InteractiveFly: GeneBrief

dendritic arbor reduction 1: Biological Overview | References

Dendrites and axons are two major neuronal compartments with differences that are critical for neuronal functions. To learn about the differential regulation of dendritic and axonal development, a genetic screen was conducted in Drosophila and the dendritic arbor reduction 1 (dar1) mutants were isolated that display defects in dendritic but not axonal growth. The dar1 gene encodes a novel transcription regulator in the Kruppel-like factor family. Neurons lacking dar1 function have severely reduced growth of microtubule- but not F-actin-based dendritic branches. In contrast, overexpression of Dar1 dramatically increased the growth of microtubule-based dendritic branches. These results suggest that Dar1 promotes dendrite growth in part by suppressing the expression of the microtubule-severing protein Spastin. This study thus uncovers a novel transcriptional program for microtubule regulation that preferentially controls dendrite growth (Ye, 2011).

Dendritic and axonal compartments have distinct morphological features that are fundamental to neuronal functions. During development, one neurite of the postmitotic neuron is specified as the axon, and then the remaining neurites are specified as dendrites. Subsequently, the developing dendrites and axons follow separate paths to form two compartments that are distinct in structure and function. The past several years have seen substantial progress in the elucidation of the molecular mechanisms underlying axon specification (Wiggin, 2005; Tahirovic, 2009). However, how the dendritic and axonal compartments of a neuron diverge in their development after the postmitotic neuron is polarized remains mostly unknown (Ye, 2011).

Both the microtubule (MT) cytoskeleton and the intracellular membrane system have been proposed to be important for the differential development of dendrites and axons. Microtubules are oriented differently in dendrites and axons. In the axons, microtubules are oriented uniformly with their plus-ends pointing distally, whereas there are microtubules with either orientation in dendrites. As microtubules are essential both for transporting molecules and organelles and for extending neurites, such a differential organization between dendrites and axons is likely to have profound impact on separating dendrite and axon development (Ye, 2011).

The secretory and endocytic pathways of the intracellular membrane system also contribute to the distinction between dendrite and axon growth. The rate of endocytosis in dendrites is much higher than that in the axon (Ye, 2007). This leads to a greater demand of membrane supply since the vast majority of the endocytosed plasma membrane are returned to the soma. Indeed, when the secretory pathway function is compromised as a result of mutations in key regulators such as Sar1, the dendritic growth is preferentially reduced (Ye, 2007). Furthermore, dendritic but not axonal growth is altered by disruption of PKD (protein kinase D) function, which regulates membrane protein exit from trans-Golgi network (Yeaman, 2004), via overexpression of a dominant-negative form. It thus seems likely that the membrane and membrane proteins required for dendritic or axonal growth are sorted in the trans-Golgi network (Ye, 2011).

How the microtubule cytoskeleton and the intracellular membrane system are regulated to contribute to the differential development of dendrites and axons is unknown. Through a genetic screen in Drosophila, the dendritic arbor reduction 1 (dar1) complementation group, which displays specific defects in dendrite development (Ye, 2007), was isolated. The dar1 gene encodes a novel member of the Krüppel-like factor (KLF) family, which regulates gene transcription. This study show that dar1 is a critical regulator of dendritic microtubule cytoskeleton. The results also suggest that Dar1 promotes dendrite growth in part by suppressing the expression of the microtubule-severing protein Spastin. These findings lend support to the notion that dendrite and axon development are controlled by partly non-overlapping genetic programs (Ye, 2011).

The da neurons in Drosophila PNS offer a wealth of features for analyzing different aspects of dendrite development, including different compositions of microtubule and actin cytoskeleton in different types of dendritic branches. The class III da neurons have characteristic dendritic filopodia (also termed dendritic spikes), which are distributed along major dendrites and are usually straight and devoid of additional branching. These dendritic spikes are enriched with filamentous actin (F-actin) and are essentially free of microtubules. In contrast, the major dendrites of the same neurons are enriched with microtubules and contain F-actin at a level much lower than that in the dendritic spikes. The separation of F-actin- and microtubule-based dendritic branches allowed investigation of the effect of dar1 mutations on these two types of cytoskeleton (Ye, 2011).

