In vivo imaging of cell behaviors and F-actin reveals LIM-HD transcription factor regulation of peripheral versus central sensory axon development
© Andersen et al; licensee BioMed Central Ltd. 2011
Received: 28 February 2011
Accepted: 27 May 2011
Published: 27 May 2011
Development of specific neuronal morphology requires precise control over cell motility processes, including axon formation, outgrowth and branching. Dynamic remodeling of the filamentous actin (F-actin) cytoskeleton is critical for these processes; however, little is known about the mechanisms controlling motile axon behaviors and F-actin dynamics in vivo. Neuronal structure is specified in part by intrinsic transcription factor activity, yet the molecular and cellular steps between transcription and axon behavior are not well understood. Zebrafish Rohon-Beard (RB) sensory neurons have a unique morphology, with central axons that extend in the spinal cord and a peripheral axon that innervates the skin. LIM homeodomain (LIM-HD) transcription factor activity is required for formation of peripheral RB axons. To understand how neuronal morphogenesis is controlled in vivo and how LIM-HD transcription factor activity differentially regulates peripheral versus central axons, we used live imaging of axon behavior and F-actin distribution in vivo.
We used an F-actin biosensor containing the actin-binding domain of utrophin to characterize actin rearrangements during specific developmental processes in vivo, including axon initiation, consolidation and branching. We found that peripheral axons initiate from a specific cellular compartment and that F-actin accumulation and protrusive activity precede peripheral axon initiation. Moreover, disruption of LIM-HD transcriptional activity has different effects on the motility of peripheral versus central axons; it inhibits peripheral axon initiation, growth and branching, while increasing the growth rate of central axons. Our imaging revealed that LIM-HD transcription factor activity is not required for F-actin based protrusive activity or F-actin accumulation during peripheral axon initiation, but can affect positioning of F-actin accumulation and axon formation.
Our ability to image the dynamics of F-actin distribution during neuronal morphogenesis in vivo is unprecedented, and our experiments provide insight into the regulation of cell motility as neurons develop in the intact embryo. We identify specific motile cell behaviors affected by LIM-HD transcription factor activity and reveal how transcription factors differentially control the formation and growth of two axons from the same neuron.
dominant negative cofactor of LIM
green fluorescent protein
Tag red fluorescent protein
utrophin calponin homology
Neurons must develop characteristic and often complex morphologies to form functional circuits of the nervous system. Development of proper neuronal structure requires precise regulation of multiple cellular processes, including axon formation, outgrowth and branching [1–4]. Axon formation creates a distinct molecular and functional compartment of the neuron and the position of axon initiation is often critical for establishing neuronal polarity . Axons initiate and extend led by highly protrusive growth cones that sense environmental guidance cues. As growth cones advance, the axon shaft undergoes consolidation, a process that suppresses protrusive activity behind the growth cone and maintains axon polarity . Axon branching and/or the extension of multiple axons allow neurons to innervate multiple targets [2, 6]. The formation of axon branches in specific locations and prevention of branching at inappropriate sites requires local regulation of signals that promote or suppress protrusions. These processes are critical for neuronal development, yet the mechanisms that regulate axon formation, consolidation and branching in the complex in vivo environment remain poorly understood.
Precise regulation of filamentous actin (F-actin) dynamics is essential for the cell motility processes underlying neuronal morphogenesis [1, 4, 7, 8]. A necessary first step in axon formation is accumulation of F-actin at the initiation site, which was first shown in situ during axonogenesis in grasshopper sensory neurons . In addition, the organization of F-actin into filopodia is a key prerequisite for neurite formation in cultured mouse cortical neurons . In Caenorhabditis elegans, the specific site of F-actin accumulation and axon formation is determined by extracellular UNC-6/netrin, which locally activates a signaling pathway that controls the actin regulatory protein MIG10/lamellipodin [11, 12]. In mammalian in vitro models of neuronal polarization, actin regulation is also a critical convergence point for polarity signals (reviewed in [13, 14]). Nonetheless, our understanding of the mechanisms controlling the process of initial axon emergence from the cell body and the underlying actin rearrangements in vivo is far from complete.
During outgrowth, the polarized structure of axons must be established and maintained by consolidation along the axon shaft and restriction of F-actin-based protrusions to the growth cone [1, 4]. In vitro, consolidation is mediated in part by Rho-kinase inhibition of F-actin at the growth cone neck . Along the axon shaft, calpain proteolytic activity is required to inhibit protrusive activity by reducing levels of cortactin, an actin regulator . Thus, active suppression of F-actin protrusive activity along the axon shaft is required for proper axon extension and to prevent ectopic branching. To date, the process of axon consolidation in vivo has not been investigated.
The formation of axon branches also relies on dynamic rearrangements of the actin cytoskeleton [4, 6, 17–19]. Axon branching can occur by at least two distinct modes: growth cone bifurcation or interstitial branching off the axon shaft [2, 6, 17]. Although bifurcation is necessary for sensory axon target innervation in vivo [20, 21], the cytoskeletal rearrangements involved in this process are largely unknown. Interstitial branching appears to be the predominant branching mode in most neuron types studied thus far [2, 6, 22–24]. During interstitial branching in cultured cortical neurons, F-actin accumulates at branch initiation points and actin polymerization is required for branch formation [25, 26]. Branch promoting factors have been shown to act through derepression of calpain-mediated consolidation, thereby releasing the inhibition of F-actin polymerization . Still, little is known about the dynamics of axon branching or the underlying F-actin rearrangements in vivo. Studies using live imaging of F-actin in vitro have been instrumental to our understanding of axon formation, outgrowth and branching [1, 4, 15, 25, 26]. Here, we image F-actin during neuronal morphogenesis in intact embryos, where axons must integrate multiple signals that influence their motility.
Recent studies have brought to light the importance of intrinsic transcription factor regulation in neuronal morphogenesis . Transcription factor expression not only defines neuronal identity, but also can dictate axon trajectory choices downstream of identity decisions. For example, specific combinations of LIM homeodomain (LIM-HD) and homeobox domain (Hox) transcription factors are expressed in subsets of spinal motor neurons, and these determine the motor axon trajectories and innervation of specific muscle targets [28–31]. In retinal ganglion cells, cell-specific expression of Zic2, a zinc-finger transcription factor, and Isl2, a LIM-HD transcription factor, regulate the midline crossing choice of retinal ganglion cell axons at the optic chiasm [32, 33]. These transcription factors mediate axon pathway choices at least in part by regulating expression of Eph family axon guidance receptors [30, 34, 35]. In general, however, the mechanisms by which transcription controls axon motility remain largely unknown.
