Michael W. Baker

RNAi of the receptor tyrosine phosphatase HmLAR2 in a single cell of an intact leech embryo leads to growth-cone collapse

Receptor protein tyrosine phosphatases (RPTPs) are important for growth-cone migration [1-5], but their specific roles have yet to be defined. Previously, we showed that the growth cones of the Comb cell, an embryonic cell in the leech, express high levels of an RPTP called HmLAR2 [6,7]. Here, we report the use of RNA interference (RNAi) to block expression of HmLAR2 in individual Comb cells in the developing embryo. HmLAR2 mRNA levels were reduced in the soma, processes and growth cones of Comb cells injected with double-stranded RNA (dsRNA) for HmLAR2, but no decrease was detected when control dsRNAs were injected. Consistent with this observation, the level of phosphotyrosine increased significantly in the growth cones of Comb cells injected with HmLAR2 dsRNA. Within 24 hours, the growth cones of treated cells showed a distinct collapsed phenotype, with sharp reductions in lamellipodial surface area and in numbers of filopodia. These experiments indicate a key role for LAR-like RPTPs in maintaining the integrity of the growth cone.

Possible role of the receptor protein tyrosine phosphatase HmLAR2 in interbranch repulsion in a leech embryonic cell

Accumulating evidence indicates that receptor protein tyrosine phosphatases (rPTPs) play major roles in growth cone migration. We have previously shown that the growth cones of the multiple parallel processes of an identified leech embryonic cell, the Comb cell (CC), express high levels of a leukocyte antigen-related (LAR)-like rPTP, HmLAR2. Embryonic injection of a polyclonal antibody to the receptors ectodomain resulted in reduced process outgrowth and in processes crossing over each other, a behavior that is seldom observed in normal or control animals. Here we present results of injecting a soluble Fc-HmLAR2 ectodomain fusion protein into embryos in order to bind the endogenous ligands of HmLAR2. Single injections of the Fc-chimeric protein into the developing embryo resulted, 12 to 24 h postinjection, in clear morphological abnormalities, ranging from abnormally directed CC processes and crossovers to apparent growth cone collapse. At later times, 2 to 5 days post injection, growth cones appeared to have recovered and processes had continued to extend, but effects of the earlier guidance errors remained, with the CCs displaying a relatively high incidence of proximal guidance errors. When injected into the germinal plate of developing embryos, the fusion protein was found to bind selectively to the processes of the CCs themselves, in contrast to control injections of Fc alone or closely related Fc-tagged proteins, which did not decorate the CCs. Double-labeling experiments revealed an early phase of Fc-HmLAR2 labeling (within 20 min after application), during which the growth cones and filopodia of the CC showed significant binding of the receptor ectodomain, and a later phase (1-2 h after injection), when most of the label was redistributed away from the growth cones and into the proximal processes of the CC. In culture, HmLAR2-transfected COS cells were found to selectively bind the Fc-recombinant protein, but not Fc-tagged proteins bearing other closely related receptor ectodomains, demonstrating that the HmLAR2 ectodomain is capable of interacting homophilically. Together, our observations demonstrate that the rPTP HmLAR2 is critically involved in CC process extension through its participation in the regulation of growth cone structure, migration, and navigation. Moreover, since our experiments also indicate that HmLAR2 can bind to itself, we hypothesize that HmLAR2 has a key role in the mechanism of mutual repulsion that maintains the parallel growth of adjacent CC projections.

In vivo imaging of growth cone and filopodial dynamics: Evidence for contact‐mediated retraction of filopodia leading to the tiling of sibling processes

In the leech embryo, the peripheral comb cell (CC) sends out many nonoverlapping, growth cone‐tipped processes that grow in parallel and serve as a scaffold for the migrating myocytes of the later‐developing oblique muscle layer. To explore how the parallel arrangement is generated we first examined the arrangement of CC cytoskeletal components by expressing a tubulin‐binding protein and actin, both tagged with fluorescent reporters. This revealed that the growth cones were compartmentalized into F‐actin‐rich filopodia and a microtubule‐rich central region. Time‐lapse analysis with a 2‐photon laser scanning microscope revealed that the growth cones of the CC are highly dynamic, undergoing rapid filopodial extension and retraction. Measurements of filopodial lifespan and length revealed that most filopodia at the leading edge of the growth cone achieved significantly longer lifespans and length than lateral filopodia. Furthermore, for the short‐lived lateral filopodia, apparent interaction with a neighboring process was found to be a significant predictor of their nearly immediate (within 2–4 minutes) retraction. When contact was experimentally prevented by ablating individual CCs, the filopodia from the growth cones of adjacent segmental neighbors were found to be significantly lengthened in the direction of the removed homolog. Treatment with low doses of cytochalasin D to disrupt F‐actin assembly led to filopodial retraction and growth cone collapse and resulted in the bifurcation of many CC processes, numerous crossover errors, and the loss of parallelism. These findings indicate the existence of a contact‐mediated repulsive interaction between processes of the CC. J. Comp. Neurol. 500:850–862, 2007.

