Fgf10-expressing tanycytes add new neurons to the appetite/energy-balance regulating centers of the postnatal and adult hypothalamus

Abstract

Increasing evidence suggests that neurogenesis occurs in the postnatal and adult mammalian hypothalamus. However, the identity and location of the putative progenitor cells is under much debate, and little is known about the dynamics of neurogenesis in unchallenged brain. Previously, we postulated that Fibroblast growth factor 10-expressing (Fgf10(+)) tanycytes constitute a population of progenitor cells in the mouse hypothalamus. Here, we show that Fgf10(+) tanycytes express markers of neural stem/progenitor cells, divide late into postnatal life, and can generate both neurons and astrocytes in vivo. Stage-specific lineage-tracing of Fgf10(+) tanycytes using Fgf10-creERT2 mice, reveals robust neurogenesis at postnatal day 28 (P28), lasting as late as P60. Furthermore, we present evidence for amplification of Fgf10-lineage traced neural cells within the hypothalamic parenchyma itself. The neuronal descendants of Fgf10(+) tanycytes predominantly populate the arcuate nucleus, a subset of which express the orexigenic neuronal marker, Neuropeptide-Y, and respond to fasting and leptin-induced signaling. These studies provide direct evidence in support of hypothalamic neurogenesis during late postnatal and adult life, and identify Fgf10(+) tanycytes as a source of parenchymal neurons with putative roles in appetite and energy balance.

Figure 1.

Fgf10⁺ tanycytes express neural stem/progenitor markers and are most abundant in the central regions of the median eminence. Images show coronal vibratome (A–E', P–R) and cryostat (F–O) sections of P60 Fgf10^nlacZ brains, at the level of ME (bregma −1.70 or −2.06, as indicated). Fgf10⁺ tanycytes, visualized by anti-βgal immunolabeling, coexpress markers of neural stem/progenitor cells: BLBP (C–D”), Musashi1 (F–J), nestin (K–O), and Sox2 (P–R), but not GFAP (A–B'). Note that expression of Sox2 is not limited to ependymal cells, but also present in some parenchymal cells. E, E', Domain of GLAST expression in the 3V ependyma as visualized by anti-GFP immunolabeling of tamoxifen-treated GLAST^creERT2:R26^GFP brain. Dashed lines in E' show the ventral limit of GLAST expression. S, T, Fgf10⁺ tanycytes are more abundant in the central regions of the ME, where only a subpopulation expresses BLBP or nestin. Scale bars: A, B', D', E, P, 50 μm; A', E', P', 25 μm; D”, G, L, 10 μm.

Figure 2.

Parenchymal descendants of Fgf10⁺ tanycytes express mature neural cell type markers and respond to acute fasting. A–D″, Expression of NeuN (A–B”), DARPP32 (D–D''), NPY (E, E'), and more rarely, GFAP (C, C') by parenchymal βgal⁺ cells, but not βgal⁺ tanycytes, in the hypothalamus of Fgf10^nlacZ mice. C, Side view of a three-dimensionally reconstructed image. E–E″, A subset of parenchymal βgal⁺ cells express the orexigenic neurotransmitter, NPY. E', E”, Cut views of a βgal⁺/NPY⁺ cell. F–G”, Upregulation of c-Fos expression and phosphorylated STAT3 in a subset of parenchymal βgal⁺ cells, in response to acute fasting alone (F–F”), or after administration of leptin to fasted mice (G–G”). Arc, Arcuate nucleus. Scale bars: A, C, 50 μm; C', E–G, 20 μm; B–B”, D-D”, F', F”, G', G”, 5 μm.

Figure 3.

Abundance and distribution of proliferating cells in the postnatal and adult hypothalamus. A, B, Cumulative exposure of P60 Fgf10^nlacZ mice to BrdU or EdU results in labeling of numerous cells in the hypothalamus with more cells being labeled after BrdU than EdU application. Most BrdU- or EdU-incorporating cells are found in the parenchymal compartment. C, A higher level of cell proliferation is observed in animals pulsed at P28–P32 (early post-weaning stage) compared with adult (P60–P70), with the difference relating mostly to parenchymally located cells than ependymal ME cells (where tanycytes reside). D, Comparable numbers of BrdU⁺/βgal⁺ tanycytes are found in P28–P32 and P60-pulsed animals (similar data were obtained with EdU; data not shown). Number of animals analyzed per condition is shown inside bars. *p < 0.05; **p < 0.01.

Figure 4.

Fgf10⁺ tanycytes can incorporate BrdU and EdU. Examples of βgal⁺ cells incorporating BrdU (A–C') or EdU (D–F') in Fgf10^nlacZ brains pulsed at P28 (A, A') or as adults (B–F'). Conical arrowheads heads point to pairs of labeled nuclei located within the ependymal lining, while larger arrows highlight pairs of labeled cells within the nearby parenchyma (in A, F). Note the parallel (C', D') versus perpendicular (A', E') planes of cell division with respect to the plane of the ventricular surface (3V). Scale bars: B, 100 μm; A, D, E, F, 50 μm; A', B', C', D', E', F', 10 μm.

