dendritic arbor reduction 1: Biological Overview | References
Gene name - dendritic arbor reduction 1
Synonyms - CG12029
Cytological map position - 63E5-63E6
Function - Transcription factor
Symbol - dar1
FlyBase ID: FBgn0263239
Genetic map position - 3L:3,626,874..3,653,443 [+]
Classification - zinc finger
Cellular location - nuclear
|Recent literature||Wu, X., Chen, Z., Gao, Y., Wang, L., Sun, X., Jin, Y. and Liu, W. (2018). The kruppel like factor Dar1 restricts the proliferation of Drosophila intestinal stem cells. FEBS J. PubMed ID: 30188612
The kruppel like factors (KLFs) are a family of transcription factor proteins that regulate a wide range of biological processes. In an RNAi-based screening experiment, this study identified Dar1, which is a KLF member in Drosophila, inhibited the proliferation of intestinal stem cells (ISCs). Suppression of Dar1 activated ISC proliferation; as a consequence, the ISCs and the young differentiated cells were increased. On the other hand, overexpression of Dar1 inhibited ISC division and blocked the formation of ISC lineages. In order to explore the molecular mechanism of the Dar1 functions, the gene expression profiles of the Dar1 knockdown and Dar1 overexpression midguts were compared, using the deep RNA sequencing (RNA-Seq) technique. This experiment revealed that Dar1 negatively regulated the expression of several critical cell cycle genes. Evidence is provided that Dar1 has a function upstream of the JAK/STAT signaling pathway, suggesting Dar1 can regulate ISC proliferation through different mechanisms. Consistent with these findings, this study discovered Dar1 was down-regulated in the wounded midguts, allowing increased ISCs proliferation to promote intestinal repair. These data suggest that Dar1 is a functional homolog of the mammalian KLF4 (Wu, 2018).
|Wu, X., Chen, Z., Gao, Y., Wang, L., Sun, X., Jin, Y. and Liu, W. (2018). The Kruppel-like factor Dar1 restricts the proliferation of Drosophila intestinal stem cells. FEBS J. PubMed ID: 30188612
The kruppel-like factors (KLFs) are a family of transcription factor proteins that regulate a wide range of biological processes. In an RNAi-based screening experiment, dendritic arbor reduction 1 (Dar1), which is a KLF member in Drosophila, inhibited the proliferation of intestinal stem cells (ISCs). Suppression of Dar1-activated ISC proliferation; as a consequence, the ISCs and the young differentiated cells were increased. On the other hand, overexpression (OE) of Dar1 inhibited ISC division and blocked the formation of ISC lineages. In order to explore the molecular mechanism of the Dar1 functions, the gene expression profiles of the Dar1 knockdown and Dar1 OE midguts was compared, using the deep RNA sequencing (RNA-Seq) technique. This experiment revealed that Dar1 negatively regulated the expression of several critical cell cycle genes. Evidence is provided that Dar1 has a function upstream of the JAK/STAT signaling pathway, suggesting Dar1 can regulate ISC proliferation through different mechanisms. Consistent with these findings, Dar1 was found to be downregulated in the wounded midguts, allowing increased ISC proliferation to promote intestinal repair. These data suggest that Dar1 is a functional homolog of the mammalian KLF4.
|Laurichesse, Q., Moucaud, B., Laddada, L., Renaud, Y., Jagla, K. and Soler, C. (2021). Transcriptomic and Genetic Analyses Identify the Kruppel-Like Factor Dar1 as a New Regulator of Tube-Shaped Long Tendon Development. Front Cell Dev Biol 9: 747563. PubMed ID: 34977007
To ensure locomotion and body stability, the active role of muscle contractions relies on a stereotyped muscle pattern set in place during development. This muscle patterning requires a precise assembly of the muscle fibers with the skeleton via a specialized connective tissue, the tendon. Like in vertebrate limbs, Drosophila leg muscles make connections with specific long tendons that extend through different segments. During the leg disc development, cell precursors of long tendons rearrange and collectively migrate to form a tube-shaped structure. A specific developmental program underlies this unique feature of tendon-like cells in the Drosophila model. This study provides for the first time a transcriptomic profile of leg tendon precursors through fluorescence-based cell sorting. From promising candidates, the Kruppel-like factor Dar1 was identified as a critical actor of leg tendon development. Specifically expressed in the leg tendon precursors, loss of dar1 disrupts actin-rich filopodia formation and tendon elongation. These findings show that Dar1 acts downstream of Stripe and is required to set up the correct number of tendon progenitors.
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 GAL44-77 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 (SpaT32), 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 SpaT32. Similarly, the number of branch points was reduced from 767.9 ± 22.8 in wild-type to 403.3 ± 43.5 in SpaT32-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 SpaT32 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 SpaT32-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).
