== (A) Single mutantlin-14(lf)animals extended axons precociously, and in doublelin-4(lf);lin-14(lf)mutants,lin-14(lf)was sufficient to suppress thelin-4(lf)retarded phenotype at the restrictive temperature (23C). of HSN axon elongation. == Author Summary == During development, nerve cells extend long structures called axons which are required for communication across distant brain regions and/or with other tissues. Many of the signals controlling the direction of axon extension are well understood, but much less is known about the factors PIK-293 that control when this growth occurs. Ultimately, failure to coordinate the direction and timing of axon elongation could lead to dramatic defects in the way our brains function. By studying the simple soil nematodeC. elegans, we have identified mutations in three timing genes that result in either premature or delayed axon growth. These genes function independently from known directional signals as well as axon position. Since two of PIK-293 the genes are shared with mammals, findings presented here will help expand our understanding of human axon development, Rabbit Polyclonal to CK-1alpha (phospho-Tyr294) and could potentially lead to novel strategies for promoting growth in injured adult nerve cells. == Introduction == During development, neurons must extend axons in the correct direction and at the proper time. Yet while several conserved families of ligands and receptors have been identified that control axon guidance[1],[2], little is known about the temporal regulation of axon growth. The observation that guidance cues can promote axon elongation as well as turning inin vitroexperiments has led to models in which the timing of receptor gene expression and/or function specifies when axons extend[1][4]. However, it is clear that additional mechanisms must ensure that axon growth and guidance are appropriately coupled. For instance, in theC. eleganshermaphrodite specific PIK-293 neurons (HSNs), the UNC-40/DCC Netrin receptor is up-regulated at the L1 larval stage and ventrally localized by the L2, yet the HSNs do not extend single axons toward the ventral UNC-6/Netrin source until early L4, nearly two stages later[5]. In addition, HSN axons grow at the correct stage in animals lacking UNC-6/Netrin or UNC-40/DCC, suggesting that the timing of axon elongation is regulated independently of Netrin signaling[5]. TheC. elegansHSNs provide a convenient system for further investigating the temporal control of axon elongation. Many of the principle molecules required forC. elegansaxon growth and guidance are conserved in PIK-293 higher animals[5][10]. In addition, the morphological features of HSN development have been very well characterized[5]. At the mid-to-late L3 larval stage, the neurons extend several short neurites ventrally, and at the L3/L4 transition, they retract all but one of these processes, the neurite selected to become the axon. In the L4, the axon completes its extension toward the ventral nerve cord (VNC) and turns anteriorly, where it forms synapses with vulval muscles and the VC4 and VC5 motor neurons before growing to the nerve ring[5],[11]. We predicted that genes which regulate the timing of cell PIK-293 divisions might also control HSN postmitotic differentiation. TheC. eleganstranscription factorlin-14has previously been shown to play a similar role during synaptic development of the DD motor neurons[12][14].lin-14is a member of the heterochronic pathway, a set of temporal patterning genes that was first characterized in mitotic hypodermal cells[15][18]. Several other heterochronic genes are also expressed in theC. elegansnervous system, including those encoding thelin-4microRNA, the cytoplasmic RNA-binding protein LIN-28, nuclear hormone receptor DAF-12 and the HBL-1 transcription factor[19][25]. However, the neuronal functions and interactions of these genes withlin-14, if any, have not yet been determined. MicroRNAs (miRNAs) represent a set of non-coding RNAs that inhibit genes post-transcriptionally by binding to partially complementary mRNA sequences[19],[20],[26],[27]. While they are dynamically expressed during the development of invertebrate and vertebrate nervous systems, only a small number have been characterizedin vivo[28][31]. Here, we show that thelin-4miRNA together withlin-14andlin-28comprise a cell-autonomous switch that promotes axon growth. These genes act independently of ventral guidance receptors to ensure that axon development is properly completed before the HSNs are required in the adult egg-laying system[32]. == Results == == Thelin-4miRNA promotes HSN axon elongation == Candidate heterochronic mutants were crossed into a transcriptional reporter strain expressing myristoylated green fluorescent protein (GFP) under the control of theuncoordinated-86 (unc-86)promoter[5].unc-86encodes a POU homeodomain transcription factor that is expressed constitutively after HSNs exit the cell cycle and controls execution of late maturation events[33],[34]. In this work, the phenotypic criterion for distinguishing HSN axons from less mature neurites was initiation of the anterior turn (Figure 1A). == Figure 1.lin-4(lf)displays delayed HSN axon extension. == (A) In late L3 wild-type (wt) animals (top), HSNs extended multiple neurites (arrow) in the ventral direction (left). By early L4 (middle) and in the adult (right), wild-type animals extended a single HSN axon ventrally and.