Abstract
The neuropeptide agouti-related protein (AgRP) is expressed in the arcuate nucleus of the mammalian hypothalamus and plays a key role in regulating food consumption and energy homeostasis. Fish express two agrp genes in the brain: agrp1, considered functionally homologous with the mammalian AgRP, and agrp2. The role of agrp2 and its relationship to agrp1 are not fully understood. Utilizing BAC transgenesis, we generated transgenic zebrafish in which agrp1- and agrp2-expressing cells can be visualized and manipulated. By characterizing these transgenic lines, we showed that agrp1-expressing neurons are located in the ventral periventricular hypothalamus (the equivalent of the mammalian arcuate nucleus), projecting throughout the hypothalamus and towards the preoptic area. The agrp2 gene was expressed in the pineal gland in a previously uncharacterized subgroup of cells. Additionally, agrp2 was expressed in a small group of neurons in the preoptic area that project directly towards the pituitary and form an interface with the pituitary vasculature, suggesting that preoptic AgRP2 neurons are hypophysiotropic. We showed that direct synaptic connection can exist between AgRP1 and AgRP2 neurons in the hypothalamus, suggesting communication and coordination between AgRP1 and AgRP2 neurons and, therefore, probably also between the processes they regulate.
Highlights
The vertebrate neuro-endocrine system regulates an array of homeostatic processes, allowing the organism to constantly adapt to its surroundings and to the changing availability of resources
To increase the utility of the generated transgenic lines, we employed the Gal4-VP16 transactivation system, generating bacterial artificial chromosome (BAC) in which Gal4-VP16 is under the control of agrp[1] and agrp[2] regulatory regions
BAC clones containing AgRP1- or AgRP2-coding sequences, as well as 45−60 kb downstream and upstream flanking regions, were modified by recombineering according to an established protocol[31] and used for transgenesis (Supplementary Fig. S1). These BACs are likely to contain all of the cis-regulatory elements needed to replicate the endogenous spatial and temporal expression of agrp[1] and agrp[2]
Summary
The vertebrate neuro-endocrine system regulates an array of homeostatic processes, allowing the organism to constantly adapt to its surroundings and to the changing availability of resources. Pineal gland expression of agrp[2] was reported in sea bass (Dicentrarchus labrax)[20], and we have detected its expression in the pineal gland of Nile tilapia (Oreochromis niloticus) and rainbow trout (Oncorhynchus mykiss; unpublished data) This suggests a conserved pineal gland expression of agrp[2] among teleosts. Focusing mainly on AgRP2, we report previously uncharacterized agrp2-expressing pineal cells, novel hypophysiotropic AgRP2 neurons, and a novel AgRP1-AgRP2 neuronal interaction Together, these observations and use of the novel transgenic fish lines may eventually lead to identification of hitherto undiscovered neuroendocrine functions for AgRP neuropeptides
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