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, 406 (2), 235-46

Rbpj-κ Mediated Notch Signaling Plays a Critical Role in Development of Hypothalamic Kisspeptin Neurons

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Rbpj-κ Mediated Notch Signaling Plays a Critical Role in Development of Hypothalamic Kisspeptin Neurons

Matthew J Biehl et al. Dev Biol.

Abstract

The mammalian arcuate nucleus (ARC) houses neurons critical for energy homeostasis and sexual maturation. Proopiomelanocortin (POMC) and Neuropeptide Y (NPY) neurons function to balance energy intake and Kisspeptin neurons are critical for the onset of puberty and reproductive function. While the physiological roles of these neurons have been well established, their development remains unclear. We have previously shown that Notch signaling plays an important role in cell fate within the ARC of mice. Active Notch signaling prevented neural progenitors from differentiating into feeding circuit neurons, whereas conditional loss of Notch signaling lead to a premature differentiation of these neurons. Presently, we hypothesized that Kisspeptin neurons would similarly be affected by Notch manipulation. To address this, we utilized mice with a conditional deletion of the Notch signaling co-factor Rbpj-κ (Rbpj cKO), or mice persistently expressing the Notch1 intracellular domain (NICD tg) within Nkx2.1 expressing cells of the developing hypothalamus. Interestingly, we found that in both models, a lack of Kisspeptin neurons are observed. This suggests that Notch signaling must be properly titrated for formation of Kisspeptin neurons. These results led us to hypothesize that Kisspeptin neurons of the ARC may arise from a different lineage of intermediate progenitors than NPY neurons and that Notch was responsible for the fate choice between these neurons. To determine if Kisspeptin neurons of the ARC differentiate similarly through a Pomc intermediate, we utilized a genetic model expressing the tdTomato fluorescent protein in all cells that have ever expressed Pomc. We observed some Kisspeptin expressing neurons labeled with the Pomc reporter similar to NPY neurons, suggesting that these distinct neurons can arise from a common progenitor. Finally, we hypothesized that temporal differences leading to premature depletion of progenitors in cKO mice lead to our observed phenotype. Using a BrdU birthdating paradigm, we determined the percentage of NPY and Kisspeptin neurons born on embryonic days 11.5, 12.5, and 13.5. We found no difference in the timing of differentiation of either neuronal subtype, with a majority occurring at e11.5. Taken together, our findings suggest that active Notch signaling is an important molecular switch involved in instructing subpopulations of progenitor cells to differentiate into Kisspeptin neurons.

Keywords: Arcuate; Kisspeptin; Neurogenesis; Notch; Pomc, Rbpj.