Although the major dendrites were dramatically reduced by ~75% in the class III da neurons mutant for the dar1 gene, the density of dendritic spikes was much less affected. This raised the possibility that dar1 preferentially regulates microtubule cytoskeleton. To test this hypothesis, Rac1 was overexpressed in class I da neurons mutant for dar1. Rac1 is a small GTPase that regulates actin cytoskeleton. Overexpression of Rac1 (OE Rac1) in many neuron types, including the da neurons, leads to hyperbranching of dendrites. The dar1 mutations did not block the branching activity of Rac1, suggesting that Dar1 is not required for regulating actin-cytoskeleton (Ye, 2011).

To further test whether Dar1 is capable of promoting MT growth, transgenic lines were generated to express Dar1 in da neurons. Overexpressing Dar1 (OE Dar1) with the driver GAL4^4-77 caused dramatic overgrowth of dendrites in the class IV da neurons in early third-instar larvae. Control neurons had 350.2 ± 14.0 branch points, excluding the dendrites around the segmental borders where dendrites of different neurons intermingle. In contrast, neurons overexpressing Dar1 had 930.7 ± 17.3 branch points. Similarly, the total dendritic length of control neurons, excluding the dendrites around the segmental borders, was 9159.0 ± 217.0 microm, whereas that of neurons overexpressing Dar1 was 18,044.0 ± 278.8 microm. These results suggest that Dar1 is not only necessary but also sufficient for promoting dendritic growth. Sholl analysis (Sholl, D.A., 1953. Dendritic organization in the neurons of the visual and motor cortices of the cat. J. Anat. 87: 387-406) showed that the increase in branch number and length took place throughout the dendritic fields rather than being restricted to particular regions. No change in the size of dendritic fields or dendritic tiling was observed (Ye, 2011).

The Flip-out technique was used to study the effects of overexpressing Dar1 on class IV da neuron axons. In contrast to its effects on dendrites, overexpression of Dar1 did not significantly affect axon growth. The axon terminal length of wild-type class IV neurons was 68.4 ± 4.2 microm, and that of class IV neuron overexpressing Dar1 was 77.7 ± 5.7 microm in dar1 mutant neurons (Ye, 2011).

Overexpressing Dar1 in class I da neurons also dramatically increased dendrite growth. Strikingly, when Dar1 was overexpressed in the class III da neurons, an increased number of long curvy terminal branches was observed that are reminiscent of microtubule-containing branches (Ye, 2011).

To directly visualize microtubule and F-actin in the class III da neurons overexpressing Dar1, use was made of tubulin-GFP and the F-actin marker GMA. GMA is a chimeric protein with the actin binding domain of Drosophila moesin fused to the C terminus of GFP and is enriched at the dendritic spikes of class III da neurons. Overexpressing Dar1 in class III da neurons led to the presence of MT in the ectopic terminal branches. No enrichment of F-actin was observed in these branches, suggesting overexpression of Dar1 promoted the growth of microtubule-containing major dendrites but not the F-actin-enriched filopodia (Ye, 2011).

Together, these results suggest that dar1 preferentially regulates microtubule cytoskeleton to mediate its specific control of dendrite growth (Ye, 2011).

The microtubule-severing protein Spastin has been found to regulate dendrite development in Drosophila da neurons. Because Dar1 preferentially regulates microtubule-based dendritic growth, it was asked whether Dar1 could regulate Spastin expression. Currently, no antibody against Drosophila Spastin provides sufficient sensitivity to detect endogenous Spastin protein level. Therefore determined the levels of Spastin transcripts was determined in dar1 mutant neurons by quantitative real-time PCR (qPCR). The qPCR technique was preferred to other techniques, such as in situ hybridization, for both consistency in quantification and sensitivity. Total RNA was extracted from purified da neurons of dar1 mutant embryos as well as from those of wild-type embryos. Real-time PCR was used to compare the amounts of Spastin transcripts between wild-type and dar1 mutant da neurons. The levels of Spastin transcripts were significantly elevated in dar1 mutant neurons. Transcripts from dar1 mutant neurons on average took 1.44 less PCR cycles to reach the threshold cycle (Ct) for amplifying Spastin than those from wild-type neurons, suggesting a 3.2-fold increase in Spastin transcript level in dar1 mutant neurons. In contrast, there is no difference in the Ct for amplifying the chromatin modifying protein 1 (Chmp1) between wild-type and dar1 mutant neurons (Ye, 2011).

