Hypothalamus Anatomical divisions Regionally elevated protein expression in human Regionally elevated protein expression in mouse Regionally elevated protein expression in pig Extended information

Hypothalamus

Summary

The hypothalamus is a part of the diencephalon, together with the thalamus, epithalamus, subthalamus and meta-thalamus. The hypothalamus develops from the embryonic diencephalon and is located at the bottom of the diencephalon. In humans, it is an almond-like structure. The hypothalamus is the center of the vegetative nervous system as well as a key regulator of the endocrine system.

The transcriptome analysis has revealed that 71% (n=14309) of all human protein-coding genes (n=20090) are expressed in the human hypothalamus. Human one-to-one orthologues were investigated in pig and mouse brain, suggesting that 12272 of all mouse one-to-one orthologues (n=16320) are expressed in the mouse hypothalamus and that 13044 of all pig orthologues (n=15829) are expressed in the pig hypothalamus.

Gene classification based on regional RNA expression in human, pig and mouse hypothalamus defines 38 genes as hypothalamus enriched in either of the three species, and 18 out of those genes show highest expression levels in hypothalamus in all three species.

Figure 1. Schematic drawing of the human brain, indicating the location of hypothalamus below the thalamus, at the bottom of diencephalon from a sagittal view.

Anatomical divisions

Anatomically, the hypothalamus can be divided into anterior, tuberal and posterior parts. The anterior part involves the preoptic nucleus (thermoregulation), the medial preoptic nucleus (releases GnRH hormone), supraoptic nucleus (releases oxytocin - OXT and vasopressin - AVP), the paraventricular nucleus (releases the thyrotropin releasing hormone - TRH, corticotropin releasing factor - CRH, oxytocin, vasopressin and somatostatin hormones), the anterior hypothalamic nucleus (involved in thermoregulation) and the suprachiasmatic nucleus (involved in the regulation of the circadian rhythms). The tuberal hypothalamus consists of a medial and a lateral part. The medial part involves the dorsomedial- (regulation of blood pressure and heart rate), ventromedial- (regulation of satiety) and arcuate nucleus (regulation of food intake, among others). The lateral part of the tuberal hypothalamus is a complex hub of neuronal networks; it contains, among others, the orexin and MCH neurons that regulate food intake and the sleep/wake cycle. The lateral hypothalamus partially extends also to the anterior and posterior hypothalamus. The posterior hypothalamus consists of the mammillary nuclei, the lateral nucleus (that is involved in the regulation of blood pressure) and the tuberomammillary nucleus (that is involved in the regulation of arousal, energy balance, learning-memory processes and sleep).

GNRH1
TRH
PMCH

Histologically, the hypothalamus could be divided into magnocellular and microcellular neurosecretory neurons and non-secretory neurons. The magnocellular neurons release oxytocin and vasopressin in the neurohypophysis. The axons of these magnocellular neurons form the suproopticohyphophyseal tract. The microcellular neurons secrete the ‘releasing’ and ‘release inhibiting’ hormones to the portal veins that will transport these hormones to the adenohypophysis. The axons of the microcellular neurons form the tuberoinfundibular tract. The non-secretory neurons are involved in the regulation of multiple and complex vegetative functions. The tuberomammillary and mammillary nuclei are important parts of the limbic-system. The hypothalamic neurons express several neuropeptides and several hypothalamic neuronal populations and sub-populations can be identified by their neuropeptide markers (e.g. orexin- and MCH-expressing neurons in the lateral hypothalamus; NPY- and POMC-expressing neurons in the arcuate nucleus etc.)

GAL
GAL
NUCB2

Galanin (GAL) is a physiologically active neuropeptide that is implicated in nociception, feeding and energy homeostasis, osmotic regulation and water balance. Galanin immunoreactivity can be observed in the tuberal hypothalamus. Nucleobindin 2 (NUCB2) is a calcium-binding protein and has a role in eating regulation in the hypothalamus NUCB2 immunoreactivity is observed in the hypothalamic paraventricular nucleus.

Regionally elevated protein expression in human

The transcriptome analysis shows that 71% (n=14309) of all human proteins (n=20090) are expressed in the hypothalamus. 78 genes show an elevated expression level in hypothalamus compared to other regions of the brain.

19 regionally enriched genes
78 regionally elevated genes in total
22 of the hypothalamus elevated genes are elevated in other tissues than the brain.
56 of the hypothalamus elevated genes are elevated in the brain.

