Evolutionarily conserved regulation of hypocretin neuron specification by Lhx9

Abstract

Loss of neurons that express the neuropeptide hypocretin (Hcrt) has been implicated in narcolepsy, a debilitating disorder characterized by excessive daytime sleepiness and cataplexy. Cell replacement therapy, using Hcrt-expressing neurons generated in vitro, is a potentially useful therapeutic approach, but factors sufficient to specify Hcrt neurons are unknown. Using zebrafish as a highthroughput system to screen for factors that can specify Hcrt neurons in vivo, we identified the LIM homeobox transcription factor Lhx9 as necessary and sufficient to specify Hcrt neurons. We found that Lhx9 can directly induce hcrt expression and we identified two potential Lhx9 binding sites in the zebrafish hcrt promoter. Akin to its function in zebrafish, we found that Lhx9 is sufficient to specify Hcrt-expressing neurons in the developing mouse hypothalamus. Our results elucidate an evolutionarily conserved role for Lhx9 in Hcrt neuron specification that improves our understanding of Hcrt neuron development.

Development of the hypothalamus: conservation, modification and innovation

“…In the anterior hypothalamus, Lhx1 and Otp have been shown to play an important role in the differentiation of neurons in the SCN and PVN/SON/aPV, respectively (Acampora et al, 1999;Wang and Lufkin, 2000;Bedont et al, 2014). The function of some factors appears to be evolutionarily conserved, as demonstrated by the role of Lhx9 in specifying Hcrt + neurons in zebrafish and mice (Liu et al, 2015). Now that effective methods have been developed to generate hypothalamic neurons from hiPSCs in vitro , it will be possible to rigorously test hypothesized transcriptional ʻcodes' for specific neuronal types, and potentially apply this knowledge to therapeutic uses.…”

QRFP and Its Receptors Regulate Locomotor Activity and Sleep in Zebrafish

“…Based on these criteria, rest in a variety of organisms, including zebrafish (Zhdanova et al, 2001;Yokogawa et al, 2007), has been shown to meet the behavioral definition of sleep. Several groups have demonstrated behavioral, anatomical, genetic, and pharmacological conservation of sleep between zebrafish and mammals, establishing zebrafish as a simple vertebrate model for sleep research (Zhdanova et al, 2001;Kaslin et al, 2004;Renier et al, 2007;Yokogawa et al, 2007;Rihel et al, 2010;Elbaz et al, 2012;Gandhi et al, 2015;Singh et al, 2015). Using zebrafish gain-of-function and loss-offunction genetic reagents, we found that QRFP/Gpr103 signaling is both necessary and sufficient to limit locomotor activity during the day, and is necessary to maintain normal daytime sleep levels.…”

“…Adult (22-month-old) Tg(qrfp:EGFP); Tg(hcrt:mRFP) zebrafish (Liu et al, 2015) were anesthetized in 0.2% tricaine, killed by incubation in ice water for 15 min, and decapitated. Intact heads were fixed in 4% PFA/PBS overnight at 4°C, then whole brains were dissected from their skulls and placed in A4P0 hydrogel monomer solution at 4°C overnight with agitation.…”

“…We occasionally observed colocalization of qrfp and crhb in one or two neurons, but never observed colocalization of qrfp and hcrt (n Ͼ 50 brains). We also examined qrfp-expressing neurons, and compared their location to that of hcrt-expressing neurons, in adult Tg(qrfp:EGFP); Tg(hcrt:mRFP) double transgenic animals (Liu et al, 2015) that were fixed and cleared using PACT (Yang et al, 2014; Fig. 3J).…”

“…However, animals lacking qrfp or the second murine qrfp receptor paralog (gpr103b) have not been reported in any animal model. Zebrafish QRFP (Liu et al, , 2015Chen et al, 2013;Ukena et al, 2014) and Gpr103 (Chen et al, 2013;Larhammar et al, 2014;Ukena et al, 2014) orthologs have been identified, but have not been functionally characterized. Here we describe the zebrafish qrfp and gpr103 orthologs, characterize qrfp-and gpr103-expressing neurons, and show that QRFP and its receptors regulate locomotor activity and sleep in zebrafish larvae.…”