Mol. Cells 2021; 44(8): 549-556
Published online August 13, 2021
https://doi.org/10.14348/molcells.2021.0129
© The Korean Society for Molecular and Cellular Biology
Correspondence to : sjeong4@jbnu.ac.kr
This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/.
Decoding the molecular mechanisms underlying axon guidance is key to precise understanding of how complex neural circuits form during neural development. Although substantial progress has been made over the last three decades in identifying numerous axon guidance molecules and their functional roles, little is known about how these guidance molecules collaborate to steer growth cones to their correct targets. Recent studies in Drosophila point to the importance of the combinatorial action of guidance molecules, and further show that selective fasciculation and defasciculation at specific choice points serve as a fundamental strategy for motor axon guidance. Here, I discuss how attractive and repulsive guidance cues cooperate to ensure the recognition of specific choice points that are inextricably linked to selective fasciculation and defasciculation, and correct pathfinding decision-making.
Keywords axon guidance, Drosophila, guidance molecule, selective defasciculation, selective fasciculation
Precise connections of neurons with their targets during neural development are responsible for the normal physiological and behavioral patterns of animals (Dorskind and Kolodkin, 2021; Engle, 2010). Neurons generated during embryonic and postnatal development extend axons that navigate along distinct paths to find their appropriate synaptic targets (Chédotal and Richards, 2010; Kolodkin and Tessier-Lavigne, 2011). Axon pathfinding is controlled by the coordinated action of attractive and repulsive cues (Tessier-Lavigne and Goodman, 1996). These opposing guidance cues act at either short-range or long-range (Kolodkin and Tessier-Lavigne, 2011; Tessier-Lavigne and Goodman, 1996). In general, long-range guidance cue molecules travel a long distance to bind to their cognate receptors expressed on growth cones, and then mediate either attractive or repulsive axon guidance (Kolodkin and Tessier-Lavigne, 2011; Tessier-Lavigne and Goodman, 1996). In contrast, short-range guidance cue molecules largely regulate axon–axon, axon–cell, and axon–extracellular matrix interactions (Kolodkin and Tessier-Lavigne, 2011; Tessier-Lavigne and Goodman, 1996). The question then arises of how these different types of axon guidance cues are integrated to steer growth cones toward their synaptic targets.
A wide range of axon guidance molecules, including four major classes of guidance cues-semaphorins, slits, netrins, and ephrins, have been discovered, and their guidance functions appear to be evolutionarily conserved across the animal kingdom (Bashaw and Klein, 2010; Dickson, 2002). Slits found in
Embryonic muscle development begins at stage 12, and somatic muscle patterning and specification are established before stage 16 (approximately 13 h after egg laying) (Bate, 1990). A repeating and identical pattern of 30 somatic muscle fibers is observed in each abdominal hemisegment A2-A7 (Fig. 1; Bate, 1990). In each hemisegment, these muscle fibers are innervated in a cell-type specific manner by 36 motor neurons that are generated and reside in the ventral nerve cord (VNC) (Fig. 1; Landgraf and Thor, 2006; Landgraf et al., 1997; Van Vactor et al., 1993). How can an individual axon, which extends from the cell bodies of motor neurons, follow the correct path to reach specific target muscle(s)? One of the most promising strategies is the control of motor axon pathfinding by the combined action of selective fasciculation at the initial step, and selective defasciculation at specific choice points (Fig. 2; Raper and Mason, 2010; Tessier-Lavigne and Goodman, 1996; Wang and Marquardt, 2013). In fact, two major nerve fascicles, namely the intersegmental nerve (ISN) and the segmental nerve (SN), and the transverse nerve (TN) as a minor nerve fascicle are observed within the VNC when late stage 16 embryos are immunostained with anti-Fas II antibody (Fig. 1; Grenningloh et al., 1991; Jeong, 2017; Van Vactor et al., 1993). This indicates that selective fasciculation of motor axons occurs before exiting the VNC. In the periphery, sequential defasciculation of the ISN at specific choice points creates three nerve branches called the ISN, ISNb, and ISNd, while selective defasciculation of the SN produces the SNa and SNc (Fig. 1; Landgraf and Thor, 2006; Van Vactor et al., 1993). Additional and sequential defasciculation of motor axons along each nerve pathway is also required to innervate particular target muscles (Fig. 2B; Jeong, 2017; Landgraf and Thor, 2006; Van Vactor et al., 1993).