The zebrafish Rohon-Beard (RB) spinal sensory neurons provide an ideal model to investigate mechanisms controlling neuronal development in vivo because of their unique and stereotyped morphology [36, 37]. RB neurons extend two types of axons, centrals and peripherals, with distinct trajectories and behaviors . Each neuron extends ascending and descending central axons that grow longitudinally within the spinal cord to form the dorsal longitudinal fasciculus, and a single peripheral axon that exits the spinal cord and branches in the skin. The differential pathways of central and peripheral RB axons are determined in part by differing responses to extracellular signals . Furthermore, LIM-HD transcription factors of the Islet family and their cofactors, CLIMs, are essential intrinsic regulators of RB neuron morphology [39, 40]. Ubiquitous expression of a dominant negative CLIM (DN-CLIM) protein disrupts LIM-HD transcriptional activity and results in the reduction or elimination of RB peripheral axons without an apparent effect on the central axons or RB cell fate [39, 40]. Thus, these transcription factors are selectively required for peripheral axon formation and/or guidance. In this study, we analyze how LIM-HD transcription factors control cell motility processes and the F-actin cytoskeleton during neuronal morphogenesis in vivo.
To investigate the mechanisms controlling RB morphology and to understand how two axons from one cell are guided differently, we used live imaging of axon behaviors and F-actin distribution in vivo. We were able to visualize changes in F-actin distribution during dynamic processes such as axon consolidation and branching. We show that extensive F-actin protrusive activity precedes formation of the peripheral axon, and that F-actin concentrates at the site of peripheral axon initiation. In addition, we found that DN-CLIM differentially affects the motility of peripheral and central axons. In most cases, peripheral axons failed to initiate in DN-CLIM-expressing embryos; however, those that formed had significantly reduced growth rates and branching. In contrast, central axon growth rates were faster in DN-CLIM-expressing embryos. Finally, we found that disruption of LIM-HD activity did not affect F-actin-based protrusive activity or the ability of F-actin to accumulate at peripheral axon initiation sites, indicating that F-actin dynamics were unperturbed by DN-CLIM. However, DN-CLIM did influence the location of F-actin accumulation and axon initiation, suggesting LIM-HD activity may function in axon positioning.
Live imaging of individual RB neuron development
The RB neurons compose the primary sensory system in the trunk of anamniotic vertebrate embryos [36, 41]. RB cell bodies lie in bilateral rows in the dorsal spinal cord and begin extending central axons between approximately 16 and 17 hours post-fertilization (hpf) . Peripheral axons form approximately 2 hours after central axon initiation and continue to extend and branch over a period of several hours. To visualize the axon morphology of individual RB neurons, we used a transient mosaic cell labeling approach . We injected plasmid DNA encoding a membrane-targeted fluorophore (GFP-CAAX or TagRFP-CAAX) driven by a cis-regulatory element from the neurogenin1 gene (-3.1 ngn1)  (see Materials and methods). This method allows visualization of the entire neuron structure during all stages of development.
During their initial outgrowth (phase I), within approximately 25 μm from the cell body, the central axons are thick and display protrusive activity along the entire length of the axon shaft (Figure 1B,D). The central growth cones extend relatively slowly during this phase (mean phase I extension rate = 12.8 ± 2.1 μm/h). In the second phase of central axon outgrowth, when axons are between approximately 25 and 50 μm long, membrane protrusions along the proximal axons are reduced and the axons display a more polarized morphology, with consolidated axon shafts and distinct protrusive growth cones (Figure 1C,D; Additional file 1). This morphological transition is accompanied by an increase in extension rates (mean phase II rate = 23.1 ± 1.1 μm/h). During this phase, central growth cones come into contact with axons from neighboring RB neurons and grow along them to form the dorsal longitudinal fasciculus (Figure 1E,F). In addition, transient filopodial protrusions continue to form along the axon shaft during this phase, and these can make lateral contact with other central axons (Figure 1F), suggesting they could mediate recognition and fasciculation. In the third phase, after central axons have reached a length of 50 μm or greater, their growth cones extend more rapidly (mean phase III rate = 50.3 ± 2.1 μm/h). Although filopodial protrusions continue to form along the central axons in phase III, their frequency in general decreases in proportion to increasing central axon length (Figure 1D).
Formation of the peripheral axon is a particularly important step of RB development because it is the point at which discrete central and peripheral compartments with different axon trajectories and behaviors are established. The peripheral axon forms orthogonally to the central axons, exits the spinal cord, and grows into a very different environment than the central pathway. Peripheral axons initiate during phase II or III of central axon outgrowth (Figure 1D), and become established during phase III. Single cell labeling allowed us to determine the site of peripheral axon formation. Like the central axons, the peripheral axon forms at the basal edge of the RB neuron; however, the precise anterior-posterior site at which it forms is variable from cell to cell. In 40% of cells (n = 47), the peripheral axon emerged directly from the cell body, and in the other 60% it arose as a branch from one of the central axons. The branches did not form preferentially from either the ascending or descending central axon, but initiated from both axons in equal proportions. However, in the majority of neurons (83%), the peripheral axon initiation site was restricted to a region near the cell body. On average, the distance from the cell body center to the peripheral axon initiation site was 10.7 ± 0.67 μm (approximately one cell body diameter). Thus, it appears there is a specific compartment of the RB cell that is capable of generating a peripheral axon.
Imaging F-actin distribution during RB development
Motility comparison between control neurons expressing a fluorophore only versus neurons expressing the UtrCH F-actin reporter
Average central growth rate (μm/h)
Average peripheral growth rate (μm/h)
Average number of filopodia per hour
23.8 ± 1.4 (n = 8)
40.9 ± 2.7 (n = 19)
40.9 ± 2.4 (n = 8)
22.4 ± 1.8 (n = 8)
40.6 ± 2.5 (n = 7)
37.1 ± 2.3 (n = 9)
Imaging RB development in embryos with disrupted LIM-HD transcription factor activity
Previous studies examining LIM-HD regulation of RB development concluded that DN-CLIM did not affect RB central axons [39, 40]. However, our live imaging showed that central axon growth rates were in fact affected in DN-CLIM-expressing embryos. We discovered that during phase II of central axon extension, when peripheral axons normally initiate outgrowth, central axon growth rates are significantly faster in DN-CLIM-expressing embryos (Figure 5G). These results indicate that DN-CLIM has specific, opposite effects on the motility of peripheral versus central axons. Moreover, these data argue that the effects of DN-CLIM on peripheral axon outgrowth are not simply due to a developmental delay or to poor health of the neurons.