Consistent with the reduced dendrite phenotype in dar1 mutants, upregulation of Spastin with the EP insertion T32 (Spa^T32), which is known to cause a modest overexpression, led to a dramatic reduction in both total dendritic length and branch number. Whereas the total dendritic length in wild-type class IV neurons was 17,789 ± 444.5 microm, it was reduced to 10,582 ± 580.3 microm in neurons overexpressing Spa^T32. Similarly, the number of branch points was reduced from 767.9 ± 22.8 in wild-type to 403.3 ± 43.5 in Spa^T32-overexpressing neurons (Ye, 2011).

These results together suggest that Dar1 restricts the expression of Spastin either directly or through a transcription repressor, allowing dendrite growth. Different from dar1 mutants, Spastin upregulation by expressing Spa^T32 also resulted in a mild yet significant reduction in axonal growth from 68.4 ± 4.2 microm in wild-type class IV neurons to 55.2 ± 2.1 microm in Spa^T32-expressing neurons (Ye, 2011).

The Collier/Olf1/EBF family transcription factor Knot has been proposed to regulate Spastin expression and promotes microtubule-based dendrite growth in class IV da neurons (Jinushi-Nakao, 2007). This study asked whether Dar1 and Knot genetically interact to control dendrite development. Overexpressing Knot (OE Knot) in class I da neurons, which have the simpler dendritic arbors, leads to an increase in dendrite growth. Knot was expressed in single class I da neurons using the MARCM technique and significant increase was observed in total dendrite length. In contrast, when Knot was expressed in neurons with dar1 mutation, the dendrites were severely reduced. To test whether Knot regulates Dar1 expression, Dar1 protein levels were examined by immunostaining in class I neurons and no difference was found between control neurons and neurons overexpressing Knot. The mean intensity of Dar1 immunofluorescence was 88.1 ± 5.7 (arbitrary unit) in control neurons and 80.6 ± 5.7 in neurons overexpressing Knot. Therefore, it is unlikely that Knot controls class IV dendrite development by regulating Dar1 expression (Ye, 2011).

This study has shown that a novel molecule in the KLF family of transcriptional regulators, Dar1, regulates the microtubule cytoskeleton during the differential development of dendrites and axons. Dar1 is specifically required for dendritic growth. Neurons lacking dar1 function exhibit reduced growth of microtubule-based dendritic branches. However, overexpression of Dar1 in neurons increased the growth of microtubule-based dendritic branches. Dar1 suppresses the expression of the microtubule-severing protein Spastin, either directly or indirectly. Upregulation of Spastin expression leads to a dendrite phenotype similar to that observed in dar1 mutant neurons (Ye, 2011).

Substantial progress has been made in the past several years on the specific regulation of axon growth. The anaphase-promoting complex (APC) and its activator Cdh1 form a multiunit complex of ubiquitin ligase in the nucleus of cerebellar granule cells to specifically restrict axon growth. This function of Cdh1-APC requires the transcriptional repressor SnoN, which is a target of the ubiquitin-dependent degradation mediated by Cdh1-APC (Stegmuller, 2006). SnoN in turn requires a scaffold protein Ccd1 to promote axon growth. The TGFβ-regulated signaling protein Smad2 plays an important role in regulating the Cdh1-APC-SnoN (Ye, 2011).

Neural activities can specifically induce dendrite growth by activating transcription factors. In cerebellar granule cells, knockdown of calcium/calmodulin-dependent protein kinase IIα (CaMKIIα) or the transcription factor NeuroD results in reduced growth of dendrites but not axons. CaMKIIα phosphorylates NeuroD in response to neural activities. Moreover, synaptic activity-induced dendritic growth can be blocked by reducing NeuroD level, suggesting that CaMKII and NeuroD mediate the dendritic growth promoted by synaptic activity (Ye, 2011).

This study has identified a novel transcriptional program that is specifically involved in the development of dendrites. Whereas whether Dar1 plays a role in activity-dependent dendritic growth remains to be determined, Dar1 is likely to be part of the activity-independent transcription program that controls dendrite growth in early development before the exposure to sensory inputs or the establishment of neuronal circuits, because the dendrite defects were observed soon after the initiation of dendritic growth in embryo. The fact that dar1 is both necessary and sufficient for dendrite growth suggests that it may determine multiple aspects required for dendrite development. Indeed, although dar1 is specifically involved in dendrite growth and is likely a regulator of Spastin expression, Spastin is involved in both dendrite and axon development. This raises the possibility that Dar1 also controls the expression of other molecules which suppresses Spastin function in the axon. It will be interesting to identify such mechanisms by systematically identifying downstream molecules of Dar1 (Ye, 2011).

