PFTK1 kinase regulates axogenesis during development via RhoA activation.

BACKGROUND: PFTK1/Eip63E is a member of the cyclin-dependent kinases (CDKs) family and plays an important role in normal cell cycle progression. Eip63E expresses primarily in postnatal and adult nervous system in Drosophila melanogaster but its role in CNS development remains unknown. We sought to understand the function of Eip63E in the CNS by studying the fly ventral nerve cord during development. RESULTS: Our results demonstrate that Eip63E regulates axogenesis in neurons and its deficiency leads to neuronal defects. Functional interaction studies performed using the same system identify an interaction between Eip63E and the small GTPase Rho1. Furthermore, deficiency of Eip63E homolog in mice, PFTK1, in a newly generated PFTK1 knockout mice results in increased axonal outgrowth confirming that the developmental defects observed in the fly model are due to defects in axogenesis. Importantly, RhoA phosphorylation and activity are affected by PFTK1 in primary neuronal cultures. We report that GDP-bound inactive RhoA is a substrate of PFTK1 and PFTK1 phosphorylation is required for RhoA activity. CONCLUSIONS: In conclusion, our work establishes an unreported neuronal role of PFTK1 in axon development mediated by phosphorylation and activation of GDP-bound RhoA. The results presented add to our understanding of the role of Cdks in the maintenance of RhoA-mediated axon growth and its impact on CNS development and axonal regeneration.

Drosophila Jing is part of the breathless fibroblast growth factor receptor positive feedback loop.

In the developing Drosophila trachea, extensive cell migration lays the foundation for an elaborate network of tubules to form. This process is controlled by the Drosophila fibroblast growth factor receptor, known as Breathless (Btl), whose expression is activated by the Trachealess (Trh) and Tango (Tgo) basic helix-loop-helix (bHLH)-PAS transcription factors. We previously identified the jing zinc finger transcription factor as a gene sensitive to the dosage of bHLH-PAS transcriptional activity and showed that its mutations interact genetically with those of trh and btl. Here, we demonstrate that jing is required for btl expression in the branching trachea and dominantly interacts with known regulators of btl expression, including the ETS and POU transcription factors, pointed, and drifter/ventral veinless, respectively. Furthermore, the zinc finger-containing C-terminus of Jing associates with a btl tracheal enhancer in a Trh/Tgo-dependent manner in chromatin immunoprecipitation assays in vitro and interferes with btl in vitro and in vivo. Together, our results support a model by which Jing/Trh/Tgo complexes regulate btl transcript levels during primary tracheal branching.

The Drosophila jing gene is a downstream target in the Trachealess/Tango tracheal pathway.

Primary branching in the Drosophila trachea is regulated by the Trachealess (Trh) and Tango (Tgo) basic helix-loop-helix-PAS (bHLH-PAS) heterodimers, the POU protein Drifter (Dfr)/Ventral Veinless (Vvl), and the Pointed (Pnt) ETS transcription factor. The jing gene encodes a zinc finger protein also required for tracheal development. Three Trh/Tgo DNA-binding sites, known as CNS midline elements, in 1.5 kb of jing 5' cis-regulatory sequence (jing1.5) previously suggested a downstream role for jing in the pathway. Here, we show that jing is a direct downstream target of Trh/Tgo and that Vvl and Pnt are also involved in jing tracheal activation. In vivo lacZ enhancer detection assays were used to identify cis-regulatory elements mediating embryonic expression patterns of jing. A 2.8-kb jing enhancer (jing2.8) drove lacZ expression in all tracheal cell lineages, the CNS midline and Engrailed-positive segmental stripes, mimicking endogenous jing expression. A 1.3-kb element within jing2.8 drove expression that was restricted to Engrailed-positive CNS midline cells and segmental ectodermal stripes. Surprisingly, jing1.5-lacZ expression was restricted to tracheal fusion cells despite the presence of consensus DNA-binding sites for bHLH-PAS, ETS, and POU domain transcription factors. Given the absence of Trh/Tgo DNA-binding sites in the jing1.3 enhancer, these results are consistent with previous observations suggesting a combinatorial basis to Trh-/Tgo-mediated transcriptional regulation in the trachea.

Glial and neuronal functions of the Drosophila homolog of the human SWI/SNF gene ATR-X (DATR-X) and the jing zinc-finger gene specify the lateral positioning of longitudinal glia and axons.

Neuronal-glial communication is essential for constructing the orthogonal axon scaffold in the developing Drosophila central nervous system (CNS). Longitudinal glia (LG) guide extending commissural and longitudinal axons while pioneer and commissural neurons maintain glial survival and positioning. However, the transcriptional regulatory mechanisms controlling these processes are not known. Previous studies showed that the midline function of the jing C2H2-type zinc-finger transcription factor was only partially required for axon scaffold formation in the Drosophila CNS. We therefore screened for gain-of-function enhancers of jing gain of function in the eye and identified the Drosophila homolog of the disease gene of human alpha-thalassemia/mental retardation X-linked (ATR-X) as well as other genes with potential roles in gene expression, translation, synaptic transmission, and cell cycle. jing and DATR-X reporter genes are expressed in both CNS neurons and glia, including the LG. Coexpression of jing and DATR-X in embryonic neurons synergistically affects longitudinal connective formation. During embryogenesis, jing and DATR-X have autonomous and nonautonomous roles in the lateral positioning of LG, neurons, and longitudinal axons as shown by cell-specific knockdown of gene expression. jing and DATR-X are also required autonomously for glial survival. jing and DATR-X mutations show synergistic effects during longitudinal axon formation suggesting that they are functionally related. These observations support a model in which downstream gene expression controlled by a potential DATR-X-Jing complex facilitates cellular positioning and axon guidance, ultimately allowing for proper connectivity in the developing Drosophila CNS.