Imaging F-actin distribution during RB development in embryos with disrupted LIM-HD transcription factor activity
Axon behaviors and F-actin dynamics in wild-type RB neurons
In this study, we were able to directly visualize F-actin during axon development in vivo using the UtrCH biosensor. This reporter has been shown to bind selectively to F-actin without stabilizing it , and has revealed F-actin dynamics during multiple cell motility processes, including wound healing and cytokinesis in Xenopus embryos [49, 50], neural crest cell or neutrophil migration in zebrafish [51, 52], and growth cone turning in cultured neurons . Here we show that neural-specific expression of UtrCH provides excellent spatial-temporal resolution of F-actin during axon development in vivo. Moreover, the UtrCH probe did not disrupt actin-based neuronal motility when expressed mosaically under control of the -3.1ngn1 cis-regulatory element. Our experiments provide the first characterization of F-actin dynamics during axon consolidation, initiation, and branching in vivo.
Consolidation is essential for establishing and maintaining axon shape and polarity, yet how this process occurs in vivo is not understood. Live imaging of newly forming RB central axons revealed that these axons undergo a dynamic transition in their consolidation state during the first few hours of outgrowth. Our results suggest that consolidation and acquisition of polar axon morphology are not immediate processes but occur gradually as axons extend. Furthermore, our finding that transient F-actin-based filopodia continue to form along the axon shaft after many hours suggests RB axons do not become completely consolidated in the in vivo environment. This state likely reflects a fine balance between signals that maintain active consolidation and those that stimulate F-actin protrusions and branching. What are the mechanisms that control consolidation in vivo? In cultured neurons, consolidation is an active process, requiring Rho-kinase-mediated inhibition of actin polymerization at the growth cone neck , and calpain-mediated degradation of actin regulatory factors in the axon shaft . Moreover, calpain inhibition in adult mice induces excess dendritic branching in hippocampal neurons , supporting a role for this pathway in vivo. Although calpain activity can be induced by low protein kinase A levels in the axon shaft , the upstream signals that activate consolidation or that control protein kinase A levels are not known. Our results suggest that the stimuli triggering consolidation are not immediately active in vivo. RB central axons initially extend along neuroepithelial cells before contacting and fasciculating with each other . Perhaps axons must reach a critical length and/or engage sufficient adhesive contacts with neuroepithelial cells to activate consolidation signals. Alternatively, contact with other central axons may stimulate consolidation. Our RB model will be useful for future studies to define the molecular signals that regulate consolidation of pioneering axons in vivo.
Axon initiation is an important first step in neuronal morphogenesis, yet few studies have examined this process in vivo. Our observations that F-actin protrusive activity and accumulation precede RB peripheral axon emergence are consistent with previous findings that remodeling of the actin cytoskeleton has a key role in neurite initiation in situ and in vitro [9, 10]. The specific position of axon initiation also is critical for neuronal morphology and polarity in vivo. We found that although F-actin-based filopodial protrusions can occur all along the central axons, only those in a cellular compartment containing the basal cell body and proximal central axons typically lead to peripheral axon initiation, suggesting this compartment is uniquely competent to generate a peripheral axon. There are several potential mechanisms controlling this axon initiation site. For example, filopodia in the compartment near the cell body may be favored for microtubule invasion because of better access to the microtubule organizing center. Indeed, both the formation of F-actin-based filopodia and their invasion by microtubules are required for neurite initiation in cultured neurons [10, 54]. In addition, the inherent apical-basal organization of the spinal neuroepithelium likely influences RB polarity and may thus define the basal position of peripheral axon initiation. Extracellular signals also likely play a role in determining the axon initiation site. Our finding that somite boundary regions are favorable for peripheral axon exit from the spinal cord suggests they may be a localized source of growth-promoting cues. Extracellular UNC-6/netrin defines axon initiation position in C. elegans by directing asymmetric activation of an actin-regulatory protein, MIG-10/lamellipodin . However, little else is known about extracellular signals that determine axon positioning in any in vivo system. On the whole, the precise location of axon initiation likely involves coordinated activity of external signals that promote and position outgrowth as well as intrinsic regulatory mechanisms that lead to localized remodeling of the cytoskeleton.
In addition to initiating axons at the correct position, neurons must regulate the number of axons they extend. In a subset of our imaging experiments, we observed transient initiation of multiple peripheral neurites from the basal cell body or central axons, all but one of which retracted after the successful axon exited the spinal cord. One potential explanation for this behavior is that axons encounter a signal upon spinal cord exit that suppresses supernumerary peripheral axons. Spinal cord exit is an important transition in the peripheral axon pathway, marked by a dramatic change in growth cone size and behavior. As they exit, peripheral axons must grow through the basal lamina, a layer of extracellular matrix enriched in laminin, which is a well-known regulator of axon outgrowth and guidance (for example, [55–61]). In zebrafish bashful (bal)/laminin-α1 mutants, supernumerary axons initiate from ectopic positions in midbrain neurons, suggesting laminin could act to regulate axon number or polarity [60, 61]. Although peripheral RB axon trajectories were normal in bal embryos, their arbors appeared denser [60, 61], suggesting they could have supernumerary peripheral axons. However, individual neuron morphologies have not been examined in these mutants. The extracellular matrix has key roles in regulating cell motility in multiple cell types, and thus is likely to influence both axon number and axon positioning.
Our live imaging has also provided insight into mechanisms underlying axon branching in vivo. Axon branches can form by two modes: growth cone bifurcation or interstitial collateral formation from the axon shaft [2, 6, 17]. We found that one of the first steps in peripheral axon branching is sustained F-actin accumulation, which is consistent with previous F-actin imaging during interstitial axon branching in vitro . Several studies of other neuron types have shown that interstitial branching begins by formation of filopodia along the axon shaft (reviewed in ). Moreover, previous work showed that in cultured neurons filopodia are preceded by F-actin patches [15, 62, 63] that form in association with localized microdomains of phosphatidylinositol-3,4,5-triphosphate (generated by phosphoinositide 3-kinase) . After the formation of F-actin accumulations and filopodia, microtubules must invade the filopodia for branch maturation [6, 19]. Microtubule invasion requires local microtubule severing [64, 65], dynamic instability , and transport of short microtubules into the branch . Moreover, interactions between actin and microtubules are required for branch development [19, 26], suggesting localized F-actin accumulations have an important function in directing microtubule capture and branch formation.