Microtargeted gene silencing and ectopic expression in live embryos using biolistic delivery with a pneumatic capillary gun.

Modulating gene expression, either by transfection or by RNA-interference (RNAi), is a powerful means for studying the functions of essential genes during nervous system development and maintenance. Both approaches depend on the successful delivery of the corresponding nucleic acids into the targeted cells. Here we demonstrate the fast, accurate and highly localized biolistic delivery of plasmid-DNA and dsRNA into central neurons and muscle cells in internal layers of living leech embryos using carrier particles launched with a recently developed pneumatic capillary gun. This delivery method is used to perform RNAi to silence the expression of the axon guidance factor netrin in muscle cells and central neurons of leech embryos, and to induce ectopic expression of actin tagged with a fluorescent protein in both types of cells. We also show the biolistic transfection and staining in monolayer cultures of mammalian cells and carry out independent injection of two different lipophilic tracking dyes with a single capillary gun.

Ectopic Expression of Select Innexins in Individual Central Neurons Couples Them to Pre-Existing Neuronal or Glial Networks That Express the Same Innexin

Fifteen of the 21 innexin (Inx) genes (Hve-inx) found in the genome of the medicinal leech, Hirudo verbana, are expressed in the CNS (Kandarian et al., 2012). Two are expressed pan-neuronally, while the others are restricted in their expression to small numbers of cells, in some cases reflecting the membership of known networks of electrically coupled and dye-coupled neurons or glial cells. We report here that when Hve-inx genes characteristic of discrete coupled networks were expressed ectopically in neurons known not to express them, the experimental cells were found to become dye coupled with the other cells in that network. Hve-inx6 is normally expressed by only three neurons in each ganglion, which form strongly dye-coupled electrical connections with each other [Shortening-Coupling interneuron (S-CI) network] (Muller and Scott, 1981; Dykes and Macagno, 2006). But when Hve-inx6 was ectopically expressed in a variety of central embryonic neurons, those cells became dye coupled with the S-CI network. Similarly, Hve-inx2 is normally uniquely expressed by the ganglions large glial cells, but when it was ectopically expressed in different central neurons, they became dye coupled to the glial cells. In contrast, overexpression of the pan-neuronal Inx genes Hve-inx1 and Hve-inx14 did not yield any novel instances of dye coupling to pre-existent neuronal networks. These results reveal that expression of certain innexins is sufficient to couple individual neurons to pre-existing networks in the CNS. We propose that a primary determinant of selective neuronal connectivity and circuit formation in the leech is the surface expression of unique subsets of gap junctional proteins.

Expression of a dominant negative mutant innexin in identified neurons and glial cells reveals selective interactions among gap junctional proteins.

Neurons and glia of the medicinal leech CNS express different subsets of the 21 innexin genes encoded in its genome. We report here that the punctal distributions of fluorescently tagged innexin transgenes varies in a stereotypical pattern depending on the innexin expressed. Furthermore, whereas certain innexins colocalize extensively (INX1 and INX14), others do not (e.g., INX1 and INX2 or INX6). We then demonstrate that the mutation of a highly conserved proline residue in the second transmembrane domain of innexins creates a gap junction protein with dominant negative properties. Coexpressing the mutated INX1 gene with its wild type blocks the formation of fluorescent puncta and decouples the expressing neuron from its normal gap junction‐coupled network of cells. Similarly, expression of an INX2 mutant transgene (a glial cell innexin), blocks endogenous INX2 puncta and wild‐type transgene puncta, and decouples the glial cell from the other glial cells in the ganglion. We show in cell culture with dye‐uptake and plasma membrane labeling experiments that the mutant innexin transgene is not expressed on the cell membrane but instead appears to accumulate in the cells perinuclear region. Lastly, we use these mutant innexin transgenes to show that the INX1 mutant transgene blocks not only INX1 puncta formation, but also puncta of INX14, with which INX1 usually colocalizes. By contrast, the formation of INX6 puncta was unaffected by the INX1 mutant. Together, these experiments suggest that leech innexins can selectively interact with one another to form gap junction plaques, which are heterogeneously located in cellular arbors.

Association of LAR-like receptor protein tyrosine phosphatases with an enabled homolog in Hirudo medicinalis.

