys and mammals suggest that the UNC5-Netrin axonal guidance system may play a role in limiting axonal regeneration after spinal cord injury.

The sea lamprey UNC5 receptors: cDNA cloning, phylogenetic analysis and expression in reticulospinal neurons at larval and adult stages of development.

Apoptosis is a major feature in neural development and important in traumatic diseases. The presence of active caspases is a widely accepted marker of apoptosis. We report here the development of a method to study neuronal apoptotic death in whole-mounted brain preparations using fluorochrome-labeled inhibitors of caspases (FLICA). As a model we used axotomy-induced retrograde neuronal death in the CNS of larval sea lampreys. Once inside the cell, the FLICA reagents bind covalently to active caspases causing apoptotic cells to fluoresce, whereas nonapoptotic cells remain unstained. The fluorescent probe, the poly caspase inhibitor FAM-VAD-FMK, was applied to whole-mounted brain preparations of larval sea lampreys 2 weeks after a complete spinal cord (SC) transection. Specific labeling occurred only in identifiable spinal-projecting neurons of the brainstem previously shown to undergo apoptotic neuronal death at later times after SC transection. These neurons also exhibited intense labeling 2 weeks after a complete SC transection when a specific caspase-8 inhibitor (FAM-LETD-FMK) served as the probe. In this study we show that FLICA reagents can be used to detect specific activated caspases in identified neurons of the whole-mounted lamprey brain. Our results suggest that axotomy may cause neuronal apoptosis by activation of the extrinsic apoptotic pathway.

Michael I. Shifman

Shriners Hospitals Pediatric Research Center (Center for Neural Repair and Rehabilitation)

Temple University School of Medicine

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

Michael I. Shifman

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Temple University School of Medicine

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

Shriners Hospitals Pediatric Research Center (Center for Neural Repair and Rehabilitation)