Regulation of neurogenesis by interkinetic nuclear migration through an apical-basal notch gradient

Abstract

The different cell types in the central nervous system develop from a common pool of progenitor cells. The nuclei of progenitors move between the apical and basal surfaces of the neuroepithelium in phase with their cell cycle, a process termed interkinetic nuclear migration (INM). In the retina of zebrafish mikre oko (mok) mutants, in which the motor protein Dynactin-1 is disrupted, interkinetic nuclei migrate more rapidly and deeply to the basal side and more slowly to the apical side. We found that Notch signaling is predominantly activated on the apical side in both mutants and wild-type. Mutant progenitors are, thus, less exposed to Notch and exit the cell cycle prematurely. This leads to an overproduction of early-born retinal ganglion cells (RGCs) at the expense of later-born interneurons and glia. Our data indicate that the function of INM is to balance the exposure of progenitor nuclei to neurogenic versus proliferative signals.

Figure 1. mok^s309 mutants have a complex retina phenotype

A, B. Whole-mount lateral views of live 4.5 dpf zebrafish larvae. In mok^s309 mutants (B), the eyes are smaller than in the wildtype (A), but external morphology is otherwise indistinguishable. C, D. Horizontal sections of 5 dpf retinas stained with DAPI (blue) and Zpr1 antibody (green). mok^s309 retinas (D) have an expanded GCL (dashed white lines), compared to wildtype (C), and no photoreceptors, as revealed by Zpr1 staining. E, F. Coronal sections of 3 dpf retinas stained by TUNEL assay. mok^s309 retinas (F) have increased apoptosis, particularly in the photoreceptor layer (stars), compared to wildtype (E). G, H. Horizontal sections of 48 hpf retinas expressing GFP driven by the atoh7 promoter (green), and stained with DAPI (blue) and HuC/D antibody (red). mok^s309 retinas (H) have increased number of GFP-expressing RGCs compared to wildtype (G). Scale bars: 500µm (A, B), 100µm (C–H).

Figure 2. mok^s309 retinas have increased RGCs and decreased bipolar and Müller glia cells due to premature neurogenesis

A–H. Horizontal 5 dpf retina sections. GFP expression under the control of atoh7 (A, E) and brn3c (Pou4f3) (B, F) promoters reveal an increase in RGCs in mok^s309 (E, F) as compared to the wildtype (A, B), with some RGCs ectopically located outside the GCL (yellow arrowheads in E, F). The Müller glia marker GS (C, G) and the bipolar cell marker PKCạ (D, H) show fewer immunoreactive cell bodies (yellow arrow heads) in mok^s309 (G, H) as compared to wildtype (C, D). I–P. Horizontal 50 hpf retina sections stained for IdU (green, injected at 26 hpf), BrdU (red, injected at 38 hpf), and DAPI (blue). mok^s309 retinas (M-P) show more IdU-positive and BrdU -negative cells than wildtype, indicating that a larger number of progenitors had exited the cell cycle between 26 and 38 hpf. Scale bars: 100µm.

Figure 3. Cell transplantation analysis reveals that mok acts cell-autonomously

A–D. Representative sections of 5 dpf chimeric retinas. The transplanted cells are clearly identified by the expression of H2A-GFP marker (green) in their nuclei. Cell transplantation shows that mok^s309 mutant clones in wildtype host retinas (B) have a higher propensity to generate neurons located in the GCL compared to control (A). Conversely, wildtype clones in mok^s309 host retinas preferentially generate INL neurons (C). mok^s309 mutant clones in mok^s309 host retinas are shown for comparison (D). Dashed lines indicate the outer limit of the GCL. Scale bars: 100µm. E. Quantification of the transplantation results showing the distribution of clones in the three retinal nuclear layers. (***) p < 0.001; (*) p < 0.01