Receptor protein tyrosine phosphatases (RPTPs) are thought to play important functions in pathfinding and target recognition by growing neuronal processes. The leech RPTPs HmLAR1 and HmLAR2 are expressed selectively by central neurons, Comb cells, and peripheral muscle tissues in the Hirudo medicinalis embryo. To explore the functions of HmLARs, we have sought to determine their physiological substrates. We report here the cloning and embryonic expression of Lena, the leech homolog of Enabled, a cytosolic protein implicated in actin-based cell motility. Lena is expressed in embryonic central neurons and in the Comb cell. We present experimental evidences indicating that Lena associates selectively with the intracellular domain of HmLAR1 and HmLAR2. Additionally, RNA interference (RNAi) of HmLAR1 in intact leech embryos leads to the hyperphosphorylation of Lena. We propose, therefore, that Lena is an in vivo substrate of HmLAR1 in neurons and perhaps of HmLAR2 in the Comb cells.

Control of neuronal morphology and connectivity: emerging developmental roles for gap junctional proteins.

Recent evidence indicates that gap junction (GJ) proteins can play a critical role in controlling neuronal connectivity as well as cell morphology in the developing nervous system. GJ proteins may function analogously to cell adhesion molecules, mediating cellular recognition and selective neurite adhesion. Moreover, during synaptogenesis electrical synapses often herald the later establishment of chemical synapses, and thus may help facilitate activity‐dependent sculpting of synaptic terminals. Recent findings suggest that the morphology and connectivity of embryonic leech neurons are fundamentally organized by the type and perhaps location of the GJ proteins they express. For example, ectopic expression in embryonic leech neurons of certain innexins that define small GJ‐linked networks of cells leads to the novel coupling of the expressing cell into that network. Moreover, gap junctions appear to mediate interactions among homologous neurons that modulate process outgrowth and stability. We propose that the selective formation of GJs between developing neurons and perhaps glial cells in the CNS helps orchestrate not only cellular synaptic connectivity but also can have a pronounced effect on the arborization and morphology of those cells involved.

The receptor phosphatase HmLAR2 collaborates with focal adhesion proteins in filopodial tips to control growth cone morphology

Receptor protein tyrosine phosphatases (RPTPs) have been shown to play key roles in regulating axon guidance and synaptogenesis. HmLAR2, one of two closely related LAR-like RPTPs in the embryonic leech, is expressed in a few central neurons and in a unique segmentally-iterated peripheral cell, the comb cell (CC). Here we show that tagged HmLAR2-EGFP has a punctate pattern of expression in the growth cones of the CC, particularly at the tips of extending filopodia. Moreover, although expression of the wild-type EGFP-tagged receptor does not affect CC growth cone morphology, expression of a putative dominant-negative mutant of the receptor, CS-HmLAR2, leads to the enlargement of the growth cones, a shortening of filopodia, and errors in cellular tiling. RNAi of several candidate substrate signaling proteins, Lena (leech Ena/Vasp), beta-integrin and paxillin, but not beta-catenin, phenocopies particular aspects of the effects of HmLAR2 RNAi. For paxillin, which co-localizes with HmLAR2 at growth cone puncta, knock-down led to a reduction in the number of such puncta. Together, our data suggests that HmLAR2 regulates the morphology of the growth cone by controlling F-actin polymerization and focal adhesion complexes.

Gap Junction-Dependent Homolog Avoidance in the Developing CNS

Oppositely directed projections of some homologous neurons in the developing CNS of the medicinal leech (Hirudo verbana), such as the AP cells, undergo a form of contact-dependent homolog avoidance. Embryonic APs extend axons within the connective nerve toward adjacent ganglia, in which they meet and form gap junctions (GJs) with the oppositely directed axons of their segmental homologs, stop growing, and are later permanently retracted (Wolszon et al., 1994a,b). However, early deletion of an AP neuron leads to resumed growth and permanent maintenance of the projections of neighboring APs. Here we test the hypothesis that a GJ-based signaling mechanism is responsible for this instance of homolog avoidance. We demonstrate that selective knockdown of GJ gene Hve–inx1 expression in single embryonic APs, by expressing a short-hairpin interfering RNA, leads to continued growth of the projections of the cell toward, into, and beyond adjacent ganglia. Moreover, the projections of the APs in adjacent ganglia also resume growth, mimicking their responses to cell deletion. Continued growth was also observed when two different INX1 mutant transgenes that abolish dye coupling between APs were expressed. These include a mutant transgene that effectively downregulates all GJ plaques that include the INX1 protein and a closed channel INX1 mutant that retains the adhesive cellular binding characteristic of INX1 GJs but not the open channel pore function. Our results add GJ intercellular communication to the list of molecular signaling mechanisms that can act as mediators of growth-inhibiting cell–cell interactions that define the topography of neuronal arbors.