Topography of Somatostatin Gene Expression Relative to Molecular Progenitor Domains during Ontogeny of the Mouse Hypothalamus

Abstract

The hypothalamus comprises alar, basal, and floor plate developmental compartments. Recent molecular data support a rostrocaudal subdivision into rostral (terminal) and caudal (peduncular) halves. In this context, the distribution of neuronal populations expressing somatostatin (Sst) mRNA was analyzed in the developing mouse hypothalamus, comparing with the expression pattern of the genes Orthopedia (Otp), Distal-less 5 (Dlx5), Sonic Hedgehog (Shh), and Nk2 homeobox 1 (Nkx2.1). At embryonic day 10.5 (E10.5), Sst mRNA was first detectable in the anterobasal nucleus, a Nkx2.1-, Shh-, and Otp-positive basal domain. By E13.5, nascent Sst expression was also related to two additional Otp-positive domains within the alar plate and one in the basal plate. In the alar plate, Sst-positive cells were observed in rostral and caudal ventral subdomains of the Otp-positive paraventricular complex. An additional basal Sst-expressing cell group was found within a longitudinal Otp-positive periretromamillary band that separates the retromamillary area from tuberal areas. Apart of subsequent growth of these initial populations, at E13.5 and E15.5 some Sst-positive derivatives migrate tangentially into neighboring regions. A subset of cells produced at the anterobasal nucleus disperses ventralward into the shell of the ventromedial hypothalamic nucleus and the arcuate nucleus. Cells from the rostroventral paraventricular subdomain reach the suboptic nucleus, whereas a caudal contingent migrates radially into lateral paraventricular, perifornical, and entopeduncular nuclei. Our data provide a topologic map of molecularly defined progenitor areas originating a specific neuron type during early hypothalamic development. Identification of four main separate sources helps to understand causally its complex adult organization.

Keywords: Dlk5; Nkx2.1; Otp; Shh; Sst; forebrain; hypothalamus; in situ hybridization.

Figure 1

Schematic model of the forebrain, illustrating hypothalamic position and main subdivisions. The rostral (R) and dorsal (D) spatial directions are indicated in (A,B). (A) Updated prosomeric model redrawn from the Allen Developing Mouse Brain Atlas (www.developingmouse.brain-map.org), showing the hypothalamo-telencephalic and hypothalamo-diencephalic boundaries, the alar–basal boundary and the main anteroposterior parts, the peduncular and terminal hypothalamic territories (PHy, THy). (B) Dorsoventral regional hypothalamic subdivisions across the color-coded PHy and THy (separated by the intrahypothalamic border; IHB). The thick blue line indicates the longitudinal alar–basal boundary. Alar territories are seen on the left [corresponding to PHyA and THyA in (A)], and basal territories on the right [PHyB and THyB in (A)]. Within the alar plate, a dashed line separates the rostral and caudal paraventricular subdomains (RPa, CPa) from the rostral and caudal subparaventricular subdomains (RSPa, CSPa). The RSPa contains the suprachiasmatic and anterior hypothalamic nuclei (SCH, AH). Within the basal plate, a thin continuous black line separates the tuberal and retrotuberal regions (Tu/RTu; dotted and striped fills, respectively) from the mamillary complex (undotted). A dashed line separates the dorsal Tu/RTu subregion, occupied by the anterobasal and posterobasal nuclei (ABa, PBa) from the main tuberal/retrotuberal region, which contains the VMH, Arc, and DMH nuclei. Another dashed line divides the mamillary body into the dorsal perimamillary/periretromamillary regions (PM, PRM) and the ventral mamillary and retromamillary areas (M, RM). The corresponding parts of the hypothalamic floor lie underneath (MF, RMF). (C) Schematic reference gene expression patterns used in our mapping, represented upon the same diagram shown in (B). Striped color patterns imply overlap.

Figure 2

Representative sagittal sections from E10. 5 (A–C) and E11.5 (D,E) embryos, showing the expression domains of Dlx5 (A), Otp (B), Dlx5 + Otp (C; digital overlap with pseudocolor of A,B), Nkx2.1 (D), and Shh (E). The transversal boundaries of the PHy and THy are indicated by dashed lines. Bars = 200 μm.

Figure 3

Sagittal sections of E10. 5 embryos taken at lateral (A–E), intermediate (F–H), and medial (I–K) levels, correlating the indicated reference markers with the presence of Sst-positive cells. Note the topographic coincidence of Sst signal with a larger surrounding population of Otp-positive cells. Bars = 200 μm.