The jing and ras1 pathways are functionally related during CNS midline and tracheal development.

The Drosophila jing gene encodes a zinc finger protein required for the differentiation and survival of embryonic CNS midline and tracheal cells. We show that there is a functional relationship between jing and the Egfr pathway in the developing CNS midline and trachea. jing function is required for Egfr pathway gene expression and MAPK activity in both the CNS midline and trachea. jing over-expression effects phenocopy those of the Egfr pathway and require Egfr pathway function. Activation of the Egfr pathway in loss-of-function jing mutants partially rescues midline cell loss. Egfr pathway genes and jing show dominant genetic interactions in the trachea and CNS midline. Together, these results show that jing regulates signal transduction in developing midline and tracheal cells.

Analysis of the transcriptional activation domain of the Drosophila tango bHLH-PAS transcription factor.

Basic-helix-loop-helix-PAS transcription factors play important roles in diverse biological processes including cellular differentiation and specification, oxygen tension regulation and dioxin metabolism. Drosophila tango is orthologous to mammalian Arnt and acts as a common dimerization partner for bHLH-PAS proteins during embryogenesis. A transient transfection assay using Drosophila S2 tissue culture cells and wild-type and mutant Drosophila tango cDNAs was used to localize the activation domain of the Tango protein. An activation domain was identified in the C-terminus of TGO consisting of poly-glutamine and histidine-proline repeats. Transcriptional activation of the fibroblast growth factor receptor (breathless) gene required an intact TGO C-terminus, in vitro. Co-expression assays of trachealess and tgo in the developing eye imaginal disc showed a requirement for the C-terminal transactivation domain of TGO for a cellular response. Genetic analysis of tgo(3) shows that the paired repeat is necessary for tracheal tubule formation in all branches. Lastly, expression of a C-terminal truncated tgo transgene specifically in the CNS midline and trachea resulted in reductions in the number of breathless-expressing cells. These results together identify TGO's transactivation domain and establish its importance for proper target gene regulation and cellular specification.

The jing gene is required for embryonic brain development in Drosophila [corrected].

Loss- and gain-of-function studies have demonstrated a crucial role for the jing zinc finger transcription factor in neuronal and glial differentiation and survival in the embryonic central nervous system midline of Drosophila. Here, we have studied the role of jing during embryonic brain development. Proper jing function is required for the formation of the primary brain axon scaffold. In homozygous jing (3) mutant embryos the preoral commissure is not pioneered and never forms. Other axon pathways are pioneered but subsequently do not form properly, including the postoral tritocerebral commissure, the circumesophageal connectives and the pathways that connect the brain with the ventral nerve cord. To understand the cellular basis of the axon phenotype the jing expression pattern in the brain was characterized using a jing-lacZ enhancer trap. jing-lacZ enhancer trap expression occurs in glia and neurons in the brain midline and lateral clusters as determined by co-localization of the lacZ gene product with Repo and Castor, respectively. In addition, the jing-lacZ enhancer trap and the basic helix-loop-helix-PAS gene, single-minded ( sim), are expressed in the only glial midline cluster present in stage-14 wild-type embryos. jing function is required for the differentiation of Repo-, Castor- and Sim-positive cells in the embryonic brain as each of these populations contain a reduced number of cells in homozygous jing (3) mutant embryos. We further find that jing is required for neuronal and glial survival as repo- and castor-expressing cells undergo cell death in homozygous jing (3) mutant embryos, as revealed by double labeling with Tunel. Expression of jing in sim-expressing cells in the brain disrupts the entire axon scaffold but most significantly results in loss of the preoral and postoral tritocerebral commissures. In addition, circumesophageal connectives are repelled after expression of two copies of UAS- jing in sim-expressing cells, suggesting the activation of axon repellent molecules. Over-expression of sim in the brain is also associated with loss of preoral and postoral tritocerebral commissures. Therefore, the proper dosage of jing and sim in the brain is critical for the formation of the primary axon scaffold. These results show that an important role for jing in the developing brain is the regulation of neuronal and glial differentiation and survival.

The jing Zn-finger transcription factor is a mediator of cellular differentiation in the Drosophila CNS midline and trachea.

We establish that the jing zinc-finger transcription factor plays an essential role in controlling CNS midline and tracheal cell differentiation. jing transcripts and protein accumulate from stage 9 in the CNS midline, trachea and in segmental ectodermal stripes. JING protein localizes to the nuclei of CNS midline and tracheal cells implying a regulatory role during their development. Loss of jing-lacZ expression in homozygous sim mutants and induction of jing-lacZ by ectopic sim expression establish that jing is part of the CNS midline lineage. We have isolated embryonic recessive lethal jing mutations that display genetic interactions in the embryonic CNS midline and trachea, with mutations in the bHLH-PAS genes single-minded and trachealess, and their downstream target genes (slit and breathless). Loss- and gain-of-function jing is associated with defects in CNS axon and tracheal tubule patterning. In jing homozygous mutant embryos, reductions in marker gene expression and inappropriate apoptosis in the CNS midline and trachea establish that jing is essential for the proper differentiation and survival of these lineages. These results establish that jing is a key component of CNS midline and tracheal cell development. Given the similarities between JING and the vertebrate CCAAT-binding protein AEBP2, we propose that jing regulates transcriptional mechanisms in Drosophila embryos and promotes cellular differentiation in ectodermal derivatives.