Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 Jan 1;143(1):45-53.
doi: 10.1242/dev.126813. Epub 2015 Nov 24.

Hypothalamic radial glia function as self-renewing neural progenitors in the absence of Wnt/β-catenin signaling

Robert N Duncan  1 , Yuanyuan Xie  1 , Adam D McPherson  1 , Andrew V Taibi  1 , Joshua L Bonkowsky  1 , Adam D Douglass  1 , Richard I Dorsky  2
Affiliations

Hypothalamic radial glia function as self-renewing neural progenitors in the absence of Wnt/β-catenin signaling

Robert N Duncan et al. Development. .

Abstract

The vertebrate hypothalamus contains persistent radial glia that have been proposed to function as neural progenitors. In zebrafish, a high level of postembryonic hypothalamic neurogenesis has been observed, but the role of radial glia in generating these new neurons is unclear. We have used inducible Cre-mediated lineage labeling to show that a population of hypothalamic radial glia undergoes self-renewal and generates multiple neuronal subtypes at larval stages. Whereas Wnt/β-catenin signaling has been demonstrated to promote the expansion of other stem and progenitor cell populations, we find that Wnt/β-catenin pathway activity inhibits this process in hypothalamic radial glia and is not required for their self-renewal. By contrast, Wnt/β-catenin signaling is required for the differentiation of a specific subset of radial glial neuronal progeny residing along the ventricular surface. We also show that partial genetic ablation of hypothalamic radial glia or their progeny causes a net increase in their proliferation, which is also independent of Wnt/β-catenin signaling. Hypothalamic radial glia in the zebrafish larva thus exhibit several key characteristics of a neural stem cell population, and our data support the idea that Wnt pathway function may not be homogeneous in all stem or progenitor cells.

Keywords: Hypothalamus; Neural progenitors; Radial glia; Wnt signaling; Zebrafish.

PubMed Disclaimer

Conflict of interest statement

Competing interests

The authors declare no competing or financial interests.

