PFTK1 kinase regulates axogenesis during development via RhoA activation

Abstract

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.

Keywords: Axogenesis; Cyclin-dependent kinases; Eip63E; Neuronal development; PFTAIRE; Rho.

Fig. 1

Eip63E deficiency leads to defasciculation and misguidance of VNC axons Ventral views of whole mount stage 14–15 embryos from both Eip63E mutant lines, co-stained for GFP and BP102 (A) or Fasciclin-II (1D4 mAb) (B). Penetrance of VNC defects was calculated as the % of embryos of a given scoring phenotype that show any defect from the total population of such phenotype (GFP + or GFP − embryos). Arrowhead points to normal-looking longitudinal connectives and arrows point to disrupted structures in deficient embryos. L63⁸¹ denotes Eip63E⁸¹. Scale bar 50 μm

Fig. 2

Eip63E deficiency leads to early embryonic developmental defects. Ventral views (unless otherwise specified) of whole-mount embryos are shown head to the left. Stage 5–8 embryos are from Eip63E⁸¹ line of unknown genotype. Panels for stages 8–9 onward show Eip63E⁸¹ homozygous mutant embryos. A Staining for single-minded at early embryonic stages (st 5–10) shows no gross difference in midline formation but cellular morphological differences are noticeable. B Staining for engrailed/ invected (4D9 mAb). 18% (n = 48) of total population of stage 8–9 embryos show acute segmentation defects. At stages 10–11, cell bodies appear disorganized (arrowheads) to the extent of loss of hemi-segments division at the midline (wavy arrow) compared to control (straight arrow). C Staining for the HRP antigen. At late stage 11, precocious axon outgrowth (arrows) seen in 36% of deficient embryos (n = 52). Scale bar 50 μm

Fig. 3

Eip63E deficiency leads to early segmentation defects. A–D Ventral views of whole-mount embryos stages 12–15 shown head to the left. CTR show GFP + embryos (wild-type and mutant heterozygous population) and Eip63E⁸¹ represent homozygous mutant embryos (GFP −). Immunostaining for Neuroglian (B), Neurotactin (C, top 2 panels), and BP102 (C, bottom 2 panels) showing axon defects (arrows). D Co-staining for glial populations (Repo) and axons (HRP) showing disorganized cell bodies (arrowheads). E VNC flat preparations of timed embryos (10–11 h, stage 14) co-stained for Futsch and HRP are shown dorsal up, head to the left. Arrowheads point to some of the structures where neuronal organization is different from control embryos. Arrows point to examples of axon growth abnormalities. Scale bar 50 μm

Fig. 4

Only ubiquitous and neuronal downregulation of Eip63E leads to embryonic axon defects. Ventral views of stage 14 whole-mount embryos stained with BP102 shown head to the left. A UAS-Gal4 approach was used to drive Eip63E downregulation in specific tissues during embryonic development. Embryos negative for GFP (present in balancers for both the Gal4 and the UAS lines) were considered experimental embryos vs the GFP positive of the same progeny. A A representative GFP + embryo is shown. B Ubiquitous downregulation led to a replica of the germline deficiency lines, with regards to CNS defective phenotype and developmental lethality (see text for details). C Neuronal-specific downregulation led to a robust and highly penetrant axonal phenotype. D–G Downregulation of Eip63E in other tested tissue (indicated in red) did not cause any detectable axonal defects. The number of embryos per genotype and the percentage of embryos per group showing axonal defects is summarized in H. Scale bar 50 μm

Fig. 5

Eip63E functionally interacts with Rho1 to regulate axogenesis. Ventral views of stage 14 whole-mount embryos stained for BP102 antigen are shown head to the left. Panels show representative images for each specified genotype, A–C representative images of A control embryos, B single heterozygous Eip63E⁸¹, and C embryo from Rho1^72F line showing defective VNC phenotype (D) GFP − embryo from a cross between Eip63E⁸¹-deficient line and dominant-negative Rho1 (Rho1^72F). B, C Images of presumed genotype (see text for details). D Axon defects of intermediate severity in 22.7% of embryos heterozygous for Rho1^72F and Eip63E⁸¹ (GFP −). E–H UAS-elavGal4 approach to drive Eip63E downregulation and constitutive active Rho1 overexpression in neurons in two independent Dmel lines. Lines were then crossed to generate the indicated genotypes. Representative images showing VNC defects from Eip63E downregulated (F) or Rho1.V14 expressing (G) embryos are presented. 86.96% of all embryos from a cross to generate both Rho1.V14 expression and Eip63E⁸¹ knockdown (H) show VNC defects. F, G, and H are representative images of presumed genotypes (marked in red, see text for details). Penetrance values from stages 13–17, n as follows: B > 100, C 86, D 22, F 89, G 126, H 69. n represents the total of screened embryos from several different pairings/collection pools. Scale bar 50 μm

Fig. 6

PFTK1 downregulation leads to faster-growing axons in murine cortical neurons. Primary cortical neurons derived from CD1 embryos (E 14-15) were infected with 100 MOI of adenoviruses expressing GFP alone, GFP and WT-PFTK1 or GFP and D228N-PFTK1. After 12 or 24 h in culture, cells were fixed and stained with Hoechst 33258 (nuclei visualization). Axons of GFP + neurons were measured using Image J. A Values are presented as averages ± SEM from 122-143 cells/treatment/timepoint. Statistics were done using two-way ANOVA followed by Tuckey’s post-hoc. B Same data as in A but presented as axon length relative to the GFP control. C Representative images of primary cortical neurons from E13–14 embryos from HET × HET crosses from WT and KO cultures fixed at the specified times and stained for Tau and Hoechst. D Axon length distribution per genotype from the indicated number of embryos (236–469 cells/embryo) at 16 h in vitro. Each column represents the percentage of cells in the specified range of length for the indicated genotype. Non-treated (NT) cultures were also fixed after 16 and 24 h in vitro. Graph represents the average axon length/genotype/treatment. t-test was used for statistical analysis whenever p value is specified. E 16-h cultures were treated for the last 8 h with 0.5ug/ml of RhoA activator (Cytoskeleton Inc. CN03). Graph represents the average axon length

Fig. 7

RhoA phosphorylation and activity are affected by PFTK1. Neuronal cultures from E13–14 embryos from HET × HET crosses were examined after 18–20 h in vitro. Total RNA (A) or protein (B) was extracted followed by RT-PCR or western blot. Representative images from RT-PCR (A) and western blot (B) assays are shown. C Bar graph represents the average band intensity from 3 KO and 6 WT for mRNA and 7 WT and KO each for protein. Non-parametric Mann-Whitney test. D Assessment of RhoA activity using an IP-based assay. Neuronal extracts were incubated with Rhotekin PBD beads to pull down active RhoA. WT extract preincubated with GTP_γS (positive) or GDP (negative) was used as control. IP was followed by western blotting for RhoA. Two representative assays of four independent assays are shown. E In vitro kinase assay. IPed protein (either with IgG control or PFTK1) from WT cortical neuronal culture extracts was used as source of kinase and incubated with human recombinant RhoA (His-tagged) with or without GDP. Results were analyzed for phospho-serine signal. F RhoA activity assay following in vitro kinase assay. Kinase reactions were analyzed by G-LISA to test for RhoA activity. Bar graph represents mean values ± SEM. Two-way ANOVA analysis of grouped data and Sidak test for multiple comparisons are indicated on bar graph. RHO1, His-RhoA human recombinant protein