Selective fasciculation of motor axons during neural development is thought to require attractive interaction and specific adhesion among axons. Therefore, cell adhesion molecules (CAMs) that primarily mediate cell–cell adhesion function may contribute to the formation of motor nerve fascicles. One of the best candidate adhesion molecules for selective fasciculation is Fasciclin II (FasII), an immunoglobulin superfamily (IgSF) protein, since panneuronal overexpression of FasII resulted in a range of motor axon defasciculation defects due to hyperfasciculation (Figs. 3A and 3B; Lin and Goodman, 1994). In addition, loss-of-function studies showed that FasII is required to recognize a specific axon pathway through axon–axon attraction in the VNC, even though no motor axon pathfinding defects in the periphery are detected in
One intriguing question remains as to what classes of guidance molecules collaborate with CAMs to control selective axon fasciculation.
Interestingly, highly penetrant fusion bypass phenotypes of the ISNb were also found in
After five nerve branches that include ISN, ISNb, and SNa are formed in the periphery, each projects to its target muscle region, and then its bundled axons sequentially defasciculate at specific choice points to innervate target muscle fibers (Fig. 1). With respect to the selective defasciculation of motor axons for targeted muscle innervation, this review will for several reasons primarily focus on the ISNb motor axon guidance and its regulation. First, when stained with anti-FasII antibody, the visibility of the axonal projection pattern of the ISNb is greater than that of other nerve branches (Jeong, 2017; Van Vactor et al., 1993). In addition, the embryonic pattern of ventrolateral muscles 7, 6, 13, and 12, which are innervated by ISNb motor axons (Fig. 2A), is also well recognized under differential interference contrast (DIC) microscopy (Van Vactor et al., 1993). These patterns appear to be very helpful for identifying more subtle axon pathfinding defects. Second, no nonmuscle mesodermal cells, which could function as guidepost cells, have yet to be recovered in the peripheral ISNb pathway (Van Vactor et al., 1993). Therefore, axon–axon and axon–muscle interactions seem to play an important role in precise navigation of the ISNb motor axons. Third, it is likely that no pioneer axon is required for axon pathfinding and growth of the ISNb (Krueger et al., 1996; Van Vactor et al., 1993). This may suggest that each growth cone of the ISNb is able to differentially respond to guidance cues emanating from the surrounding, and also able to recognize its own target muscle(s) (Krueger et al., 1996). Fourth, loss-of-function studies have identified a relatively larger number of guidance molecules required for correct axon pathfinding in the ISNb pathway, compared to other nerve pathways (Arzan Zarin and Labrador, 2019). This could in part be ascribed to the absence of guidepost cell and pioneer axon in the ISNb (Krueger et al., 1996; Van Vactor et al., 1993). Finally, compared to other peripheral nerve branches, more severe and diverse guidance phenotypes seem to be observed in the ISNb (Desai et al., 1996; Fambrough and Goodman, 1996; Krueger et al., 1996; Sink et al., 2001; Van Vactor et al., 1993; Winberg et al., 1998a; Yu et al., 1998). Therefore, when one single gene or one copy of a gene is absent, subtle axon guidance errors could easily be detected. This is probably due to relatively low levels of genetic redundancy, which indicate that the individual guidance molecule retains distinct non-overlapping guidance functions.
