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. 2010 Jun;13(6):767-75.
doi: 10.1038/nn.2545. Epub 2010 May 2.

A Genomic Atlas of Mouse Hypothalamic Development

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Free PMC article

A Genomic Atlas of Mouse Hypothalamic Development

Tomomi Shimogori et al. Nat Neurosci. .
Free PMC article

Abstract

The hypothalamus is a central regulator of many behaviors that are essential for survival, such as temperature regulation, food intake and circadian rhythms. However, the molecular pathways that mediate hypothalamic development are largely unknown. To identify genes expressed in developing mouse hypothalamus, we performed microarray analysis at 12 different developmental time points. We then conducted developmental in situ hybridization for 1,045 genes that were dynamically expressed over the course of hypothalamic neurogenesis. We identified markers that stably labeled each major hypothalamic nucleus over the entire course of neurogenesis and constructed a detailed molecular atlas of the developing hypothalamus. As a proof of concept of the utility of these data, we used these markers to analyze the phenotype of mice in which Sonic Hedgehog (Shh) was selectively deleted from hypothalamic neuroepithelium and found that Shh is essential for anterior hypothalamic patterning. Our results serve as a resource for functional investigations of hypothalamic development, connectivity, physiology and dysfunction.

Conflict of interest statement

COMPETING FINANCIAL INTERESTS

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1
Microarray-based identification of developmentally dynamic transcripts in mouse hypothalamus. (a) Schema for microarray analysis in developing mouse hypothalamus. Hypothalamic tissue from a minimum of six individuals was collected for each of the four indicated genotypes at each of the time points indicated. (b) Anatomical landmarks used for dissection of hypothalamic and preoptic tissue at each indicated time point are shown. Arrows indicate tissue that was collected for RNA extraction. 3V, third ventricle; AC, anterior commissure; BST, bed nucleus of stria terminalis; CGE, caudal ganglionic eminence; Di, diencephalon; DTh, dorsal thalamus; Hy, hypothalamus; LV, lateral ventricle; MB, midbrain; POA, preoptic area; PTh, prethalamus; Tel, telencephalon; Thal, thalamus. (c) Self-organizing map–based cluster analysis of the 25,471 probe sets that showed significantly different expression levels (<1% false discovery rate and >twofold change) over the course of development is shown. The signal intensity indicates the difference between the median log2-scaled signal intensity at the indicated time point and the median log2-scaled signal intensity observed for that probe set across all time points tested. Magnified images of clusters enriched for genes involved in neuronal progenitor proliferation, hypothalamic neuronal differentiation and terminal markers of hypothalamic neuronal fate are shown. Genes whose expression in developing hypothalamus has been previously examined are shown in black and marked by a double asterisk, whereas transcripts showing similar expression, but which have not been previously functionally analyzed in hypothalamus are shown in gray.
Figure 2
Figure 2
Marker genes that delineate the telencephalic-diencephalic border. (a,b) Sagittal and coronal views of two-color ISH analysis of Gdf10 (blue) and Foxg1 (brown) at E12.5. Gdf10 demarcated the anterior border of prethalamus (red arrow). (c,d) Sagittal and coronal views of two-color ISH analysis of Foxd1 (blue) and Foxg1 (brown) at E12.5. Foxg1 and Foxd1 were expressed in adjacent, but nonoverlapping, zones in telencephalon and anterior diencephalon, respectively. The green arrows indicate the ventral telencephalic-diencephalic boundary. (e,f) Sagittal and coronal views of two-color ISH analysis of Gdf10 (blue) and Foxg1 (brown) at E15.5. Gdf10 expression continued to demarcate the anterior border of prethalamus at this later stage of development. Schematic figures indicate zones of gene expression at all of the stages shown. Di, diencephalon; Tel, telencephalon. Scale bar represents 0.2 mm.
Figure 3
Figure 3
Regional patterning of prethalamus and anterior hypothalamus is revealed by analysis of marker gene expression. (a) Schematic indicating Shh expression in the developing diencephalon at E12.5. (b) Two-color ISH analysis of Shh (blue) and Arx (brown) indicated that Arx was expressed in prethalamus (black arrow) and two distinctive regions in hypothalamic neuroepithelium (black arrowheads and red arrowhead). (c,d) Nkx2.1 (blue) expression partially overlapped with Arx (brown) expression in the hypothalamic zone running parallel to the basal plate (yellow arrow) and in posterior hypothalamus (yellow arrowhead). (e) Lhx6 expression (purple) demarcated the intrahypothalamic diagonal and the tuberomamillary terminal, overlapping with Arx (brown). (f,g) Lhx9 expression (brown) marked a region ventral to the intrahypothalamic diagonal (red arrow) that was devoid of Nkx2.1 (purple). (h) Arx (2/3, blue) and Lhx9 (EmThal (1), brown) defined nonoverlapping domains of gene expression in prethalamus and hypothalamus. Lhx9 was expressed immediately ventral to the Arx-positive zone in the intrahypothalamic diagonal (yellow arrow). (i) Olig2 expression (blue, in combination with Lhx9) selectively marked the most ventral portion of the prethalamus (3). (j) Lhx6 expression (brown) overlapped with Nkx2.1 (blue) in the intrahypothalamic diagonal and tuberomamillary terminal. (k) Gsh2 (blue) and Rax (brown) were expressed on the border of prethalamus and hypothalamus (orange arrow). (l) Sim1 (blue) was expressed immediately anterodorsal to the zone of Arx expression (brown) in intrahypothalamic diagonal (blue arrowhead). (m) A domain of ventral anterior hypothalamic neuroepithelium was labeled by Nkx6.2 (green arrow). Scale bar represents 0.2 mm (b, c, e, f, h, i and km) and 0.10 mm for (d, g and j). ID, intrahypothalamic diagonal; TT, tuberomamillary terminal.