Table 1: Number of genes within the different categories of regionally elevated expression, in human hypothalamus.

Specificity	Number of Human elevated genes
Region enriched	19
Group enriched	39
Region enhanced	20
Elevated	78

Elevated expression in hypothalamus compared to other brain regions is divided into three different categories; regionally enriched (at least four-fold higher mRNA levels in hypothalamus compared to all other regions), group enriched (at least four-fold higher mRNA levels in a group of 2-5 regions) and regionally enhanced (at least four-fold higher mRNA levels in hypothalamus compared to the average of all regions), The number of genes in the individual category is shown in Table 1. In Table 2, the 12 genes with the highest level of regional specificity among the 19 enriched genes are listed.

Table 2. The 12 genes with the highest level of enriched expression in human hypothalamus. "Predicted localization" shows the classification of each gene into three main classes: Secreted, Membrane, and Intracellular, where the latter consists of genes without any predicted membrane and secreted features. RS-score (Regional Specificity score) corresponds to the score calculated as the fold change to the second highest region.

Gene	Description	Predicted location	RS-score
OXT	Oxytocin/neurophysin I prepropeptide	Secreted	141
AVP	Arginine vasopressin	Secreted	131
AGRP	Agouti related neuropeptide	Secreted	60
PRG4	Proteoglycan 4	Secreted	33
GAL	Galanin and GMAP prepropeptide	Secreted	13
GHRH	Growth hormone releasing hormone	Secreted	12
NPVF	Neuropeptide VF precursor	Secreted	10
GSC	Goosecoid homeobox	Intracellular	9
HCRT	Hypocretin neuropeptide precursor	Secreted	7
NPY	Neuropeptide Y	Secreted	7
ETV3L	ETS variant transcription factor 3 like	Intracellular	6
HDC	Histidine decarboxylase	Intracellular	6

HCRT
AVP
OTP

AGRP
HDC

The (HCRT) gene produces two neuropeptides, orexin A and orexin B, and are implicated in the regulation of sleep and might also play a role in feeding behavior and homeostasis. Vasopressin (AVP) is synthesized in the supraoptic nucleus and paraventricular nucleus of the hypothalamus and is transported to the nerve endings in the neurohypophysis. When secreted into the bloodstream, vasopressin acts as an antidiuretic hormone in the kidneys. OTP is a transcription factor involved in the development of the hypothalamus. The agouti related neuropeptide (AGRP) has a role in feeding control and weight homeostasis. The HDC protein controls several processes such as gastric acid secretion and smooth muscle contraction.

Regionally elevated protein expression in mouse

The mouse hypothalamus is anatomically similar to the human hypothalamus, with similar topography of nuclei and similar neurochemical character (e.g. neuropeptide-expression) of the homologous nuclei.

The transcriptome analysis shows that 61% (n=12272) of all mouse one-to-one human orthologue genes (n=16320) are expressed in the hypothalamus and 97 genes show an elevated expression in hypothalamus compared to other regions of the brain.

Table 3: Number of genes within the different categories of regionally elevated expression, in mouse hypothalamus

Specificity	Number of Mouse elevated genes
Region enriched	25
Group enriched	39
Region enhanced	33
Elevated	97

Figure 2. Schematic drawing of the mouse brain, indicating the location of hypothalamus from a sagittal perspective.

Elevated expression in hypothalamus compared to other brain regions is divided into three different categories; regionally enriched (at least four-fold higher mRNA levels in hypothalamus compared to all other regions), group enriched (at least four-fold higher mRNA levels in a group of 2-5 regions) and regionally enhanced (at least four-fold higher mRNA levels in hypothalamus compared to the average of all regions). The number of genes in the individual category is shown in Table 3. In Table 4, the 12 genes with the highest level of regional specificity among the 25 enriched genes are listed.

Table 4: The 12 genes with the highest level of enriched expression in mouse hypothalamus. "Predicted localization" shows the classification of each gene into three main classes: Secreted, Membrane, and Intracellular, where the latter consists of genes without any predicted membrane and secreted features. RS-score (Regional Specificity score) corresponds to the score calculated as the fold change to the second highest tissue.