The question then arises as to how motor axons can recognize defasciculation choice points. The recognition of specific choice points should be a prerequisite for the selective defasciculation of motor axons. Therefore, the fusion bypass phenotype observed in genetically muscle-ablated embryos indicates that muscle founder cells and muscle-derived cues seem to play an essential role in recognizing defasciculation choice points (Landgraf et al., 1999; Prokop et al., 1996). Moreover, the presence of a single founder cell in muscle-ablated embryos is sufficient to induce defasciculation of motor axons, further supporting this idea (Landgraf et al., 1999). The absence of both Dptp69D and Dptp99A results in highly penetrant fusion bypass phenotypes of the ISNb, which are like those of genetic muscle ablation (Fig. 3B; Desai et al., 1996; Landgraf et al., 1999). These findings may suggest that RPTP proteins expressed on motor axons both serve as signaling molecules required for the recognition of specific choice points, and seemingly the selective axon–axon defasciculation. In addition, Sema-1a/PlexA signaling may also be involved in the recognition of choice points due to the fusion bypass phenotypes, even though these phenotypes are occasionally observed in either
In addition to fusion bypass phenotypes, different types of pathfinding defects that include U-turn, split/detour, and split/stall phenotypes (Figs. 3E and 3F), were observed in
Highly penetrant bypass phenotypes of the ISNb were also observed in either
One of the most prominent mechanisms underlying motor axon guidance is the selective fasciculation and defasciculation that occurs at specific choice points. At least two criteria for selective fasciculation should be fulfilled. First, the individual growth cones of motor neurons born at different locations must be guided to meet each other or pioneer axon(s). Second, these axons associate to form a fascicle via axon–axon attraction. Selective defasciculation of motor axons preferentially requires the recognition of defasciculation choice points in response to muscle-derived guidance cues. Multiple muscle-derived guidance cues should not only induce selective axon–axon repulsion, but also mediate attractive axon guidance in a differential manner. Once motor axons selectively defasciculate at specific choice points, additional guidance receptor molecules mainly expressed on the growth cones are involved in making a correct pathfinding decision for targeted muscle innervation in response to muscle-derived guidance cues.
One unsolved question in neural development is how the complex and stereotyped patterns of neural circuits observed in many higher organisms can be shaped by a limited number of guidance molecules (Dickson, 2002). This could be explained by several mechanisms that include combinatorial codes of guidance molecules, differential regulation of guidance receptors and their ligands, and the reiterative use of a limited set of guidance molecules (Bonanomi and Pfaff, 2010; Dickson, 2002; Pasterkamp, 2012). In addition, I here propose that selective fasciculation and defasciculation contribute much to the formation of complex and precise neural circuits with a relatively small number of guidance molecules, based on the following reasons. First, selective fasciculation of growing follower axons ensures their correct pathfinding between fasciculation and defasciculation points, even in the absence of the signaling mechanisms necessary for axon pathfinding. In the
This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korea government (MIST) (2018R1A2B6008037), and research funds of Jeonbuk National University in 2019.
S.J. contributed to the manuscript preparation.
The author has no potential conflicts of interest to disclose.
Mol. Cells 2021; 44(8): 549-556
Published online August 31, 2021 https://doi.org/10.14348/molcells.2021.0129
Copyright © The Korean Society for Molecular and Cellular Biology.
Division of Life Sciences (Molecular Biology Major), Department of Bioactive Material Sciences, and Research Center of Bioactive Materials, Jeonbuk National University, Jeonju 54896, Korea
Correspondence to:sjeong4@jbnu.ac.kr
This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/.
Decoding the molecular mechanisms underlying axon guidance is key to precise understanding of how complex neural circuits form during neural development. Although substantial progress has been made over the last three decades in identifying numerous axon guidance molecules and their functional roles, little is known about how these guidance molecules collaborate to steer growth cones to their correct targets. Recent studies in Drosophila point to the importance of the combinatorial action of guidance molecules, and further show that selective fasciculation and defasciculation at specific choice points serve as a fundamental strategy for motor axon guidance. Here, I discuss how attractive and repulsive guidance cues cooperate to ensure the recognition of specific choice points that are inextricably linked to selective fasciculation and defasciculation, and correct pathfinding decision-making.
Keywords: axon guidance, Drosophila, guidance molecule, selective defasciculation, selective fasciculation
Precise connections of neurons with their targets during neural development are responsible for the normal physiological and behavioral patterns of animals (Dorskind and Kolodkin, 2021; Engle, 2010). Neurons generated during embryonic and postnatal development extend axons that navigate along distinct paths to find their appropriate synaptic targets (Chédotal and Richards, 2010; Kolodkin and Tessier-Lavigne, 2011). Axon pathfinding is controlled by the coordinated action of attractive and repulsive cues (Tessier-Lavigne and Goodman, 1996). These opposing guidance cues act at either short-range or long-range (Kolodkin and Tessier-Lavigne, 2011; Tessier-Lavigne and Goodman, 1996). In general, long-range guidance cue molecules travel a long distance to bind to their cognate receptors expressed on growth cones, and then mediate either attractive or repulsive axon guidance (Kolodkin and Tessier-Lavigne, 2011; Tessier-Lavigne and Goodman, 1996). In contrast, short-range guidance cue molecules largely regulate axon–axon, axon–cell, and axon–extracellular matrix interactions (Kolodkin and Tessier-Lavigne, 2011; Tessier-Lavigne and Goodman, 1996). The question then arises of how these different types of axon guidance cues are integrated to steer growth cones toward their synaptic targets.