Figure 5.

Abundance and contribution of Fgf10⁺ tanycytes to hypothalamic parenchymal cells. A–I, Apparent descendants of Fgf10⁺ tanycytes, revealed by βgal immunolabeling, occupy the arcuate (Arc), ventromedial (VM), and to a lesser degree the dorsomedial (DM) and lateral (LH) hypothalamic nuclei at P60. J–N, Cross age comparison shows a conserved presence of such descendants in the arcuate nucleus. Note the reduction in the intensity of βgal label in parenchymal, but not ependymal (tanycytic) cells at P150 and P400. O–Q, Quantification of βgal⁺ cells in parenchymal and ependymal/ME compartments. Data point on each graph represent P4, P30, P60, P150, and P400, respectively. n = 3 for each age group. *p < 0.05; **p < 0.01. Scale bars: A–I, 75 μm; J–N, 50 μm.

Figure 6.

Direct lineage tracing of Fgf10⁺ tanycytes using Fgf10^creERT2 and R26^LacZ reporter mice. A, Schematic of Fgf10 gene locus showing its coding exons (hatched bars), the knock-in creERT2 transgene, as well as the position of genotyping primers (P1–P3) and the resulting PCR products, used to distinguish between the two alleles. B, Strategy for tamoxifen application and time course analysis of tamoxifen-treated animals. C–K', Images of recombined (Xgal⁺) cells in the hypothalamus of tamoxifen-treated Fgf10^creERT2:R26^LacZ mice analyzed at short (C–D') or long (E–K') time-points after the last tamoxifen dose. Large white arrow in J points to the absence of recombined tanycytes at bregma −1.06, contrasted with the presence of Xgal⁺ cells in the neighboring parenchyma (black arrows). Large open arrows in I and K point to pairs of Xgal⁺ parenchymal cells, magnified in I' and K', respectively. L–L”, In situ hybridization using DIG-labeled probes shows expression of Fgf10 by ventral ependymal cells (L, L', arrows) but neither by dorsal ependymal (L”) nor by neighboring parenchymal cells at P28. M–N′, Very short term recombination analysis after 3 (M, M') or 8 (N, N′) d of tamoxifen-treatment of Fgf10^creERT2:R26^TOM mice. Recombination is restricted to tanycytes after a 3 d treatment (large arrows and insets in M') while both recombined tanycytes (large arrows) and parenchymal cells (small arrows) are evident following an 8 d pulse (N′). Scale bars: C, D, H, L, M', N′, 100 μm; C', D', E–G, I–K, L', 50 μm; L”, 25 μm; I', K', 5 μm.

Figure 7.

Quantification and spatial distribution of Fgf10^creERT2 lineage-traced cells in early postnatal and young adult mice. A, In general, more Xgal⁺ recombined cells are detected in animals treated with tamoxifen at P28–P32 (a week after weaning) versus adulthood (P53–P83), whether examined after short (24–27 d) or long (39–83 d) survival time points. B, The number of recombined tanycytes is not significantly different in postweaned and adult animals analyzed at short time points. A significant difference emerges with longer time-point analysis. C, There is a dramatic amplification of parenchymally located recombined cells in postweaned (P28–P32) treated animals when compared with adults. D, D', Quantifications under shorter tamoxifen-treatment/survival time periods using R26^Tom allele. D, After a 3 d treatment of P28 Fgf10^creERT2:R26^tomato mice, the vast majority of recombined (Tom⁺) cells are composed of tanycytes, residing in the ependymal layer. D', Longer treatment/survival of P28-treated animals results in a significant amplification of parenchymally located recombined cells. Number within bars represent the number of animals analyzed per treatment. *p < 0.05; ***p < 0.001.

Figure 8.

Fgf10^creERT2 lineage-traced cells generate neurons in multiple hypothalamic nuclei and can respond to acute fasting. A–G, Immunoperoxidase labeling for NeuN (A–D') or GFAP (E–G) shows that adult lineage traced (Xgal⁺) cells can differentiate into neurons of the arcuate and ventromedial nuclei. Lineage-traced cells do not express GFAP (E–G). H–K, Better visualization of P28 lineage-traced cells through the use of Fgf10^creERT2:R26^tomato line confirms the radial morphology of the recombined tanycytes (long arrows) as well as the elaborate axonal/dendritic arborization (H, short arrows) of their parenchymally located neural descendants, whose neuronal identity is confirmed by NeuN labeling (short arrows in I–K). L–O, An example of a P28 lineage-traced (Tom⁺) arcuate neuron that has responded to fasting and in vivo leptin treatment by expressing phospho-STAT3. Scale bars: A–D, E–H, 75 μm; L, 30 μm; A', B', C', D', I, M, 15 μm.