Gene	Description	Predicted location	RS-score
NR5A1	Nuclear receptor subfamily 5 group A member 1	Intracellular	52
NKX2-4	NK2 homeobox 4	Intracellular	23
NPVF	Neuropeptide VF precursor	Secreted	18
AGRP	Agouti related neuropeptide	Secreted	16
SIX6	SIX homeobox 6	Intracellular	14
FEZF1	FEZ family zinc finger 1	Intracellular	11
FOXB2	Forkhead box B2	Intracellular	11
AVP	Arginine vasopressin	Secreted	10
HIGD1C	HIG1 hypoxia inducible domain family member 1C	Membrane	10
GHRH	Growth hormone releasing hormone	Secreted	9
SIM1	SIM bHLH transcription factor 1	Intracellular	8
OTP	Orthopedia homeobox	Intracellular	7

HCRT
AVP
OTP

Orexin (HCRT) immunostaining in the lateral hypothalamus. Vasopressin (AVP) peptide immunostaining in the paraventricular hypothalamic nucleus. Orthopedia homeobox protein (OTP) localization in the hypothalamic paraventricular nucleus.

Regionally elevated protein expression in pig

The pig hypothalamus is anatomically similar to the human hypothalamus, with similar topography of nuclei and similar neurochemical character (e.g. neuropeptide-expression) of the homologous nuclei.

The transcriptome analysis shows that 65% (n=13044) of all pig one-to-one human orthologue genes (n=15829) are expressed in the hypothalamus. 42 genes show an elevated expression in hypothalamus compared to other regions of the brain.

Table 5: Number of genes within the different categories of regionally elevated expression, in pig hypothalamus.

Specificity	Number of Pig elevated genes
Region enriched	11
Group enriched	20
Region enhanced	11
Elevated	42

Figure 3. Schematic drawing of the pig brain, indicating the location of hypothalamus from a sagittal perspective.

Elevated expression in hypothalamus compared to other brain regions is divided into three different categories; regionally enriched (at least four-fold higher mRNA levels in hypothalamus compared to all other regions), group enriched (at least four-fold higher mRNA levels in a group of 2-5 regions) and regionally enhanced (at least four-fold higher mRNA levels in hypothalamus compared to the average of all regions), The number of genes in the individual category is shown in Table 5. In Table 6, the 12 genes with the highest level of regional specificity among the 11 enriched genes are listed.

Table 6: The 12 genes with the highest level of enriched expression in pig hypothalamus. "Predicted localization" shows the classification of each gene into three main classes: Secreted, Membrane, and Intracellular, where the latter consists of genes without any predicted membrane and secreted features. RS-score (Regional Specificity score) corresponds to the score calculated as the fold change to the second highest region.

Gene	Description	Predicted location	RS-score
CGA	Glycoprotein hormones, alpha polypeptide	Secreted	40
PMCH	Pro-melanin concentrating hormone	Secreted	34
HCRT	Hypocretin neuropeptide precursor	Secreted	21
FEZF1	FEZ family zinc finger 1	Intracellular	14
SIX6	SIX homeobox 6	Intracellular	12
NKX2-4	NK2 homeobox 4	Intracellular	11
BSX	Brain specific homeobox	Intracellular	10
GPR50	G protein-coupled receptor 50	Membrane	10
NPVF	Neuropeptide VF precursor	Secreted	8
NR5A1	Nuclear receptor subfamily 5 group A member 1	Intracellular	8
POMC	Proopiomelanocortin	Secreted	5

Extended information

3D imaging of mouse brain

This video demonstrates the 3D distribution of orexin immunostaining (HCRT) in the mouse lateral hypothalamus (iDISCO volume immunostaining). A descriptive movie including more details and an interview with Dr. Csaba Adori, Karolinska Institute, related Orexin and Narcolepsy is found here.

List of proteins stained on human hypothalamus tissue section

The standard setup in the Tissue Atlas, which profiles the human tissues, is based on Tissue Micro array technique, thus saving valuable tissue material and reagents. The TMA technique provides a wide tissue representation for protein profiling. Cerebral cortex, caudate nucleus, hippocampus and cerebellum are the four brain regions in the standard human brain profiling. In-depth analysis of the elevated genes in hypothalamus and literature search for hypothalamus-associated proteins served as a strategy when selecting which proteins to analyze in human hypothalamus samples.

Table 7. The following 41 genes have been analyzed using extended hypothalamus samples.