A wide range of axon guidance molecules, including four major classes of guidance cues-semaphorins, slits, netrins, and ephrins, have been discovered, and their guidance functions appear to be evolutionarily conserved across the animal kingdom (Bashaw and Klein, 2010; Dickson, 2002). Slits found in
Embryonic muscle development begins at stage 12, and somatic muscle patterning and specification are established before stage 16 (approximately 13 h after egg laying) (Bate, 1990). A repeating and identical pattern of 30 somatic muscle fibers is observed in each abdominal hemisegment A2-A7 (Fig. 1; Bate, 1990). In each hemisegment, these muscle fibers are innervated in a cell-type specific manner by 36 motor neurons that are generated and reside in the ventral nerve cord (VNC) (Fig. 1; Landgraf and Thor, 2006; Landgraf et al., 1997; Van Vactor et al., 1993). How can an individual axon, which extends from the cell bodies of motor neurons, follow the correct path to reach specific target muscle(s)? One of the most promising strategies is the control of motor axon pathfinding by the combined action of selective fasciculation at the initial step, and selective defasciculation at specific choice points (Fig. 2; Raper and Mason, 2010; Tessier-Lavigne and Goodman, 1996; Wang and Marquardt, 2013). In fact, two major nerve fascicles, namely the intersegmental nerve (ISN) and the segmental nerve (SN), and the transverse nerve (TN) as a minor nerve fascicle are observed within the VNC when late stage 16 embryos are immunostained with anti-Fas II antibody (Fig. 1; Grenningloh et al., 1991; Jeong, 2017; Van Vactor et al., 1993). This indicates that selective fasciculation of motor axons occurs before exiting the VNC. In the periphery, sequential defasciculation of the ISN at specific choice points creates three nerve branches called the ISN, ISNb, and ISNd, while selective defasciculation of the SN produces the SNa and SNc (Fig. 1; Landgraf and Thor, 2006; Van Vactor et al., 1993). Additional and sequential defasciculation of motor axons along each nerve pathway is also required to innervate particular target muscles (Fig. 2B; Jeong, 2017; Landgraf and Thor, 2006; Van Vactor et al., 1993).
Selective fasciculation of motor axons during neural development is thought to require attractive interaction and specific adhesion among axons. Therefore, cell adhesion molecules (CAMs) that primarily mediate cell–cell adhesion function may contribute to the formation of motor nerve fascicles. One of the best candidate adhesion molecules for selective fasciculation is Fasciclin II (FasII), an immunoglobulin superfamily (IgSF) protein, since panneuronal overexpression of FasII resulted in a range of motor axon defasciculation defects due to hyperfasciculation (Figs. 3A and 3B; Lin and Goodman, 1994). In addition, loss-of-function studies showed that FasII is required to recognize a specific axon pathway through axon–axon attraction in the VNC, even though no motor axon pathfinding defects in the periphery are detected in
One intriguing question remains as to what classes of guidance molecules collaborate with CAMs to control selective axon fasciculation.