Final branch morphology does not necessarily reflect how a branch formed. Thus, distinguishing between bifurcation versus interstitial branching mechanisms requires live imaging with high temporal resolution. Previous live imaging of axon branching in vivo in mammalian cortex , in zebrafish or Xenopus optic tectum [46, 47], or of zebrafish trigeminal sensory axons  showed that interstitial branching appears to be the predominant mode, although occasional growth cone splitting was observed in some studies [46, 67]. We found that both growth cone bifurcations and interstitial branches commonly occur in RB peripheral axons as they arborize in vivo. RB neurons may employ bifurcation to lay out their overall arbor territory, and interstitial branching to fully arborize and fill the territory. In addition, we found that the two branching modes occur simultaneously within one neuron. In contrast, DRG sensory neurons, which serve a function analogous to RBs, display bifurcation and interstitial branching at distinct stages of their outgrowth [20, 21], and recent evidence suggests the two branching modes may be controlled by different molecular mechanisms [6, 20]. It will be important to determine the extent to which these branching modes employ common versus distinct molecular mechanisms. Multiple extracellular cues have been identified that can promote axon branches in other systems (reviewed in [2, 6]). To date, the only branching factor identified that affects RB axons is Slit2, which can promote excess secondary branching of peripheral RB arbors when overexpressed . The ability to combine live imaging with molecular manipulation makes the zebrafish RB model ideal for future studies to determine mechanisms of axon branching and the molecular differences in bifurcation versus interstitial branching.
LIM-HD transcription factor activity differentially regulates RB axons
The importance of transcriptional regulation in axon development has only recently come to light, and little is known about the molecular and cellular steps between transcription and axon behavior. Previous studies implicated LIM-HD transcription factors in RB peripheral axon formation [39, 40]. Here we show that these transcription factors function in several cell motility processes during RB morphogenesis, and we show how they differentially affect peripheral versus central axon behavior. LIM-HD activity is required for the initiation and outgrowth of the peripheral axon but not the central axons. Although most RB neurons did not initiate a peripheral axon in DN-CLIM embryos, F-actin accumulations and filopodia still formed in these neurons, suggesting LIM-HD activity does not affect the actin remodeling underlying axon initiation. Instead, our results suggest that LIM-HD activity regulates a subsequent step in axon development, perhaps controlling the ability of microtubules to invade filopodia and/or mediating interactions between the microtubule and actin cytoskeleton. Furthermore, our finding that F-actin accumulations and initiating neurites can form in ectopic apical locations on the RB cell body in DN-CLIM embryos suggests that LIM-HD activity contributes to the basal positioning of peripheral axons. The fact that DN-CLIM specifically affects peripheral and not central axon initiation suggests that distinct mechanisms may direct initiation of these two axon types.
DN-CLIM affects not only peripheral axon initiation, but also secondary branching in the periphery. This finding suggests that LIM-HD transcription factor activity could control multiple processes that occur at different stages of RB development. Alternatively, axon initiation and branching may be variations of the same process and may share similar underlying mechanisms mediated by LIM-HD activity. Indeed, in many cases the peripheral axon forms as a branch from the central axon. Both axon initiation and branching require precise control over actin rearrangement and microtubule invasion into the developing branch [4–8, 26, 54]. Moreover, several extracellular signals, such as netrin [11, 25], are known to stimulate both processes in other systems. Axon branching and the underlying cytoskeletal changes are influenced both by branch promoting cues [2, 6, 18] and by suppressive signals that prevent ectopic branching [16, 69]. LIM-HD activity could potentially modulate axon responses to either branch-stimulating or branch-suppressing signals.
LIM-HD transcription factor activity also differentially affects the motility of RB axons during outgrowth. In contrast to peripheral axons, whose growth was slowed by DN-CLIM, central axon growth rates were increased when LIM-HD activity was disrupted. This result could indicate that LIM-HD activity has a specific function in suppressing central outgrowth. Alternatively, the change in central axon growth rate may result from a general increase in available cellular resources when a peripheral axon does not extend. Consistent with the former hypothesis, differential neurite outgrowth ability of cultured Xenopus spinal neurons was shown to be mediated by an active intracellular signaling mechanism, and not simply by limited cellular resources . Although we cannot distinguish between these possibilities, our results support the idea that an intrinsic transcriptional mechanism can selectively regulate the behavior of two axons from a single neuron.
How might LIM-HD transcription factors control the differential behavior of central versus peripheral axons? One possibility is that LIM-HD transcription factors regulate differential localization or trafficking of receptor or signaling molecules to one RB axon versus the other. Some evidence exists for molecular differences between central and peripheral RB axons. For instance, the cell surface glycoprotein TAG-1 (transiently expressed axonal glycoprotein 1)/Contactin-2 is required specifically for central axon extension, while extracellular Sema3D is repulsive to peripheral but not central axons . In Xenopus RB neurons, focal adhesion kinase, which regulates adhesions and F-actin dynamics, is required for peripheral but not central RB axon growth . To date, few downstream targets of LIM-HD transcription factors have been identified in zebrafish: plexinA4 (a semaphorin receptor) , dpysl3/crmp4 (a semaphorin signaling component) , and contactin-1 . Dpysl3 cooperates with Sema3D to regulate peripheral axon outgrowth , PlexinA4 plays a role in mediating Slit2-induced secondary branching of peripheral RB axons , and contactin-1 functions in regulating Xenopus RB axon fasciculation . These latter processes - branching and contact-mediated axon behaviors - represent key differences between central and peripheral axons, further supporting the idea that LIM-HD transcription factors control differential behavior of the axons. There are likely multiple downstream genes regulated by LIM-HD transcription factors that function together to control axon behavior. Indeed, inhibition of any one of the known target genes does not phenocopy the DN-CLIM effect, consistent with this idea. Future work to identify the essential downstream targets of LIM-HD transcription factors, and how they cooperate in RB neurons will be critical for understanding mechanisms of transcriptional regulation of neuronal morphogenesis.
Our live imaging experiments provide insight into the cell motility processes underlying neuronal morphogenesis in vivo. We characterize cell behaviors and the dynamics of F-actin distribution during axon initiation, consolidation and branching in the natural in vivo environment. Our results demonstrate that LIM-HD transcription factor activity differentially regulates the motile behavior of two axons from one neuron. In addition, we show that LIM-HD activity does not affect F-actin protrusive activity or the ability of F-actin to accumulate during axon initiation and branching, but may have a role in peripheral axon positioning. The zebrafish RB model will be ideal for future studies to investigate molecular mechanisms that control these processes in vivo.