Figure 4

Sagittal sections of E12. 5 embryos taken at lateral (A–G) and medial (H–N) levels, correlating the indicated reference markers with the presence of Sst-positive cells. The transversal boundaries of the PHy and THy are indicated by dashed lines. Note there are abundant Sst cells within basal and alar regions of the prethalamus [PTh; p3Tg in (N)], which need to be distinguished from the hypothalamic elements, found rostral to the hypothalamo-diencephalic boundary (caudal dashed line). The black arrowheads mark the incipient ventralward subpial displacement of some ABa-derived Sst cells. The white arrowheads in (G) indicate the sparse Otp-positive cells present within the CSPa subdomain. Bars = 200 μm.

Figure 5

Sagittal (A–K) and transversal (L–O) sections of E13. 5 embryos taken at lateral (A–H) and medial (I–K) paramedian levels, correlating the indicated reference markers (Otp, Nkx2.1, Dlx5) with the presence of Sst-positive cells. (A–G) these images reveal advanced migratory displacement of ABa-derived Sst cells into the underlying VMHs and Arcs formations. (H) This nearly median section shows the ventricular domain expressing Nkx2.1, as well as its caudal extension along the PM/PRM band. The black arrowheads in (E–G) mark the area of overlap between Nkx2.1-expressing mantle cells and the sparse Sst-positive cell population found within the CSPa. Note also in (F,G) a few Sst-positive cells lying immediately ventral to the CSPa band, identified by us as the posterobasal nucleus (PBa), where Nkx2.1 signal is absent. This peculiar retrotuberal basal plate locus (compare Figure 1B) corresponds to an area selectively expressing Lhx9, recently described by Shimogori et al. (2010). (I–K) Dashed lines indicate the HDB and IHB limits demarcating the RM area from the prethalamic tegmentum (p3Tg) and the M area, next to the floor plate. (L–N) Correlation of Otp and Sst signals in adjacent transversal sections (orthogonal to the alar–basal boundary, marked by the black dashed line; white dashed lines indicate the main alar and basal subdivisions). The telencephalic Otp-positive migration stream of the CPa area – containing isolated Sst cells (black arrowheads in M,N) – enters the medial amygdalar area (MA), after passing underneath the Sst-positive elements of the subpallial bed nucleus striae terminalis complex (white asterisk; BST). The CPa area itself shows radial stratification of Otp-positive derivatives within its mantle layer (L,N). At intermediate levels of this domain, superficially migrated Sst cells form an aggregate interpreted as a perifornical population [PFx (M,N)], though it may contain as well elements destined to reach the entopeduncular nucleus, more superficially (not shown). A higher magnification detail of this area is shown in (O). Within the basal plate, both Otp and Sst cells are observed at the VMHs and Arcs formations, as well as in the overlying ABa. Corresponding overlap between Nkx2.1 and Sst signals is shown in (O). Bars = 200 μm.

Figure 6

Sagittal sections of E15. 5 embryos, correlating the indicated reference markers with the presence of Sst-positive cells in adjacent sections. (A–I) This series of sections is a medio-lateral progression, where (A–F) represent adjacent sections compared to each other by digital overlap (C,F), and the analogous overlaps shown in (G–I) lie progressively more laterally. The dashed lines in (A–F) indicate the alar–basal boundary. Note non hypothalamic Dlx5 and Sst expression in the prethalamus (PTh), but restriction of Otp to hypothalamus. The VMHs and Arcs formations express Dlx5, whereas the VMHc does not (C). Note as well close neighborhood of Dlx5 signal to Otp signal at the Pa and PRM domains (G). (J–L) Sagittal adjacent sections from a different E15.5 embryo, correlating NKX2.1 immunoreaction with the distribution of Sst cells. Bars = 200 μm.

Figure 7

Schematic color-coded mapping (upon our basic schema of Figures 1B,C) of the described Sst source areas and the apparent Sst recipient areas, involving postulated tangential migrations (red arrows). The migration moving cells potentially from the ABa to the CSPa is marked with an interrogant because the intrinsic versus extrinsic origin of these cells is not clearly distinguishable in our material. Note that we described radial migrations toward intermediate or subpial loci at the ventral subdomains of CPa and RPa (CPaV, RPaV) and PRM areas. Only the radial migration finishing at the suboptic nucleus (SbO; or retrochiasmatic supraoptic nucleus) is represented.