Apical polarity protein PrkCi is necessary for maintenance of spinal cord precursors in zebrafish

Abstract

During development, neural precursors divide to produce new precursors and cells that differentiate as neurons and glia. In Drosophila, apicobasal polarity and orientation of the mitotic spindle play important roles in specifying the progeny of neural precursors for different fates. We examined orientation of zebrafish spinal cord precursors using time-lapse imaging and tested the function of protein kinase C, iota (PrkCi), a member of the Par complex of proteins necessary for apicobasal polarity in the nervous system. We found that nearly all precursors divide within the plane of the neuroepithelium of wild-type embryos even when they must produce cells that have different fates. In the absence of PrkCi function, neural precursor divisions become oblique during late embryogenesis and excess oligodendrocytes form concomitant with loss of dividing cells. We conclude that PrkCi function and planar divisions are necessary for asymmetric, self-renewing division of spinal cord precursors.

Fig. 1

Zebrafish spinal cord cells have apical polarity, which requires protein kinase C, iota (PrkCi) function. All panels show transverse sections through trunk spinal cord, dorsal up. Dashed circle marks the perimeter of the spinal cord. Arrowheads and arrows indicate central canal and medial septum, respectively. A–F: Sections labeled with anti-PrkCi/z antibody. A,B: PrkC is localized to the medial septum and central canal of wild-type embryos at 24 and 48 hours postfertilization (hpf). C: At 72 hpf, PrkC is absent from the medial septum but remains around the central canal of wild-type larvae. D: At 24 hpf, PrkC localization is normal in has^−/− embryos. E: However, by 48 hpf very little PrkC is evident at the medial septum and, although PrkC remains around the central canal, the central canal is reduced in size (E) or entirely absent (not shown). F: Sections of 72 hpf has^−/− larvae similarly reveal PrkC localization around an abnormally small and discontinuous central canal. The apical protein ZO-1 has a similar localization pattern to PrkC in wild-type and has^−/− embryos and larvae (G–L). Scale bar = 20 µM.

Fig. 2

Protein kinase C, iota (PrkCi) maintains planar divisions of cells that divide along the central canal. A–D: Frames captured from time-lapse movies, from a dorsal view, of wild-type and has^−/− embryos carrying the Tg(h2afv:egfp) transgene. Numbers in upper right corners indicate time elapsed from beginning of imaging at 27 hours postfertilization (hpf). Dashed circles outline dividing cells, arrows point to the central canal and bi-directional arrows indicate orientation of the mitotic spindle and angle of division. A,B: In both wild-type and has^−/− embryos, divisions that occur before 30 hpf are planar. The central canal is less distinct in has^−/− embryos than in wild-type. C,D: Divisions after 30 hpf remain planar in wild-type embryos (C) but become oblique in has^−/− embryos (D). The central canal is indistinct and appears to have been replaced by cells. E,F: Quantification of angles of division in wild-type and has^−/− embryos (E). In wild-type most division planes are within 15° of the plane of the epithelium, indicated by the central canal. has^−/− embryos have numerous divisions greater than 15°. Scale bar = 24 µM.

Fig. 3

Loss of protein kinase C, iota (PrkCi) function produces excess oligodendrocyte progenitor cells (OPCs). All images are of transverse sections through trunk spinal cord, dorsal up. Outlined circle marks the perimeter of the spinal cord. A–F: The number and distribution of neurons, marked by anti-Hu labeling, are similar in wild-type and has^−/− embryos at 24, 48, and 72 hours postfertilization (hpf). G–L: Isl⁺ motor neurons (brackets), interneurons (solid arrowheads), and Rohon-Beard sensory neurons (open arrowheads) are similar in wild-type and has^−/− embryos and larvae through 72 hpf. M,N: Anti-Sox10 labeling reveals excess OPCs in 72 hpf has−/− larva (N) compared with wildtype (M). O,P: cldnk RNA expression marking differentiating oligodendrocytes. O: In wild-type, cldnk⁺ cells (arrows) are at the pial surface in dorsal and ventral spinal cord. P: has^−/− larvae have excess cldnk⁺ cells and some occupy ectopic positions in medial spinal cord. Q: Quantification of spinal cord Isl⁺ motor neurons in the spinal cord between wild-type and has^−/− embryos at 48 and 72 hpf. R: Quantification of spinal cord Sox10⁺ OPCs. Error bars represent SEM. Statistical significance was determined using Student’s t-test. Scale bar = 20 µM.