Materials and methods
Adult zebrafish (Danio rerio) were maintained in a laboratory breeding colony on a 14:10 h light:dark cycle. Wild-type AB strain and transgenic Tg(-3.1ngn1:gfp) (provided by U Strähle)  or Tg(-3.1ngn1:gfp-caax) lines with labeled primary sensory neurons were used for experiments. Embryos were raised at 23 to 28.5°C and staged as described previously . Animals were handled in accordance with guidelines set forth by NIH and IACUC. Our animal use protocol was approved by the University of Wisconsin Animal Care and Use Committee.
DNA expression constructs were generated with the Multisite Gateway® Cloning System (Invitrogen, Carlsbad, CA, USA) using zebrafish-compatible Tol2 transposon vectors [76–78] (Tol2 kits provided by K Kwan, C-B Chien and N Lawson), as described .
To drive expression in Rohon-Beard neurons, a 3.1-kb cis-regulatory element from the zebrafish neurogenin1 gene (-3.1ngn1)  was subcloned from pCS2:ngn3.1-GFP (provided by U Strähle) into the SpeI/MslI sites of p5E-MCS (Tol2 kit). The resulting construct, p5E-ngn(-koz), lacks the kozak translational start site sequence, and was subsequently used to generate all expression vectors in this study.
For transgenesis and cell labeling, we used fluorophores GFP or TagRFP (Axxora LLC, San Diego, CA, USA) fused to a CAAX box prenylation sequence, which targets proteins to the plasma membrane. Tol2 kit middle entry vectors  were used to generate expression vectors. Two-way gateway recombination cloning using pTolDestR4-R2pA  generated RB-specific expression constructs pEXP-3.1ngn1:gfp-caax or pEXP-3.1ngn1:tagrfp-caax, referred to as GFP-CAAX and TagRFP-CAAX, respectively.
For F-actin labeling, the F-actin-binding calponin homology domain of utrophin with amino-terminal mCherry was amplified from pCS2+mCh-utrCH  (gift from B Burkel and W Bement) and cloned by Gateway recombination into pDONR221 (Invitrogen). The resulting middle entry vector (pME-mCherry-UtrCH) was subsequently used in a two-way gateway recombination reaction to generate pEXP-3.1ngn1:mCherry-UtrCH (referred to as mCh-UtrCH).
5'-Capped RNA was synthesized from template DNA in vitro using the mMessage mMachine kit (Ambion, Austin, TX, USA). Tol2 transposase RNA was generated using plasmid pCS2FA-transposase DNA (Tol2 kit), as described . DN-CLIM RNA was generated using plasmid pCS2+DN-CLIM DNA (gift from I Bach), as described .
A transgenic line, Tg(-3.1ngn1:gfp-caax), expressing membrane-targeted GFP in all RB neurons was generated by co-injecting 50 pg (pEXP-3.1ngn1:gfp-caax) plasmid DNA along with 25 pg of Tol2 transposase mRNA into wild-type AB embryos at the one-cell stage. Injected G0 embryos were screened by epifluorescence and raised to adulthood. F1 progeny were screened for stable transmission of the gfp-caax transgene and GFP-positive embryos were raised to establish the line. F2 or F3 embryos were used for experiments.
Cell and F-actin labeling by transient transgenesis
Membrane-targeted fluorophores and mCh-UtrCH were used to visualize cell motility and F-actin, respectively, in developing RB neurons. Injection of plasmid DNA at the one-cell stage results in transient mosaic expression. Embryos were microinjected with 10 to 25 pg of DNA encoding GFP-CAAX, TagRFP-CAAX or mCh-UtrCH in RB-specific expression vectors. Embryos containing individually labeled RB neurons were dechorionated and sorted under epifluorescence illumination using a Nikon AZ100 dissecting microscope equipped with a 4× objective (at 40× magnification). RB neurons in the central region of the trunk (somites 3 to 14) were selected for imaging.
LIM-HD activity was disrupted by ubiquitous expression of a dominant negative cofactor of LIM (DN-CLIM) . Approximately 150 pg of DN-CLIM mRNA was injected into wild-type or transgenic embryos at the one-cell stage. Embryos were sorted at approximately 16 hpf for DN-CLIM-associated eye and midbrain-hindbrain boundary phenotypes , and those with morphological defects were selected for further analysis. Efficacy of DN-CLIM was assessed after imaging by examining all RB neurons and confirming the peripheral RB axon outgrowth phenotype.
Fixed embryo imaging
To analyze mature RB morphology and the extent of peripheral axon branching, selected embryos with individually labeled RB neurons were raised to 24 hpf. Embryos were fixed in 4% paraformaldehyde overnight at 4°C and washed stepwise into 70% glycerol in phosphate-buffered saline. Embryos were whole mounted and imaged on an Olympus Fluoview1000, IX81 confocal microscope equipped with a 60× oil immersion objective (NA 1.35).
Live embryo imaging
Embryos were anesthetized with 0.02% 3-amino benzoic acid ethylester (tricaine) then mounted in 1% low melting point agarose in 10 mM HEPES-buffered E3 embryo medium, as described . Images were captured using an Olympus Fluoview1000, IX81 confocal microscope equipped with a 60× oil immersion objective (NA 1.35).
Z-stacks of 0.5- to 2-μm step sizes encompassing a total of approximately 25 to 30 μm into the trunk were taken of lateral or dorsal-lateral mounts to visualize the RB peripheral and central axon pathways. For time-lapse imaging, embryos ranged in age from 16 to 19 hpf at the beginning of the experiment and were imaged for durations of 2 to 8 hours at 28°C, with Z-stacks captured at 1 to 2 minute intervals.
Image analysis and quantifications
Images were processed and analyzed using Volocity software (Perkin Elmer, Waltham, MA, USA), and figures assembled with Adobe Photoshop (Adobe Systems, Inc., San Jose, CA, USA). Movies were processed and built with Volocity and ImageJ software, and are played at a rate of 4, 6 or 9 frames per second depending on the time intervals used during acquisition. In figures with time-lapse images, time is displayed relative to the first image in the series shown.
