Protein tyrosine phosphatase receptor type O (Ptpro) regulates cerebellar formation during zebrafish development through modulating Fgf signaling

Abstract

Protein activities controlled by receptor protein tyrosine phosphatases (RPTPs) play comparably important roles in transducing cell surface signals into the cytoplasm by protein tyrosine kinases. Previous studies showed that several RPTPs are involved in neuronal generation, migration, and axon guidance in Drosophila, and the vertebrate hippocampus, retina, and developing limbs. However, whether the protein tyrosine phosphatase type O (ptpro), one kind of RPTP, participates in regulating vertebrate brain development is largely unknown. We isolated the zebrafish ptpro gene and found that its transcripts are primarily expressed in the embryonic and adult central nervous system. Depletion of zebrafish embryonic Ptpro by antisense morpholino oligonucleotide knockdown resulted in prominent defects in the forebrain and cerebellum, and the injected larvae died on the 4th day post-fertilization (dpf). We further investigated the function of ptpro in cerebellar development and found that the expression of ephrin-A5b (efnA5b), a Fgf signaling induced cerebellum patterning factor, was decreased while the expression of dusp6, a negative-feedback gene of Fgf signaling in the midbrain-hindbrain boundary region, was notably induced in ptpro morphants. Further analyses demonstrated that cerebellar defects of ptpro morphants were partially rescued by inhibiting Fgf signaling. Moreover, Ptpro physically interacted with the Fgf receptor 1a (Fgfr1a) and dephosphorylated Fgfr1a in a dose-dependant manner. Therefore, our findings demonstrate that Ptpro activity is required for patterning the zebrafish embryonic brain. Specifically, Ptpro regulates cerebellar formation during zebrafish development through modulating Fgf signaling.

Fig. 1

Spatial and temporal expression patterns of the zebrafish ptpro gene. Expressions of ptpro mRNAs were detected by antisense RNA whole-mount in situ hybridization (WISH). Images showing dorsal (a–d) or lateral views (a′–d′) of embryos collected at 16, 22, 48, 72, and 96 h post-fertilization (hpf). Anterior side is to the left and dorsal side is to the top. Small inserted image in (a) is from our double in situ staining to demonstrate the co-localization of the ptpro and ephA4a transcripts in the rhombomere 3 and 5. (f, g) Images of RT-PCR results for ptpro transcripts obtained from embryos at different developmental stages (f) or different adult tissues (g). Lower panels show RT-PCR results of α-actin for the controls. ce cerebellum, fb forebrain, hb hindbrain, MHB midbrain-hindbrain boundary, mb midbrain, re retina, r3/5 rhombomere 3/5, tb tailbud

Fig. 2

Inhibition of ptpro translation caused developmental defects in the central nervous system. A Sequence around the translation start site (in red) of zebrafish ptpro cDNA and the corresponding sequence of ptpro MO. B Representative images showing control (panel a) or MO-injected embryos (panel b) at 24 hpf. The head is always to the left. C Representative images showing normal (panel a), mild (panel b), and severe (panel c) phenotypes at 48 hpf. D Chart showing the statistical analysis of embryos injected with various doses of the ptpro MO or p53 MO. E Western blot of embryos injected with different doses of MOs against ptpro at 24 hpf using an anti-ptpro antibody. GAPDH was used as the loading control. F Quantitative analysis of ptpro protein levels in ptpro morphants. Values from treated ptpro MOs were normalized to matched GAPDH measurements and then expressed as a ratio of normalized values to the control. G–I Images showing results from MO effectiveness tests using the corresponding zptpro-egfp expression construct with a control (G), with the ptpro MO (H), or using the mismatched construct, MM-zptpro-egfp, with a ptpro MO (I). Construct descriptions are given in “Materials and methods”. Images were taken at 24 hpf. All embryos were injected with either 1 nl 1 % phenol red as injection control or 2 ng MO unless specified otherwise (such as in D)

Fig. 3

ptpro morphants exhibit defects in neuronal cell fate determination. (A-H’) Images of WMISH results from control (a–h) and ptpro MO-injected (a′–h′) embryos at 24 (a–g) and (a′–g′) and 72 hpf (h) and (h′). Each specific mRNA detected by WMISH is shown in the bottom right corner of each image. Lateral views with the anterior to the left and dorsal to the top in (a–g) and (a′–g′); dorsal views with the anterior to the left and right to the top in (h) and (h′). Arrow in (h′) indicates developing cerebellum. I Schematic drawing indicating the locations of dorsal thalamus (dt), diencephalon (die), epiphysis (e), hypothalamus (ht), isthmic organizer (IsO), metencephalon (met), pallial domain (p), prethalamus (pt), rhombencephalon (rho), subpallial domain (sub), telencephalon (tel), thalamus (t), zona limitans intrathalamica (ZLI). All embryos were injected with either 1 nl 1 % phenol red as injection control or 2 ng MO. The fraction of embryos displaying each phenotype is labeled on the corresponding image

Fig. 4

ptpro morphants exhibit defects in cerebellar development. (a–f′) Images of WMISH results from control (a-f) and ptpro MO-injected (a′–f′) embryos at various stages as indicated in the top right corner of each image. Each specific mRNA detected by WMISH is shown in the bottom right corner of each image. Dorsal views with the anterior to the left and right to the top in (a–e) and (a′–e′); lateral views with the anterior to the left and dorsal to the top in (f) and (f′). Arrows indicate locations of the developing cerebellum. All embryos were injected with either 1 nl 1 % phenol red as injection control or 2 ng MO. The fractions of embryos were labeled on each corresponding image

Fig. 5

Ptpro modulates the Fgf signaling pathway. a–d′ Images of WMISH results from control (a–d) and ptpro MO-injected (a′–d′) embryos at 24 hpf. e–h Images of WMISH results from control (e) and ptpro MO-injected (f–h) embryos treated with DMSO (f) or 5–10 μM SU5402 (refer to “Results“) at 76 hpf. Each specific mRNA detected by WMISH is shown in the bottom right corner of each image. Lateral views with the anterior to the left and dorsal to the top in (a–d) and (a′–d′); dorsal views with the anterior to the left and right to the top in (e and h). i Chart showing the fraction of oligo2 positive embryos injected with different combinations of the ptpro MO and SU5402. j Image showing co-immunoprecipitation (IP) analysis of the interaction between zebrafish Ptpro and Fgfr1a. An anti-flag IP was conducted with cells transfected with either an empty vector, or the flag-tagged fgfr1a plasmid, or the HA-tagged ptpro plus flag-tagged fgfr1a plasmids as indicated at the top of the image. K Top image showing the tyrosine phosphorylation analysis of ectopically expressed Fgfr1a detected with anti-phosphotyrosine antibodies (‘pY99’). Anti-flag IP was conducted with cells transfected with either empty vectors, or 0.4 μg flag-tagged fgfr1a plasmid combined with 0–0.4 μg HA-tagged ptpro and 0–0.2 Myc-tagged fgf8a plasmids as indicated at the top of the image. The bottom image shows the amount of total Fgfr1a precipitated with anti-flag antibodies in each reaction. l Top image shows the tyrosine phosphorylation analysis of ectopically expressed Fgfr1a under fgf8 stimulation. Anti-flag IP was conducted with cells transfected with 0.4 μg of the Flag-tagged fgfr1a plasmid combined with 0–0.4 μg of HA-tagged ptpro, followed by 0, 30, or 60 min of fgf8 stimulation as indicated at the top of the image. Bottom image shows the amount of total fgfr1a precipitated with anti-flag antibodies in each reaction