Figure 4. Expression of Dnct1 and its function in INM

A. Western blotting of extracts from 4 dpf embryos shows that Dnct1 is undetectable in mok^s309. B. Time course analysis of Dnct1 expression by western blot in wildtype zebrafish (numbers on top indicate hours after fertilization). C, D. Whole-mount in situ hybridization shows dnct1 enriched in the head and eye region (C) and in the notochord (D) E, F. Coronal sections of 2 dpf retinas stained for the mitotic marker PH3. In mok^s309 (F), a number of mitotic cells are sparsely located throughout the retina, while in the wildtype (E) they are confined within 2–3 cell diameters from the ventricular surface. Dashed lines demarcate the apical (right) and basal (left) domains. Asterisks indicate mitotic cells residing in the developing lens. G. Scatter plot of wildtype (gray triangles) and mok^s309 mutant (black squares) circles, showing the maximum basal distance of nuclei during INM from 30–48 hpf. H. Histogram showing that these populations are statistically different (p=0.001, Wilcoxon two sample test). Scale bars (C, D): 100µm.

Figure 5. A gradient of Notch signaling along the apical-basal axis of the developing retina

A–C. Coronal sections of 26 hpf retinas showing mRNA expression levels of components of the Notch/Delta signaling pathway. In situ hybridization shows higher levels of notch1a close to the apical surface of the retina (A), deltaB and deltaC close to the basal surface (B, C). D. Optical section of a 33 hpf retina expressing her4:dRFP and H2A-GFP transgenes in a mosaic manner. E, F. Coronal sections of 26 hpf retinas stained with anti-DeltaC antibody, showing punctuated cytoplasmic staining distributed in the basal half of the tissue both in wildtype and mok^s309 retinas. G, H. Coronal sections of 26 hpf retinas stained with anti-Dnct1 antibody, showing an enrichment at the apical surface in wildtype retinas (G), which is virtually absent in mutants (H). I. Coronal section of 26 hpf retina; α–Tubulin staining reveals the parallel orientation of microtubules to the apical-basal axis. J–L. Sections of mouse retina. Activated-Notch1 antibody labels a subset of nuclei at the apical surface (J). Anti-Dnct1 and anti-BBS4 antibodies show a cytoplasmic, punctated staining enriched at the apical surface γ–Tubulin staining (G, H, K, L) reveals the apical localization of the centrioles in retinal progenitors. In panels D-L, DAPI (blue) stains the nuclei. In all panels, apical surface is on the left. Scale bars: 25µm (A-I), 50 µm (J-L).

Figure 6. Atoh7 loss of function and Notch activation can rescue late cell fates in mok^s309

A–D. Compared to wildtype (A), mok mutants (B) have an excess of RGCs and few bipolar cells, labeled by PKCa immunoreactivity. lak (atoh7) mutants fail to generate RGCs and overproduce bipolar neurons (C). lak/mok double mutants have rescued bipolar cell production. E–H. Heat shock induction of an activated form of Notch receptor NICD at 32 hpf drives progenitors towards a Müller glia fate (GS-positive) in both wildtype (G) and mok^s309 retinas (H). The presence of the transgenes (hsp:Gal4, UAS:NICD) has no effect on Müller glia differentiation in the absence of heat shock (E,F). Scale bars: 100µm.

Figure 7. Disruption of the nuclear anchor to dynactin phenocopies the mok^s309 mutation

A, B. Expression of the Müller glia marker GS is reduced in the retina of larvae injected with an syne2a-MO (B) compared to control MO-injected larvae (A). C, D. Representative examples of sections of 5 dpf retinas overexpressing a control vector (C), or a dominant negative Syne2a (KASH, D), under the control of a heat-shock promoter. The cells that express the constructs are identified by the expression of GFP marker (green). Clones of cells that express the syne2a dominant-negative construct preferentially generate GCL neurons. Scale bars: 100µm. E. Quantification of the KASH overexpression results, showing the distribution of clones in the three retinal nuclear layers. (*) p < 0.01; (**) p < 0.005. F. A model of the mechanism that couples INM with graded Notch activation.