Fig. 4

Protein kinase C, iota (PrkCi) is required to maintain ventral spinal cord cells with precursor characteristics. All images are of transverse sections through trunk spinal cord, dorsal up. Outlined circle marks the perimeter of the spinal cord. A–F: Enhanced green fluorescent protein (EGFP) expression driven by the Tg(olig2:egfp) promoter. A,B: In wild-type embryos, EGFP marks motor neurons and pMN precursors through 48 hours postfertilization (hpf). C: By 72 hpf, EGFP⁺ fibers (arrowheads), marking precursors that persist into larval stage, become evident. D,E: EGFP expression appears normal in has^−/− embryos at 24 and 48 hpf. F: At 72 hpf, few EGFP⁺ radial fibers are evident. Asterisks mark dorsally migrated OPCs. G–L: Zrf-1 immunocytochemistry to label radial glial fibers. G–I: In wild-type embryos and larvae, radial fibers are distributed uniformly through the spinal cord. In ventral spinal cord, the apical membrane of some radial glia surround the central canal (arrowheads). J–L: In has mutants, radial fibers initially appear normal, but by 72 hpf, a gap appears in ventral spinal cord (bracket) and the central canal is reduced or absent. M,N: sox19b RNA expression. M: At 72 hpf in wild-type, sox19b expression marks cells near the central canal (arrow). N: At 72 hpf sox19b is not expressed at its normal position in has^−/− larva (bracket). Instead, ventral and dorsal spinal cord cells express sox19b. Scale bar = 20 µM.

Fig. 5

Loss of PrkCi function causes a transient excess and then deficit of dividing spinal cord precursors. A–D: Images, from dorsal view and focused on the trunk spinal cord, of wild-type and has^−/− embryos and larvae carrying the Tg(olig2:egfp) reporter and labeled with anti-PH3 antibody to mark M phase cells. In wild-type most mitotic cells (arrowheads) are adjacent to the central canal (arrow). A,B: More cells divide at 2 days postfertilization (dpf; A) than at 3 dpf (B). C,D: In has^−/− embryos, many EGFP⁺ cells form rosettes (brackets) surrounding discontinuous portions of central canal (asterisks). PH3⁺ are usually only found within rosettes. E–H: Images, from dorsal view and focused on the trunk spinal cord, of wild-type and has^−/− embryos and larvae labeled with anti-PH3 antibody (red) and anti–ZO-1 antibody (blue) to mark apical membrane. E,F: In wild-type, ZO-1 localization outlines a continuous central canal closely associated with PH3⁺ mitotic cells. G,H: In has^−/− embryos and larvae, ZO-1 labeling is discontinuous but mitotic cells are nearly always associated with remaining apical membrane. I: Quantification of PH3⁺ cells at 2 and 3 dpf over a 288 µm length of trunk spinal cord. Error bars represent SEM. Statistical significance was determined using Student’s t-test.

Fig. 6

Loss of protein kinase C, iota (PrkCi) function causes formation of excess oligodendrocyte progenitor cells (OPCs) from pMN precursors. Panels show frames captured from time-lapse videos of wild-type and has^−/− embryos carrying the Tg(olig2:egfp) reporter. Dorsal is up and time elapsed from beginning of imaging at 50 hours postfertilization (hpf) is indicated in upper right corners of panels. Brackets mark pMN precursor domain in ventral spinal cord. A: Wild-type embryo. Arrowheads mark OPCs migrating dorsally from the ventral pMN precursor domain. B: has−/− embryo. More OPCs (arrowheads) emerge from the pMN domain than in wild-type over a similar period of time. C: Quantification of dorsally migrating OPCs. Data were obtained from time-lapse movies of 6 wild-type and 5 has^−/− embryos. Error bars represent SEM. Statistical significance was determined using Student’s t-test.