Microtubule severing by Katanin and Spastin is important for axon and dendrite development, possibly by keeping microtubules sufficiently short to be efficiently transported into developing neurites or by creating more free ends of microtubules to interact with other proteins in developing processes. The Spastin gene, which encodes a microtubule-severing protein, is mutated at high frequency in autosomal dominant hereditary spastic paraplegia. RNA interference (RNAi)-mediated knockdown of Spastin leads to reduced axon length in cultured hippocampal neurons from rats and reduced dendrite growth in Drosophila da neurons in vivo. Overexpression of Spastin in cultured hippocampal neurons has no effect in total axon length although results in increased branch numbers. In Drosophila neuromuscular junction, loss-of-function mutant of spastin exhibits an increase in synaptic bouton number but a reduction in bouton size. In the present studies, it was found that overexpressing Spastin dramatically reduced dendrite growth in Drosophila da neurons. The fact that both RNAi knockdown and overexpression of Spastin lead to reduced dendrite growth is consistent with the notion that proper Spastin levels are important for neurite growth. A slight reduction was observed in axonal length in da neurons on overexpressing Spastin. The in vivo technique does not provide enough resolution to determine whether there is an increase in the number of fine branches of the axons. It is possible that PNS and CNS neurons require distinct levels of Spastin for proper axon development. Alternatively, there might be a difference between mammalian and fly neurons in their requirement of Spastin for axon development (Ye, 2011).

The different phenotypes in axon development between dar1 mutants and Spastin overexpression may be explained by additional factors regulated by Dar1, which inhibits Spastin functions in the axon. It is known that microtubule-associated proteins (MAPs), such as Tau, can shield microtubules from being severed by the severing proteins. In addition, microtubule-severing abilities of the severing proteins are also influenced by posttranslational modifications of tubulin. It is possible that Dar1 also regulates MAPs and/or posttranslational modification of tubulin in concert with Spastin expression (Ye, 2011).

Little is known about how the expression of Spastin is controlled. The current results suggest that Dar1, either directly or indirectly, suppresses the transcription of Spastin. Since proper Spastin levels are important for neurite growth (Riano, 2009), it is likely that Dar1 is required for maintaining proper levels of Spastin in neurons for dendrite growth. In Drosophila, the Collier/Olf1/EBF (COE) family transcription factor Knot, which is expressed in the class IV but not other classes of da neurons, has been proposed to positively regulate Spastin expression as overexpressing Knot increases Spastin transcription (Jinushi-Nakao, 2007). Ectopic expression of Knot in the class I da neurons, which normally have the simplest dendritic arbors among the four classes of da neurons, leads to dendrite overgrowth. However, overexpressing Spastin in class I neurons did not result in dendrite overgrowth that resembles that caused by Knot overexpression. This raises the possibility that, in the class I neurons, Knot induces the expression of other factors together with Spastin to cause dendrite overgrowth. The current results show that the effects of Knot overexpression in class I neurons requires Dar1. Knot overexpression might require factors that are positively regulated by Dar1 to cause dendrite overgrowth. Alternatively, the absence of Dar1 function in that scenario probably results in Spastin levels that are too high to allow dendrite growth (Ye, 2011).

The KLF family of transcriptional regulators has been implicated in a variety of biological processes. At least 17 members of the KLF family have been identified in mammals to date. In contrast, the Drosophila genome encodes only three KLFs: Luna and two predicted molecules, CG12029 and CG9895. Luna has been reported to be a potential fly homolog of KLF6 and 7. RNAi and overexpression studies found that luna is required for embryonic development and cell differentiation in adult eyes; no luna mutant is currently available for additional genetic analysis (Ye, 2011).