Neurons typically assume multipolar, bipolar, or unipolar morphologies. Little is known about the mechanisms underlying the development of these basic morphological types. This study shows that the Kruppel-like transcription factor Dar1 determines the multipolar morphology of postmitotic neurons in Drosophila. Dar1 is specifically expressed in multipolar neurons and loss of dar1 gradually converts multipolar neurons into the bipolar or unipolar morphology without changing neuronal identity. Conversely, misexpression of Dar1 or its mammalian homolog in unipolar and bipolar neurons causes them to assume multipolar morphologies. Dar1 regulates the expression of several dynein genes and nuclear distribution protein C (nudC), which is an essential component of a specialized dynein complex that positions the nucleus in a cell. These genes were shown to be required for Dar1-induced multipolar neuron morphology. Dar1 likely functions as a terminal selector gene for the basic layout of neuron morphology by regulating both dendrite extension and the dendrite-nucleus coupling (Wang, 2015).
Ramon y Cajal placed neurons into three major morphological types based on the number of dendrites connected to the soma (i.e., primary dendrites): unipolar, bipolar, and multipolar and this classification system is universally applicable to different species throughout evolution. Multipolar neurons, like mammalian pyramidal neurons, develop more than one primary dendrite. In contrast, bipolar neurons are defined as having a single primary dendrite that may (e.g., cerebellar Purkinje cells) or may not (e.g., photoreceptors) branch out into an elaborate dendritic arbor. Finally, unipolar neurons such as DRG neurons in vertebrates and the majority of CNS neurons in invertebrates extend a single primary neurite, which usually bifurcates into dendritic and axonal branches (Wang, 2015).
Multipolar morphology separates the dendritic arbor into distinct fields around the soma, which has an impact, not only on the passive current spread and processing of electrical signals in the neuron), but also on the types of synaptic or sensory inputs that the neuron receives . In addition, the three basic morphologies of neurons are relevant to the distinct organizational principles used in both the nervous systems of different animal species and in different parts of a single nervous system. Although all three morphological types are found in different species throughout evolution, the majority of neurons in invertebrates are unipolar, whereas the majority of those in vertebrates are multipolar (Wang, 2015).
In the insect CNS, unipolar neurons extend a single process from the soma to a synapse-enriched neuropil and then bifurcate into dendrites that arborize locally and an axon that typically projects to other neuropil areas or target tissues. Unipolar organization of neuronal processes allows the formation of synaptic connections away from the location of the neuronal cell body, so it is likely an alternative strategy for neuronal migration, which is rare in the insect CNS but common in the vertebrate CNS. Despite the importance of these fundamental organizations of neuronal processes, very little progress has been made toward understanding the molecular and cellular programs that lead postmitotic neurons to develop multipolar, bipolar, or unipolar morphologies since their description a century ago (Wang, 2015).
This study shows that the transcription factor Dar1 determines the multipolar morphology of postmitotic neurons in Drosophila. Dar1 is selectively expressed in postmitotic multipolar neurons and is required for these neurons to assume the multipolar morphology. Ectopic expression in unipolar or bipolar neurons leads to multipolar morphology. Dar1 regulates the expression of several dynein genes and nudC, which is an essential component of a specialized dynein complex that positions the nucleus in a cell. It is further shown that this evolutionarily conserved complex is required for multipolar morphology of neurons. These results suggest that dar1 likely functions as a terminal selector gene for the basic layout of neuron morphology (Wang, 2015).
The universal morphological organization of neuronal dendrites and axons-in the form of the unipolar, bipolar, and multipolar morphologies-is important for information processing in neurons and for the wiring of neural circuits. It has generally been assumed that the formation of the basic morphological types of neurons is determined by the number of dendrites growing out from the cell body (the 'outgrowth model'). This study shows that this model alone is insufficient to explain the formation of multipolar morphology. Nuclear positioning is introduced as a factor in determining the multipolar neuron morphology, and it is proposed that Dar1 determines multipolar morphology by regulating both dendrite extension and primary dendrite-nucleus coupling (Wang, 2015).
A novel, instructive role is reported for Dar1 in determining the multipolar morphology of postmitotic neurons without changing cell fate. First, despite the dendritic defects, axon morphology (Ye, 2011) and targeting are unchanged in dar1 mutant neurons. Second, ectopic expression of Dar1 in postmitotic neurons leads to supernumerary primary dendrites. Third, the remaining dendrites in dar1-/- neurons still follow the branching pattern assumed by wild-type neurons (Ye, 2011). Fourth, dar1 mutations do not affect the expression of neuron type-specific markers. Based on extensive studies in C. elegans, Hobert proposed the concept of terminal selector genes (Hobert, 2008). A terminal selector gene is required for determining specific aspects of a neuron's identity by regulating the expression of genes responsible for these characteristics such as those encoding neurotransmitter receptors, enzymes in a neurotransmitter synthesis pathway, and structural proteins. Loss of a terminal selector gene results in the loss of a specific aspect of the neuron type without affecting the overall neuronal identity. Dar1 plays such a function in the basic layout of neuronal morphology and thus is likely a 'terminal selector gene' for neuronal morphology (Wang, 2015).