Interestingly, highly penetrant fusion bypass phenotypes of the ISNb were also found in
After five nerve branches that include ISN, ISNb, and SNa are formed in the periphery, each projects to its target muscle region, and then its bundled axons sequentially defasciculate at specific choice points to innervate target muscle fibers (Fig. 1). With respect to the selective defasciculation of motor axons for targeted muscle innervation, this review will for several reasons primarily focus on the ISNb motor axon guidance and its regulation. First, when stained with anti-FasII antibody, the visibility of the axonal projection pattern of the ISNb is greater than that of other nerve branches (Jeong, 2017; Van Vactor et al., 1993). In addition, the embryonic pattern of ventrolateral muscles 7, 6, 13, and 12, which are innervated by ISNb motor axons (Fig. 2A), is also well recognized under differential interference contrast (DIC) microscopy (Van Vactor et al., 1993). These patterns appear to be very helpful for identifying more subtle axon pathfinding defects. Second, no nonmuscle mesodermal cells, which could function as guidepost cells, have yet to be recovered in the peripheral ISNb pathway (Van Vactor et al., 1993). Therefore, axon–axon and axon–muscle interactions seem to play an important role in precise navigation of the ISNb motor axons. Third, it is likely that no pioneer axon is required for axon pathfinding and growth of the ISNb (Krueger et al., 1996; Van Vactor et al., 1993). This may suggest that each growth cone of the ISNb is able to differentially respond to guidance cues emanating from the surrounding, and also able to recognize its own target muscle(s) (Krueger et al., 1996). Fourth, loss-of-function studies have identified a relatively larger number of guidance molecules required for correct axon pathfinding in the ISNb pathway, compared to other nerve pathways (Arzan Zarin and Labrador, 2019). This could in part be ascribed to the absence of guidepost cell and pioneer axon in the ISNb (Krueger et al., 1996; Van Vactor et al., 1993). Finally, compared to other peripheral nerve branches, more severe and diverse guidance phenotypes seem to be observed in the ISNb (Desai et al., 1996; Fambrough and Goodman, 1996; Krueger et al., 1996; Sink et al., 2001; Van Vactor et al., 1993; Winberg et al., 1998a; Yu et al., 1998). Therefore, when one single gene or one copy of a gene is absent, subtle axon guidance errors could easily be detected. This is probably due to relatively low levels of genetic redundancy, which indicate that the individual guidance molecule retains distinct non-overlapping guidance functions.
The question then arises as to how motor axons can recognize defasciculation choice points. The recognition of specific choice points should be a prerequisite for the selective defasciculation of motor axons. Therefore, the fusion bypass phenotype observed in genetically muscle-ablated embryos indicates that muscle founder cells and muscle-derived cues seem to play an essential role in recognizing defasciculation choice points (Landgraf et al., 1999; Prokop et al., 1996). Moreover, the presence of a single founder cell in muscle-ablated embryos is sufficient to induce defasciculation of motor axons, further supporting this idea (Landgraf et al., 1999). The absence of both Dptp69D and Dptp99A results in highly penetrant fusion bypass phenotypes of the ISNb, which are like those of genetic muscle ablation (Fig. 3B; Desai et al., 1996; Landgraf et al., 1999). These findings may suggest that RPTP proteins expressed on motor axons both serve as signaling molecules required for the recognition of specific choice points, and seemingly the selective axon–axon defasciculation. In addition, Sema-1a/PlexA signaling may also be involved in the recognition of choice points due to the fusion bypass phenotypes, even though these phenotypes are occasionally observed in either
In addition to fusion bypass phenotypes, different types of pathfinding defects that include U-turn, split/detour, and split/stall phenotypes (Figs. 3E and 3F), were observed in
Highly penetrant bypass phenotypes of the ISNb were also observed in either
One of the most prominent mechanisms underlying motor axon guidance is the selective fasciculation and defasciculation that occurs at specific choice points. At least two criteria for selective fasciculation should be fulfilled. First, the individual growth cones of motor neurons born at different locations must be guided to meet each other or pioneer axon(s). Second, these axons associate to form a fascicle via axon–axon attraction. Selective defasciculation of motor axons preferentially requires the recognition of defasciculation choice points in response to muscle-derived guidance cues. Multiple muscle-derived guidance cues should not only induce selective axon–axon repulsion, but also mediate attractive axon guidance in a differential manner. Once motor axons selectively defasciculate at specific choice points, additional guidance receptor molecules mainly expressed on the growth cones are involved in making a correct pathfinding decision for targeted muscle innervation in response to muscle-derived guidance cues.
One unsolved question in neural development is how the complex and stereotyped patterns of neural circuits observed in many higher organisms can be shaped by a limited number of guidance molecules (Dickson, 2002). This could be explained by several mechanisms that include combinatorial codes of guidance molecules, differential regulation of guidance receptors and their ligands, and the reiterative use of a limited set of guidance molecules (Bonanomi and Pfaff, 2010; Dickson, 2002; Pasterkamp, 2012). In addition, I here propose that selective fasciculation and defasciculation contribute much to the formation of complex and precise neural circuits with a relatively small number of guidance molecules, based on the following reasons. First, selective fasciculation of growing follower axons ensures their correct pathfinding between fasciculation and defasciculation points, even in the absence of the signaling mechanisms necessary for axon pathfinding. In the
This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korea government (MIST) (2018R1A2B6008037), and research funds of Jeonbuk National University in 2019.
S.J. contributed to the manuscript preparation.
The author has no potential conflicts of interest to disclose.
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