Axonal growth rates were calculated as distance over time, and are reported as μm/h. Distance was determined by measuring axon length in XYZ dimensions at two time points of axon extension and calculating their difference. Only axons that extended through the field of view for at least 20 minutes were measured. Central axon lengths were measured from their initiation site on the cell body to the growth cone tip. To compensate for drift during imaging, fixed embryonic features, such as RB cell body or somite boundary position, were used as positional landmarks. Peripheral axon lengths were measured from their initiation site (on the cell body or central axon) to the leading growth cone of the peripheral arbor. For peripheral axons, growth rates were measured starting at the time that the axon reaches the skin and grows ventrally while branching.
Peripheral branching was measured by counting the number of axon termini per axon arbor/neuron in mosaically labeled or transgenic embryos, and expressed as number of axon tips per neuron. The extent of peripheral RB axon branching was assessed in both fixed and live wild-type embryos, and exclusively in live DN-CLIM embryos. To remain consistent, only peripheral axon arbors that extended past the horizontal myoseptum (approximately 80 μm in the ventral direction) were quantified for extent of branching, generally between 21 and 24 hpf depending on the anterior-posterior axial position of the RB neuron within the trunk.
F-actin protrusive activity was measured by counting the number of F-actin-containing filopodia that formed along RB central axons during the time when a peripheral axon formed, or would be expected to form in the case of DN-CLIM-expressing embryos (phase II). Filopodia were defined as visible protrusions, labeled with mCh-UtrCH, with a length of at least 1.5 μm, and were counted over a 20-minute time window. Protrusive activity is reported as number of filopodia per hour.
Statistical and graphical analyses were performed using Microsoft Excel software. Axon growth rates, extent of axon branching, and protrusive activity are reported as mean ± standard error of the mean. Statistical significance was assessed using a two-tailed t-test.
This work was supported by NIH grant NS042228 (MCH). The confocal microscope was acquired with an NIH shared instrumentation grant (S10RR023717, PI Bill Bement, Department of Zoology, University of Wisconsin). We would like to thank Dustin Tryggestad and John Irwin for fish care, and Brennan Boettcher and Xuejiao Tian for technical assistance. We thank Bill Bement and Brian Burkel for the mCh-UtrCH actin probe, Ingolf Bach, Catherina Becker and Thomas Becker for the DN-CLIM construct, Kristen Kwan, Chi-Bin Chien and Nathan Lawson for Tol2 kit vectors, Uwe Strähle for the ngn1 promoter and transgenic fish.
- Dent EW, Gertler FB: Cytoskeletal dynamics and transport in growth cone motility and axon guidance. Neuron 2003, 40:209–227.PubMedView Article
- Gibson DA, Ma L: Developmental regulation of axon branching in the vertebrate nervous system. Development 2011, 138:183–195.PubMedView Article
- Lowery LA, Van Vactor D: The trip of the tip: understanding the growth cone machinery. Nat Rev Mol Cell Biol 2009, 10:332–343.PubMedView Article
- Luo L: Actin cytoskeleton regulation in neuronal morphogenesis and structural plasticity. Annu Rev Cell Dev Biol 2002, 18:601–635.PubMedView Article
- Polleux F, Snider W: Initiating and growing an axon. Cold Spring Harb Perspect Biol 2010, 2:a001925.PubMedView Article
- Schmidt H, Rathjen FG: Signalling mechanisms regulating axonal branching in vivo . Bioessays 2010, 32:977–985.PubMedView Article
- da Silva JS, Dotti CG: Breaking the neuronal sphere: regulation of the actin cytoskeleton in neuritogenesis. Nat Rev Neurosci 2002, 3:694–704.PubMedView Article
- Dent EW, Gupton SL, Gertler FB: The growth cone cytoskeleton in axon outgrowth and guidance. Cold Spring Harb Perspect Biol 2010, 3:pii a001800.
- Lefcort F, Bentley D: Organization of cytoskeletal elements and organelles preceding growth cone emergence from an identified neuron in situ. J Cell Biol 1989, 108:1737–1749.PubMedView Article
- Dent EW, Kwiatkowski AV, Mebane LM, Philippar U, Barzik M, Rubinson DA, Gupton S, Van Veen JE, Furman C, Zhang J, Alberts AS, Mori S, Gertler FB: Filopodia are required for cortical neurite initiation. Nat Cell Biol 2007, 9:1347–1359.PubMedView Article
- Adler CE, Fetter RD, Bargmann CI: UNC-6/Netrin induces neuronal asymmetry and defines the site of axon formation. Nat Neurosci 2006, 9:511–518.PubMedView Article
- Quinn CC, Wadsworth WG: Axon guidance: asymmetric signaling orients polarized outgrowth. Trends Cell Biol 2008, 18:597–603.PubMedView Article
- Tahirovic S, Bradke F: Neuronal polarity. Cold Spring Harb Perspect Biol 2009, 1:a001644.PubMedView Article
- Arimura N, Kaibuchi K: Neuronal polarity: from extracellular signals to intracellular mechanisms. Nat Rev Neurosci 2007, 8:194–205.PubMedView Article
- Loudon RP, Silver LD, Yee HF Jr, Gallo G: RhoA-kinase and myosin II are required for the maintenance of growth cone polarity and guidance by nerve growth factor. J Neurobiol 2006, 66:847–867.PubMedView Article
- Mingorance-Le Meur A, O'Connor TP: Neurite consolidation is an active process requiring constant repression of protrusive activity. EMBO J 2009, 28:248–260.PubMedView Article
- Acebes A, Ferrus A: Cellular and molecular features of axon collaterals and dendrites. Trends Neurosci 2000, 23:557–565.PubMedView Article
- Dent EW, Tang F, Kalil K: Axon guidance by growth cones and branches: common cytoskeletal and signaling mechanisms. Neuroscientist 2003, 9:343–353.PubMedView Article
- Gallo G: The cytoskeletal and signaling mechanisms of axon collateral branching. Dev Neurobiol 2011, 71:201–220.PubMedView Article