Based on the findings in this study, it is proposed that generating neuronal multipolar morphology requires, not only dendritic extension, but also a coupling mechanism between the nucleus and the dendrites. Dar1 promotes both dendritic growth and dendrite-nucleus coupling. Therefore, its misexpression converts unipolar neurons into neurons with multipolar morphology. The results presented in this study raise the interesting possibility that a specialized dynein complex in multipolar neurons with components that are transcriptionally regulated by Dar1 couples the nucleus with the primary dendrites. If the primary dendrite-nucleus coupling is weakened, then the nucleus may move to a different location in relation to the dendrites and axons. It is speculated that there might also be an active or passive force that pulls the nucleus toward the axon, opposing the force that couples the primary dendrites and the nucleus. Consistent with this model, the remaining single primary dendrites of all bipolar-shaped da neurons-caused by loss of dar1, reduced functions of dynein, or nuclear positioning complex-are those that project in the direction opposite the axon. This observation again rules out the possibility that the reduction in number of primary dendrites of dar1-/- da neurons is the result of reduction in dendrite growth. If that were the case, then the remaining single primary dendrites would likely project in random directions and not solely away from the axon. Further studies are needed to determine the cellular and molecular basis of the dendrite-nucleus coupling (Wang, 2015).
Several prior studies have demonstrated that neurons switch between different morphological types during development. These observations suggest that the acquisition of basic morphological types in many neurons includes intermediate morphologies with nucleus-primary dendrite relationships that are different from those seen in the mature neurons. It will be interesting to investigate whether the activity of the nuclear positioning complex and its regulators play a role in these developmental changes in morphological type (Wang, 2015).
In summary, this study offers a novel model for understanding the establishment of the three basic morphological types of neurons. Starting from genetic analysis of the KLF transcription factor Dar1, the study not only uncovers an instructive factor that determines the multipolar morphology of neurons, but also provide a mechanistic model showing that the position of the nucleus is critical for establishing multipolar neuron morphology. This study also demonstrates that the basic morphological types are determined by intrinsic molecular mechanisms in postmitotic neurons rather than in precursor cells. The model presented in this study may also be applied to explaining the changes in basic morphological type during neuron development. This study therefore opens the door for a unifying theory of basic structural organization in neurons (Wang, 2015).
Kruppel-like factor 5 (Klf5) (see Drosophila dar1) is a zinc-finger transcription factor that controls various biological processes, including cell proliferation and differentiation. This study shows that Klf5 is also an essential mediator of skeletal muscle regeneration and myogenic differentiation (see myogenesis in Drosophila). During muscle regeneration after injury (cardiotoxin injection), Klf5 is induced in the nuclei of differentiating myoblasts and newly formed myofibers expressing myogenin in vivo. Satellite cell-specific Klf5 deletion severely impairs muscle regeneration, and myotube formation is suppressed in Klf5-deleted cultured C2C12 myoblasts and satellite cells. Klf5 knockdown suppresses induction of muscle differentiation-related genes, including myogenin. Klf5 ChIP-seq revealed that Klf5 binding overlaps that of MyoD (see Drosophila nau) and Mef2 (see Drosophila Mef2), and Klf5 physically associates with both MyoD and Mef2. In addition, MyoD recruitment is greatly reduced in the absence of Klf5. These results indicate that Klf5 is an essential regulator of skeletal muscle differentiation, acting in concert with myogenic transcription factors such as MyoD and Mef2 (Hayashi, 2016).
Search PubMed for articles about Drosophila Dar1
Hayashi, S., Manabe, I., Suzuki, Y., Relaix, F. and Oishi, Y. (2016). Klf5 regulates muscle differentiation by directly targeting muscle-specific genes in cooperation with MyoD in mice. Elife [Epub ahead of print]. PubMed ID: 27743478
Hobert, O. (2008). Regulatory logic of neuronal diversity: terminal selector genes and selector motifs. Proc Natl Acad Sci U S A 105: 20067-20071. PubMed ID: 19104055
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 ID: 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 ID: 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 ID: 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 ID: 17011544
Moore, D. L., et al. (2009). KLF family members regulate intrinsic axon regeneration ability. Science 326: 298-301. PubMed ID: 19815778
Riano, E., et al. (2009). Pleiotropic effects of spastin on neurite growth depending on expression levels. J. Neurochem. 108: 1277-1288. PubMed ID: 19141076
Stegmuller, J., et al. (2006). Cell-intrinsic regulation of axonal morphogenesis by the Cdh1-APC target SnoN. Neuron 50(3): 389-400. PubMed ID: 16675394
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date revised: 4 April 2022
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