Figures

Fig. 1.
Fig. 1.
Radial glia in the hypothalamic posterior recess are not Wnt-responsive. (A) Diagram of the hypothalamic posterior recess from a ventral view of a 5 dpf zebrafish brain. Radial glia are in green; the red box indicates the area depicted in B. (B) High-magnification optical section of her4.3:EGFP expression, showing the position of cell soma and radial processes. (C) Radial glial cells in the 5 dpf posterior recess can be labeled with anti-Glutamine synthetase (GS, blue), a her4.3:EGFP transgene (green), and a Gal4 enhancer trap line (red). Yellow box indicates the region shown beneath. A triple-labeled cell is indicated by red circles. (D) Most cells expressing GS or her4.3:EGFP also express the other marker. Error bars indicate s.e.m.; n=22 optical sections from two brains. (E) Most GS+ and her4.3:EGFP+ radial glia do not express the Wnt reporter transgene 7xTCF-Xla.Siam:GFP. Yellow box indicates region shown beneath. A reporter-negative radial glial cell is indicated by green circles, and a reporter-expressing non-glial cell is indicated by red circles. (F) GS+ cells are not reduced in number in lef1 mutants at 5 dpf. Images are single optical sections from ventral views of whole-mount brains. WT, wild type; nuclei are stained with Hoechst 33342 (blue). Scale bars: 10 µm.
Fig. 2.
Fig. 2.
Hypothalamic radial glia are self-renewing neural progenitors. (A) Timeline and schematic of experiments. Cells expressing −3her4.1:ERT2-Cre-ERT and ubi:loxP-eGFP-loxP-mCherry were genetically labeled by the addition of 5 µM 4-OHT from 5-6 dpf. Labeled progeny (red) comprise expanding radial units spanning hemispheres of the posterior recess. Processes of GS+ radial glia (green) span the tissue, and nuclei occupy variable positions from the ventricle to the outer edge. Neurons (blue) are located either adjacent to the ventricle or at the outer edge. (B) Immediately following conversion, the few labeled mCherry+ cells are also GS+. Yellow boxes indicate regions shown beneath. (C) Six days after conversion, labeled cells extend radially and include GS+ (yellow box) and GS− (green box) cells. (D) Five weeks after 4-OHT addition, discrete groups of mCherry+ cells extend from the ventricle (dashed line). (E-H) mCherry+ progeny 5 weeks after recombination include GS+ radial glia (E), HuC/D+ neurons (F), 5HT+ neurons (G) and th2:gfp+ dopaminergic neurons (H). Yellow boxes indicate regions shown beneath, and double-labeled cells are indicated by red circles. Green signal in E,F is ubi:GFP from unconverted cells. Images are single optical sections from ventral views of whole-mount brains. Scale bars: 10 µm.
Fig. 3.
Fig. 3.
Wnt/β-catenin signaling is only required for the differentiation of a specific subset of ventricular neurons. (A,B) Following recombination of −3her4.1:ERT2-Cre-ERT+ progeny from 5-6 dpf, expression of Dkk1 from 6-9 dpf does not affect the average number of labeled cells (A) or the percentage of GS+ cells (B) in the lineage. (C) The differentiation of HuC/D+ neurons from labeled radial glia is not inhibited by Dkk1 expression. (D) Representative images of labeling for lineage and GS in control and Dkk1-expressing larvae. (E) In lef1 mutants there is no change in the percentage of GS+ cells after conversion from 5-6 dpf and analysis at 9 dpf. (F,G) lef1-dependent ventricular neurons fail to arise from the radial glial lineage, whereas other non-ventricular neurons are not significantly affected. Yellow boxes indicate areas shown to the right. Images are single optical sections from ventral views of whole-mount brains. Error bars indicate s.e.m.; n=50 confocal slices from five brains for each experiment. Scale bars: 10 µm.
Fig. 4.
Fig. 4.
Hypothalamic radial glia respond to partial ablation by increasing proliferative activity. (A,B) Partial ablation of NTR-mCherry-expressing radial glia (red) by incubation in 1 mM MTZ from 5-6 dpf; DMSO provides a control. (C-F) After partial ablation from 5-6 dpf and BrdU labeling from 7-8 dpf (C,D; yellow box indicates region shown beneath), there is a significant increase in the number of BrdU+ radial glia labeled either by GS (C-E) or by her4.3:EGFP (F) expression. (G,H) Inhibition of Wnt signaling by Dkk1 expression (G), or lef1 mutation (H), does not block the increase in BrdU labeling following partial ablation. Images are maximum-intensity z-projections (A,B) or single optical sections (C,D) from ventral views of whole-mount brains. Error bars indicate s.e.m.; n=40 optical sections from four brains for each experiment. Scale bars: 10 µm.
Fig. 5.
Fig. 5.
Hypothalamic radial glia proliferate in response to ablation of dopaminergic progeny. (A,B) The th2:GFP (5 dpf, A) and th2:Gal4 (9 dpf, B) transgenes do not label GS+ radial glia. Yellow boxes indicate regions shown beneath. (C) Ablation of th2:Gal4+ cells from 5-6 dpf leads to increased BrdU labeling only at 8-9 dpf. (D,E) Ablation of th2:Gal4+ cells increases the number and percentage of GS+ radial glia, as well as the number of GS− cells, labeled with BrdU from 8-9 dpf. (F) The overall number of GS+ radial glia is decreased at 9 dpf following ablation of th2:Gal4+ cells. Images are single optical sections from ventral views of whole-mount brains. Error bars indicate s.e.m.; n=50 optical sections from five brains for each experiment. Scale bars: 10 µm.
Fig. 6.
Fig. 6.
Wnt/β-catenin signaling activation blocks expansion of the radial glial population. (A) Induction of Wnt8a at 5 dpf leads to a decrease in the number of GS+ radial glia at 6 dpf. (B) Addition of 4 µM BIO, a Gsk3β inhibitor, daily from 6-9 dpf leads to a decrease in GS+ radial glia. (C) Representative images of labeling for GS in control and BIO-treated larvae. Images are single optical sections from ventral views of whole-mount brains. Scale bar: 10 µm. (D) Following recombination from 5-6 dpf, incubation in BIO causes a significant decrease in the number of labeled progeny at 9 dpf. (E) Incubation in BIO causes a significant increase in the percentage of GS+ cells within the labeled lineage. Error bars indicate s.e.m.; n=40 optical sections from four brains for each experiment.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Agathocleous M., Iordanova I., Willardsen M. I., Xue X. Y., Vetter M. L., Harris W. A. and Moore K. B. (2009). A directional Wnt/beta-catenin-Sox2-proneural pathway regulates the transition from proliferation to differentiation in the Xenopus retina. Development 136, 3289-3299. 10.1242/dev.040451 - DOI - PMC - PubMed
    1. Barbosa J. S., Sanchez-Gonzalez R., Di Giaimo R., Baumgart E. V., Theis F. J., Gotz M. and Ninkovic J. (2015). Neurodevelopment. Live imaging of adult neural stem cell behavior in the intact and injured zebrafish brain. Science 348, 789-793. 10.1126/science.aaa2729 - DOI - PubMed
    1. Blanpain C. and Fuchs E. (2009). Epidermal homeostasis: a balancing act of stem cells in the skin. Nat. Rev. Mol. Cell Biol. 10, 207-217. 10.1038/nrm2636 - DOI - PMC - PubMed
    1. Boniface E. J., Lu J., Victoroff T., Zhu M. and Chen W. (2009). FlEx-based transgenic reporter lines for visualization of Cre and Flp activity in live zebrafish. Genesis 47, 484-491. 10.1002/dvg.20526 - DOI - PMC - PubMed
    1. Briona L. K. and Dorsky R. I. (2014). Radial glial progenitors repair the zebrafish spinal cord following transection. Exp. Neurol. 256, 81-92. 10.1016/j.expneurol.2014.03.017 - DOI - PMC - PubMed

Publication types

MeSH terms

LinkOut - more resources