- Schmidt H, Stonkute A, Juttner R, Koesling D, Friebe A, Rathjen FG: C-type natriuretic peptide (CNP) is a bifurcation factor for sensory neurons. Proc Natl Acad Sci USA 2009, 106:16847–16852.PubMedView Article
- Zhao Z, Ma L: Regulation of axonal development by natriuretic peptide hormones. Proc Natl Acad Sci USA 2009, 106:18016–18021.PubMedView Article
- O'Leary DD, Terashima T: Cortical axons branch to multiple subcortical targets by interstitial axon budding: implications for target recognition and "waiting periods". Neuron 1988, 1:901–910.PubMedView Article
- Portera-Cailliau C, Weimer RM, De Paola V, Caroni P, Svoboda K: Diverse modes of axon elaboration in the developing neocortex. PLoS Biol 2005, 3:e272.PubMedView Article
- Szebenyi G, Callaway JL, Dent EW, Kalil K: Interstitial branches develop from active regions of the axon demarcated by the primary growth cone during pausing behaviors. J Neurosci 1998, 18:7930–7940.PubMed
- Dent EW, Barnes AM, Tang F, Kalil K: Netrin-1 and semaphorin 3A promote or inhibit cortical axon branching, respectively, by reorganization of the cytoskeleton. J Neurosci 2004, 24:3002–3012.PubMedView Article
- Dent EW, Kalil K: Axon branching requires interactions between dynamic microtubules and actin filaments. J Neurosci 2001, 21:9757–9769.PubMed
- Polleux F, Ince-Dunn G, Ghosh A: Transcriptional regulation of vertebrate axon guidance and synapse formation. Nat Rev Neurosci 2007, 8:331–340.PubMedView Article
- Dalla Torre di Sanguinetto SA, Dasen JS, Arber S: Transcriptional mechanisms controlling motor neuron diversity and connectivity. Curr Opin Neurobiol 2008, 18:36–43.PubMedView Article
- Dasen JS, Tice BC, Brenner-Morton S, Jessell TM: A Hox regulatory network establishes motor neuron pool identity and target-muscle connectivity. Cell 2005, 123:477–491.PubMedView Article
- Kania A, Jessell TM: Topographic motor projections in the limb imposed by LIM homeodomain protein regulation of ephrin-A:EphA interactions. Neuron 2003, 38:581–596.PubMedView Article
- Kania A, Johnson RL, Jessell TM: Coordinate roles for LIM homeobox genes in directing the dorsoventral trajectory of motor axons in the vertebrate limb. Cell 2000, 102:161–173.PubMedView Article
- Herrera E, Brown L, Aruga J, Rachel RA, Dolen G, Mikoshiba K, Brown S, Mason CA: Zic2 patterns binocular vision by specifying the uncrossed retinal projection. Cell 2003, 114:545–557.PubMedView Article
- Pak W, Hindges R, Lim YS, Pfaff SL, O'Leary DD: Magnitude of binocular vision controlled by islet-2 repression of a genetic program that specifies laterality of retinal axon pathfinding. Cell 2004, 119:567–578.PubMedView Article
- Garcia-Frigola C, Carreres MI, Vegar C, Mason C, Herrera E: Zic2 promotes axonal divergence at the optic chiasm midline by EphB1-dependent and -independent mechanisms. Development 2008, 135:1833–1841.PubMedView Article
- Lee R, Petros TJ, Mason CA: Zic2 regulates retinal ganglion cell axon avoidance of ephrinB2 through inducing expression of the guidance receptor EphB1. J Neurosci 2008, 28:5910–5919.PubMedView Article
- Bernhardt RR, Chitnis AB, Lindamer L, Kuwada JY: Identification of spinal neurons in the embryonic and larval zebrafish. J Comp Neurol 1990, 302:603–616.PubMedView Article
- Kuwada JY, Bernhardt RR, Nguyen N: Development of spinal neurons and tracts in the zebrafish embryo. J Comp Neurol 1990, 302:617–628.PubMedView Article
- Liu Y, Halloran MC: Central and peripheral axon branches from one neuron are guided differentially by Semaphorin3D and transient axonal glycoprotein-1. J Neurosci 2005, 25:10556–10563.PubMedView Article
- Becker T, Ostendorff HP, Bossenz M, Schluter A, Becker CG, Peirano RI, Bach I: Multiple functions of LIM domain-binding CLIM/NLI/Ldb cofactors during zebrafish development. Mech Dev 2002, 117:75–85.PubMedView Article
- Segawa H, Miyashita T, Hirate Y, Higashijima S, Chino N, Uyemura K, Kikuchi Y, Okamoto H: Functional repression of Islet-2 by disruption of complex with Ldb impairs peripheral axonal outgrowth in embryonic zebrafish. Neuron 2001, 30:423–436.PubMedView Article
- Clarke JD, Hayes BP, Hunt SP, Roberts A: Sensory physiology, anatomy and immunohistochemistry of Rohon-Beard neurones in embryos of Xenopus laevis . J Physiol 1984, 348:511–525.PubMed
- Andersen E, Asuri N, Clay M, Halloran M: Live imaging of cell motility and actin cytoskeleton of individual neurons and neural crest cells in zebrafish embryos. J Vis Exp 2010., 36: pii 1726
- Blader P, Plessy C, Strahle U: Multiple regulatory elements with spatially and temporally distinct activities control neurogenin1 expression in primary neurons of the zebrafish embryo. Mech Dev 2003, 120:211–218.PubMedView Article
- Alsina B, Vu T, Cohen-Cory S: Visualizing synapse formation in arborizing optic axons in vivo : dynamics and modulation by BDNF. Nat Neurosci 2001, 4:1093–1101.PubMedView Article
- Bastmeyer M, O'Leary DD: Dynamics of target recognition by interstitial axon branching along developing cortical axons. J Neurosci 1996, 16:1450–1459.PubMed
- Meyer MP, Smith SJ: Evidence from in vivo imaging that synaptogenesis guides the growth and branching of axonal arbors by two distinct mechanisms. J Neurosci 2006, 26:3604–3614.PubMedView Article
- Harris WA, Holt CE, Bonhoeffer F: Retinal axons with and without their somata, growing to and arborizing in the tectum of Xenopus embryos: a time-lapse video study of single fibres in vivo . Development 1987, 101:123–133.PubMed
- Burkel BM, von Dassow G, Bement WM: Versatile fluorescent probes for actin filaments based on the actin-binding domain of utrophin. Cell Motil Cytoskeleton 2007, 64:822–832.PubMedView Article
- Clark AG, Miller AL, Vaughan E, Yu HY, Penkert R, Bement WM: Integration of single and multicellular wound responses. Curr Biol 2009, 19:1389–1395.PubMedView Article