Figure 4
Figure 4
Characterization of molecular markers of regional identity in posteroventral hypothalamus. Selective and nucleus-specific expression of markers is indicated at both E12.5 and E16.5. (a,b) Foxb1 (blue) marked the mamillary nucleus (MM), which was next to the Lhx6-expressing tuberomamillary terminal (brown) at E12.5 (a). Their spatial relationship was conserved at E16.5 (b). (c,d) Irx5 expression demarcated the supramamillary nucleus (SMM), which is located posterior to the tuberomamillary terminal marked by Lhx6 (brown) at E12.5 (c). A possible cell migration made the Irx5-expressing domain crossed the Lhx6-expressing tuberomamillary terminal at E16.5 (d). (e,f) Lef1 (blue) expression marked the premamillary (PM) region at E12.5 and E16.5, demonstrating a conserved domain of expression relative to the domain of Lhx6 expression (brown). (g,h) Nr5a1 was expressed in the ventromedial hypothalamic nucleus (VMH) at E12.5 and E16.5. (i,j) Pomc was expressed in the arcuate nucleus (ARC) at E12.5 and E16.5. To aid in the identification of these structures, we used Lhx6 as a second ISH color (brown) in ah and Lhx1 (blue) for i,j. Scale bar represents 0.2 mm. ID, intrahypothalamic diagonal; TT, tuberomamillary terminal.
Figure 5
Figure 5
Lhx family members delineate discrete regions of the developing hypothalamus. Schematic figures indicate the patterns of Lhx gene expression at E12.5 and E16.5. (a,b) Lhx6 (blue) was expressed in the posterior portion of the intrahypothalamic diagonal immediately dorsal to the zone of Lhx9 expression (brown) (a). This pattern of Lhx6 and Lhx9 expression was preserved at E16.5 (b). (d,e) Lhx8 (blue) was expressed anterior to the Lhx6-positive region of the intrahypothalamic diagonal (brown). (g,h) Lhx8 (blue) was expressed anterior and dorsal to Lhx9 expression (brown). (k,l) Lhx1 (blue) was expressed anterior and dorsal to Lhx9 expression (brown), but overlapped with Lhx8 and the anterior domain of Lhx6 expression (data not shown). (c,f,i,m) Postnatal expression patterns of intrahypothalamic diagonal–expressed Lhx6 overlapped with Cart. A coronal section is shown in c, indicating that there is a conserved spatial relationship in lateral hypothalamic expression of Lhx9 (purple) and Lhx6 (brown) at P6. Lhx6-positive cells (brown) were found medial and dorsal to galanin (Gal)-positive neurons (purple) (f). Lhx6-positive cells (brown) were found medial and dorsal to hypocretin (Hcrt)-positive neurons (blue) (i). Lhx8 (brown) was expressed medial to Cart (blue) in dorsomedial hypothalamic nucleus (m). Schematic drawings indicating expression of neuropeptides and Lhx genes in postnatal hypothalamus, along with regions of overlap, are shown at the top of each column. (j,n) The anterior terminus of the zone of Lhx1 expression corresponded to the Rorα-positive SCN at E16.5. However, expression also extended posterodorsal from the SCN into the ventral subparaventricular zone (yellow arrow). Scale bar represents 0.2 mm (a, b, d, e, g, h, k and l) and 0.3 mm (c, f, i, j, m and n).
Figure 6
Figure 6
Diencephalic phenotype of Nkx2.1-Cre × Shh loxP/loxP mice at E10.5 and E12.5, as indicated by analysis of region-specific marker genes. (a,b) Shh was not expressed in the basal hypothalamus at E10.5 in Nkx2.1-Cre × Shh loxP/loxP mice, as visualized by Shh probe (red bracket). (c,d) Expression of Foxb1 (blue) in mamillary neuroepithelium (red arrow) was preserved in Nkx2.1-Cre × Shh lox/lox mice. (e,f) Sim1 (blue) was not expressed in mamillary neuroepithelium (green arrow), but was not expressed in paraventricular nucleus (PVN) in mutant mice (orange arrow). (g,h) Arx was not expressed in the intrahypothalamic diagonal and tuberomamillary terminal (blue) in Nkx2.1-Cre × Shh lox/lox mice, but was expressed in the posterior prethalamic region. (i,j) Lhx9 (blue) was not expressed in ventral hypothalamus, but its expression in anterior prethalamus was expanded anteriorly (yellow arrow). (k,l) Lhx1 was not expressed in the intrahypothalamic diagonal, but it was expressed in anterior and posterior prethalamus, as well as in the mamillary hypothalamus. Lhx1 was expressed in a prominent anterior-located region (green arrow), but this region was dorsal to the optic recess (blue arrow) and resided in preoptic neuroepithelium. (m,n) Hypothalamic Lhx6 expression (blue) was absent in Nkx2.1-Cre × Shh lox/lox mice. Shh (brown) was expressed in Zli (asterix) and posterior to the diencephalon in mutant mice (d,f,n), but not in ventral diencephalon and telencephalon. (o) Schematic drawings of gene expression in wild-type and mutant mice with color combination. Scale bar represents 0.2 mm.

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