Gene	Gene description	Staining pattern
ADCYAP1	Adenylate cyclase activating polypeptide 1	Synaptic staining.
AGRP	Agouti related neuropeptide	Strong staining of synapses and neuronal projections
ARHGAP36	Rho GTPase activating protein 36	Strong in neuronal cells, synapses and projections.
AVP	Arginine vasopressin	Strong cytoplasmic staining in magnocellular neurosecretory neurons.
CALCR	Calcitonin receptor	Strong membranous staining in neurons.
CALY	Calcyon neuron specific vesicular protein	Cytoplasmic staining in neurons.
CARTPT	CART prepropeptide	Neuronal projections and synaptic positivity.
CCK	Cholecystokinin	Strong neuronal projection and extracellular staining.
CNGA3	Cyclic nucleotide gated channel subunit alpha 3	Stains in neural projections and neuronal cell bodies.
CRABP1	Cellular retinoic acid binding protein 1	Strong in a subset of hypothalamic neurons.
DDX25	DEAD-box helicase 25	Strong staining processes and in subsets of astrocyte-like cells.
ECEL1	Endothelin converting enzyme like 1	Strong in neurons.
FEZF1	FEZ family zinc finger 1	Distinct positivity in processes and neuropil of hypothalamus.
GAL	Galanin and GMAP prepropeptide	Strong cytoplasmic positivity in neuronal cells and projections.
GATA2	GATA binding protein 2	Nuclear staining in a few neurons.
GNRH1	Gonadotropin releasing hormone 1	Strong cytoplasmic staining in a subset of hypothalamic neuronal cells.
GRIN2D	Glutamate ionotropic receptor NMDA type subunit 2D	Membranous and cytoplasmic staining in neurons. Projections and synapses are also positive.
HCRT	Hypocretin neuropeptide precursor	Strong staining in a subset of hypothalamic neurons.
HCRTR1	Hypocretin receptor 1	Positive projections and synapses.
HDC	Histidine decarboxylase	Strong staining in neurons of hypothalamus.
HMX2	H6 family homeobox 2	Strong staining in neurons and projections/synapses.
HTR7	5-hydroxytryptamine receptor 7	Cytoplasmic in a subset of neurons. Distinct staining in neuronal projections.
KISS1R	KISS1 receptor	Distinct neuronal projections and staining of neuronal cells.
LUZP2	Leucine zipper protein 2	Strong staining in subsets neuronal and glial projections.
MC3R	Melanocortin 3 receptor	Cytoplasmic positivity in a subset of hypothalamic neurons.
NPVF	Neuropeptide VF precursor	Moderate positivity mainly in glial cells in hypothalamus.
OTP	Orthopedia homeobox	Strong nuclear staining in subsets of neurons.
OXT	Oxytocin/neurophysin I prepropeptide	Strong cytoplasmic staining in magnocellular neurosecretory neurons.
PABPC1L2A	Poly(A) binding protein cytoplasmic 1 like 2A	Strong staining in processes and nerve fibers in hypothalamus.
PABPC1L2B	Poly(A) binding protein cytoplasmic 1 like 2B	Strong staining in processes and nerve fibers in hypothalamus.
PCSK2	Proprotein convertase subtilisin/kexin type 2	Cytoplasmic staining in a subset of glial cells.
PMCH	Pro-melanin concentrating hormone	Distinct cytoplasmic positivity in subsets of hypothalamic neurons and neural processes.
PPP1R17	Protein phosphatase 1 regulatory subunit 17	Cytoplasmic staining in subset of neurons and cell projections in hypothalamus.
PTPRN	Protein tyrosine phosphatase receptor type N	Cytoplasmic staining in neuronal cells. Synaptic positivity.
QRFPR	Pyroglutamylated RFamide peptide receptor	Synaptic positivity.
RTP5	Receptor transporter protein 5 (putative)	Cytoplasmic staining in neuronal cells.
SCG2	Secretogranin II	Granular cytoplasmic staining in neurons. Positive axons.
SST	Somatostatin	Strong secreted and axonal and synapse staining in hypothalamus.
TPPP3	Tubulin polymerization promoting protein family member 3	Staining in in subsets of glial cells and ependymal cells in ventricle walls.
TRH	Thyrotropin releasing hormone	Strong positivity in subsets of hypothalamic neuronal cells and processes.
VGF	VGF nerve growth factor inducible	Strong positivity was observed in neuronal cells and neuronal processes. Glial cells were negative.
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