- Miller AL, Bement WM: Regulation of cytokinesis by Rho GTPase flux. Nat Cell Biol 2009, 11:71–77.PubMedView Article
- Berndt JD, Clay MR, Langenberg T, Halloran MC: Rho-kinase and myosin II affect dynamic neural crest cell behaviors during epithelial to mesenchymal transition in vivo . Dev Biol 2008, 324:236–244.PubMedView Article
- Yoo SK, Deng Q, Cavnar PJ, Wu YI, Hahn KM, Huttenlocher A: Differential regulation of protrusion and polarity by PI3K during neutrophil motility in live zebrafish. Dev Cell 2010, 18:226–236.PubMedView Article
- Marsick BM, Flynn KC, Santiago-Medina M, Bamburg JR, Letourneau PC: Activation of ADF/cofilin mediates attractive growth cone turning toward nerve growth factor and netrin-1. Dev Neurobiol 2010, 70:565–588.PubMedView Article
- Dehmelt L, Smart FM, Ozer RS, Halpain S: The role of microtubule-associated protein 2c in the reorganization of microtubules and lamellipodia during neurite initiation. J Neurosci 2003, 23:9479–9490.PubMed
- Adams DN, Kao EY, Hypolite CL, Distefano MD, Hu WS, Letourneau PC: Growth cones turn and migrate up an immobilized gradient of the laminin IKVAV peptide. J Neurobiol 2005, 62:134–147.PubMedView Article
- Bonner J, O'Connor TP: The permissive cue laminin is essential for growth cone turning in vivo . J Neurosci 2001, 21:9782–9791.PubMed
- Garcia-Alonso L, Fetter RD, Goodman CS: Genetic analysis of Laminin A in Drosophila : extracellular matrix containing laminin A is required for ocellar axon pathfinding. Development 1996, 122:2611–2621.PubMed
- Halfter W: The behavior of optic axons on substrate gradients of retinal basal lamina proteins and merosin. J Neurosci 1996, 16:4389–4401.PubMed
- Hopker VH, Shewan D, Tessier-Lavigne M, Poo M, Holt C: Growth-cone attraction to netrin-1 is converted to repulsion by laminin-1. Nature 1999, 401:69–73.PubMedView Article
- Paulus JD, Halloran MC: Zebrafish bashful/laminin-alpha 1 mutants exhibit multiple axon guidance defects. Dev Dyn 2006, 235:213–224.PubMedView Article
- Wolman MA, Sittaramane VK, Essner JJ, Yost HJ, Chandrasekhar A, Halloran MC: Transient axonal glycoprotein-1 (TAG-1) and laminin-alpha1 regulate dynamic growth cone behaviors and initial axon direction in vivo . Neural Dev 2008, 3:6.PubMedView Article
- Ketschek A, Gallo G: Nerve growth factor induces axonal filopodia through localized microdomains of phosphoinositide 3-kinase activity that drive the formation of cytoskeletal precursors to filopodia. J Neurosci 2010, 30:12185–12197.PubMedView Article
- Korobova F, Svitkina T: Arp2/3 complex is important for filopodia formation, growth cone motility, and neuritogenesis in neuronal cells. Mol Biol Cell 2008, 19:1561–1574.PubMedView Article
- Yu W, Ahmad FJ, Baas PW: Microtubule fragmentation and partitioning in the axon during collateral branch formation. J Neurosci 1994, 14:5872–5884.PubMed
- Yu W, Qiang L, Solowska JM, Karabay A, Korulu S, Baas PW: The microtubule-severing proteins spastin and katanin participate differently in the formation of axonal branches. Mol Biol Cell 2008, 19:1485–1498.PubMedView Article
- Gallo G, Letourneau PC: Different contributions of microtubule dynamics and transport to the growth of axons and collateral sprouts. J Neurosci 1999, 19:3860–3873.PubMed
- Sagasti A, Guido MR, Raible DW, Schier AF: Repulsive interactions shape the morphologies and functional arrangement of zebrafish peripheral sensory arbors. Curr Biol 2005, 15:804–814.PubMedView Article
- Miyashita T, Yeo SY, Hirate Y, Segawa H, Wada H, Little MH, Yamada T, Takahashi N, Okamoto H: PlexinA4 is necessary as a downstream target of Islet2 to mediate Slit signaling for promotion of sensory axon branching. Development 2004, 131:3705–3715.PubMedView Article
- Homma N, Takei Y, Tanaka Y, Nakata T, Terada S, Kikkawa M, Noda Y, Hirokawa N: Kinesin superfamily protein 2A (KIF2A) functions in suppression of collateral branch extension. Cell 2003, 114:229–239.PubMedView Article
- Zheng JQ, Zheng Z, Poo M: Long-range signaling in growing neurons after local elevation of cyclic AMP-dependent activity. J Cell Biol 1994, 127:1693–1701.PubMedView Article
- Robles E, Gomez TM: Focal adhesion kinase signaling at sites of integrin-mediated adhesion controls axon pathfinding. Nat Neurosci 2006, 9:1274–1283.PubMedView Article
- Tanaka H, Nojima Y, Shoji W, Sato M, Nakayama R, Ohshima T, Okamoto H: Islet1 selectively promotes peripheral axon outgrowth in Rohon-Beard primary sensory neurons. Dev Dyn 2011, 240:9–22.PubMedView Article
- Gimnopoulos D, Becker CG, Ostendorff HP, Bach I, Schachner M, Becker T: Expression of the zebrafish recognition molecule F3/F11/contactin in a subset of differentiating neurons is regulated by cofactors associated with LIM domains. Gene Expr Patterns 2002, 2:137–143.PubMedView Article
- Fujita N, Saito R, Watanabe K, Nagata S: An essential role of the neuronal cell adhesion molecule contactin in development of the Xenopus primary sensory system. Dev Biol 2000, 221:308–320.PubMedView Article
- Kimmel CB, Ballard WW, Kimmel SR, Ullmann B, Schilling TF: Stages of embryonic development of the zebrafish. Dev Dyn 1995, 203:253–310.PubMedView Article
- Kawakami K: Transgenesis and gene trap methods in zebrafish by using the Tol2 transposable element. Methods Cell Biol 2004, 77:201–222.PubMedView Article
- Kwan KM, Fujimoto E, Grabher C, Mangum BD, Hardy ME, Campbell DS, Parant JM, Yost HJ, Kanki JP, Chien CB: The Tol2kit: a multisite gateway-based construction kit for Tol2 transposon transgenesis constructs. Dev Dyn 2007, 236:3088–3099.PubMedView Article
- Villefranc JA, Amigo J, Lawson ND: Gateway compatible vectors for analysis of gene function in the zebrafish. Dev Dyn 2007, 236:3077–3087.PubMedView Article
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.