Several KLFs are known to be involved in neurite development. KLF7 is required for dendrite and axon development. In the CNS of KLF7-null mice, both dendrite growth and axon growth are reduced (Laub, 2005). In the PNS of these mice, TrkA expression is reduced in the DRG neurons (Lei, 2005). Consequently, NGF-dependent nociceptive neurons undergo increased apoptosis. In trigeminal ganglion neurons, KLF7 cooperates with the POU homeodomain protein Brn3a to control TrkA expression (Lei, 2006). KLF9 also serves important functions in the nervous system. Its expression is strongly induced by thyroid hormone. Furthermore, it is required for neurite growth. It remains to be determined whether KLF9 differentially regulates dendrite or axon development. KLF4 has recently been identified as a suppressor of neurite growth in mammalian CNS neurons. Overexpression of KLF4 reduces both dendritic and axonal length. Consistently, axon length is increased both in cultured neurons and in injured optic nerve of KLF4 knock-out mice (Ye, 2011).

The closest mammalian homolog of Dar1 is KLF5. Overexpression of KLF5 in cultured retinal ganglion cells leads to a modest reduction of neurite length (Moore, 2009); the functions of KLF5 in neuronal development have not been explored using loss-of-function studies or in vivo studies. KLF5 has been reported to be expressed in many tissues, including the brain, and is downregulated in schizophrenia patients. KLF5 homozygous mutant mice die before embryonic day 8.5 and the heterozygous mutant mice have defects in angiogenesis, cardiovascular remodeling, gut development, and adipogenesis. In light of the involvement of Dar1 in Drosophila dendrite development, it will be interesting to examine these KLF5 mutant mice with respect to neural development (Ye, 2011).

It is unknown whether any of the mammalian KLFs function specifically in either dendrite or axon development. Whereas the simplest scenario of such evolutionary conservation is that one of the mammalian KLFs is the ortholog of Dar1, it is equally possible that multiple KLFs coordinate their activities (as proposed by Moore, 2009) to perform the equivalent of Dar1 function (Ye, 2011).

In summary, this study has identified a novel transcription factor and demonstrated its requirement for dendritic but not axonal growth in Drosophila. Future studies that elucidate the regulatory mechanisms of Dar1 and its downstream effector genes will shed light on the genetic program that differentiates dendrite and axon development (Ye, 2011).

Search PubMed for articles about Drosophila Dar1

Jinushi-Nakao, S., et al. (2007). Knot/Collier and Cut control different aspects of dendrite cytoskeleton and synergize to define final arbor shape. Neuron 56: 963-978. PubMed Citation: 18093520

Laub F., et al. (2005) Transcription factor KLF7 is important for neuronal morphogenesis in selected regions of the nervous system. Mol. Cell Biol. 25: 5699-5711. PubMed Citation: 15964824

Lei L., et al. (2005). The zinc finger transcription factor Klf7 is required for TrkA gene expression and development of nociceptive sensory neurons. Genes Dev. 19: 1354-1364. PubMed Citation: 15937222

Lei L., Zhou, J., Lin, L. and Parada, L. F. (2006). Brn3a and Klf7 cooperate to control TrkA expression in sensory neurons. Dev. Biol. 300: 758-769. PubMed Citation: 17011544

Moore, D. L., et al. (2009). KLF family members regulate intrinsic axon regeneration ability. Science 326: 298-301. PubMed Citation: 19815778

Riano, E., et al. (2009). Pleiotropic effects of spastin on neurite growth depending on expression levels. J. Neurochem. 108: 1277-1288. PubMed Citation: 19141076

Stegmuller, J., et al. (2006). Cell-intrinsic regulation of axonal morphogenesis by the Cdh1-APC target SnoN. Neuron 50(3): 389-400. PubMed Citation: 16675394

Tahirovic, S. and Bradke, F. (2009). Neuronal polarity. Cold Spring Harb. Perspect. Biol. 1: a001644. PubMed Citation: 20066106

Wiggin, G. R., Fawcett, J. P. and Pawson, T. (2005) Polarity proteins in axon specification and synaptogenesis. Dev. Cell 8: 803-816. PubMed Citation: 15935771

Ye, B., et al. (2007). Growing dendrites and axons differ in their reliance on the secretory pathway. Cell 130: 717-729. PubMed Citation: 17719548

Ye, B., et al. (2011). Differential regulation of dendritic and axonal development by the novel Kruppel-like factor dar1. J. Neurosci. 31(9): 3309-19. PubMed Citation: 21368042

Yeaman, C., et al. (2004). Protein kinase D regulates basolateral membrane protein exit from trans-Golgi network. Nat. Cell Biol. 6: 106-112. PubMed Citation: 14743217

date revised: 10 May 2011

Home page: The Interactive Fly © 2009 Thomas Brody, Ph.D.