Figures

Fig. 1
Fig. 1
Both loss and persistent expression of Notch signaling results in a reduction in Kisspeptin expression. Coronal sections at p0 of both control female (A) and male (D) mice show robust Pomc expression lateral to the 3V dispersed through the ARC. Rbpj cKO female (B) and male (E) mice show a more disperse Pomc expression pattern and loss of the HVZ. NICD persistent expressor female (C) and male (F) mice show an expanded third ventricle (3V) and HVZ and block of neural differentiation. Kisspeptin expression for both female (G) and male (J) mice appears more modest and restricted more laterally than Pomc to the 3V. Both sexes show a visual reduction in Kisspeptin expression in both Rbpj cKO (H, K) and NICD tg (I, L) mice. Image magnification = 100×. n=3-4. Scale bar = 50 μm.
Fig. 2
Fig. 2
Quantification of Kiss1-positive neurons of the ARC. (A) Kiss1 neurons detected by in situ hybridization in control female mice.. (B) Rbpj cKO mice show a drastic reduction in Kiss1 expression, however positive neurons were consistently observed (arrows). (C) Quantification of Kiss1 positive neurons shows a rostrocaudal gradient within control Rbpj mice, and very few cells are detected in Rbpj cKO mice. (D) Control littermates to NICD tg mice show a similar Kiss1 expression pattern to Rbpj control mice. (E) NICD tg mice show very few positive Kiss1 cells. (F) Similar to Rbpj mice, NICD control mice show a rostrocaudal gradient of Kiss1 expression and NICD tg mice show virtually no detectable cells.. Image magnification = 100×. n=3-4. Scale bar = 50 μm. * = P < 0.05. P-value = 3.1×10−7 for Rbpj cKO and 1.1×10−7 for NICD tg.
Fig. 3
Fig. 3
Both NPY and Kisspeptin neurons arise from a common Pomc lineage. (A) 200× magnification of the ARC. Many NPY-positive neurons of the ARC (green) also co-label with the fluorescent tdTomato reporter. (B) 800× confocal microscopy reveals that while many NPY-positive neurons label with the reporter protein (white arrowheads), a number of NPY-positive cells are not tdTomato positive (black arrowheads). (C) 200× magnification also reveals that a subset of Kisspeptin-positive neurons (green) label with the tdTomato reporter. (D) 800× confocal microscopy reveals that some Kisspeptin cells label with the reporter (white arrowheads), while many positive neurons do not (black arrowheads). n=3. Scale bar in C = 50 μm, D =25 μm.
Fig. 4
Fig. 4
NPY neurogenesis within the ARC occurs between e11.5 and e12.5. (A) Single BrdU injection paradigm on either e11.5, e12.5, or e13.5. Pups were collected on the day of birth (p0) and histology was performed. BrdU distribution when exposed at e11.5 (B) or e12.5 (F) appeared abundant and relatively evenly distributed throughout the ARC (N, O). When exposed at e13.5 (J), a rostrocaudal gradient was observed and far fewer cells retained the BrdU label (P). (C, G, K) NPY distribution was uniform throughout in the ventromedial region of the ARC at p0 in each of the days of embryonic exposure to BrdU. (D, H, L) A number of neurons visually double-labeled with BrdU and appeared to be born on e11.5 (D) and e12.5 (H), however few, if any, appeared to differentiate on e13.5 (L). (E, I, M) 800× magnification clearly shows several BrdU positive NPY neurons (white arrowheads) and some BrdU negative NPY neurons (black arrowheads) when injected at e11.5 (E) and e12.5 (I), but few at e13.5 (M). (Q) Quantification of double-labeled neurons show that most of neurogenesis occurs at e11.5 to e12.5 and sharply declines by e13.5. Image magnification for B-D, F-H, J-L = 200×, E, I, M = 800×. n=3. Scale bar in L = 50 μm, M = 10 μm.
Fig. 5
Fig. 5
Kisspeptin neurogenesis also occurs between e11.5 and e12.5 within the ARC. (A) An identical injection paradigm was utilized to birthdate Kisspeptin neurogenesis within the ARC. Injections occurred on either e11.5, e12.5 or e13.5 and pups were collected on p0. (B, F, J) BrdU expression patterns mimicked what was observed when birthdating NPY neurons. (C, G, K) Kisspeptin expression appeared to be more localized more laterally and dorsally with respective to NPY neurons. (D, H, L) Similar to observations made when birthdating NPY neurons, a number of Kisspeptin neurons differentiated at e11.5 (D) and e12.5 (H), and again nearly none differentiated by e13.5 (L). (E, I, M) 800× magnification clearly shows several BrdU positive Kisspeptin neurons (white arrowheads) when injected at e11.5 (E) and e12.5 (I), but again very few at e13.5 (M). BrdU negative Kisspeptin neurons were also detected at all ages of injection (black arrowheads). (Q) Quantification revealed differentiation of Kisspeptin neurons, peaking between e11.5 and e12.5, and sharply declining by e13.5. Image magnification = 200× for B-D, F-H, J-L, E, I, M = 800×. n=3. Scale bar in L = 50 μm. M = 10 μm.
Fig. 6
Fig. 6
Conditional loss of Rbpj-κ in Nkx2.1 positive cells reduces Kisspeptin expression in the ARC as late as p35. (A) NPY-positive neurons and fibers extend lateral to the 3V throughout the ARC in control mice. (B) In cKO mice, the 3V is still absent in the ARC and NPY-positive neurons are scattered throughout the entire region. (C) Kisspeptin-positive neurons also lie lateral to the 3V and appear to have a similar rostrocaudal gradient as observed at p0. (D) cKO mice persistently exhibit a loss of Kisspeptin neurons within the ARC as far out as 5 weeks of age. (E) TH-positive neurons of the periventricular nucleus are present in the RP3V and extend into the AVPV in control mice. (F) TH-positive neurons are also present within the RP3V of Rbpj cKO mice and found throughout the defined region. (G) Kisspeptin-positive cell bodies and fibers are found just lateral to the rostral 3V in the RP3V/AVPV in control mice. (H) Similar to within the ARC, virtually no Kisspeptin-postitive cells or fibers are found throughout the RP3V/AVPV. Image magnification = 100×. n=4. Scale bar in H = 50 μm.
Fig. 7
Fig. 7
Female and male Rbpj cKO mice face severe reproductive challenges. (A) Results of a single mating challenge of Rbpj control of cKO males and females with age-matched CD-1 mice of proven reproductive success. Data are represented as a percentage of mice who successfully produced litters and average litter size has been reported. 1 Denotes the litter size of the single male Rbpj cKO who sired a litter. (B) Representative cyclicity of 3 Rbpj control (left) or cKO (right) females cycled for 14 consecutive days. Note that Rbpj cKO females entered the proestrus/metestrus phase, but never fully reached estrus. n=8-11 individuals.
Fig. 8
Fig. 8
Proposed model of neural differentiation of SOX2-positive neurons of the HVZ. Previous studies have suggested that nearly all cell-types found within the ARC arise from SOX2-positive progenitors of the HVZ. Many of these cells will differentiate into a Pomc expressing immature progenitor cell (Pomc Prog.), and can then further mature into at least 3 other neural subtypes. While a subset of Kiss1 neurons appear to arise from a Pomc lineage, a number may come from another progenitor pool. There is the formal possibility that all Kisspeptin and NPY neurons are derived from a Pomc expressing progenitor as well. Persistent Notch signaling appears to completely block differentiation of each neural subtype, but Rbpjκ-dependent Notch signaling appears necessary for development of Kisspeptin neurons.

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