The blood–brain barrier (BBB) is critical in maintaining a physical and metabolic barrier between the blood and the brain. The BBB consists of brain microvascular endothelial cells (BMECs) that line the brain vasculature and combine with astrocytes, neurons and pericytes to form the neurovascular unit. We hypothesized that astrocytes and neurons generated from human‐induced pluripotent stem cells (iPSCs) could induce BBB phenotypes in iPSC‐derived BMECs, creating a robust multicellular human BBB model. To this end, iPSCs were used to form neural progenitor‐like EZ‐spheres, which were in turn differentiated to neurons and astrocytes, enabling facile neural cell generation. The iPSC‐derived astrocytes and neurons induced barrier tightening in primary rat BMECs indicating their BBB inductive capacity. When co‐cultured with human iPSC‐derived BMECs, the iPSC‐derived neurons and astrocytes significantly elevated trans‐endothelial electrical resistance, reduced passive permeability, and improved tight junction continuity in the BMEC cell population, while p‐glycoprotein efflux transporter activity was unchanged. A physiologically relevant neural cell mixture of one neuron: three astrocytes yielded optimal BMEC induction properties. Finally, an isogenic multicellular BBB model was successfully demonstrated employing BMECs, astrocytes, and neurons from the same donor iPSC source. It is anticipated that such an isogenic facsimile of the human BBB could have applications in furthering understanding the cellular interplay of the neurovascular unit in both healthy and diseased humans. image Read the Editorial Highlight for this article on page 843.

Sleep is an essential biological need of all animals studied to date. The sleep disorder narcolepsy is characterized by excessive daytime sleepiness, fragmentation of nighttime sleep, and cataplexy. Narcolepsy is caused by selective degeneration of hypothalamic hypocretin/orexin (HCRT) neurons. In mammals, HCRT neurons primarily regulate the sleep/wake cycle, feeding, reward-seeking, and addiction. The role of HCRT neurons in zebrafish is implicated in both sleep and wake regulation. We established a transgenic zebrafish model enabling inducible ablation of HCRT neurons and used these animals to understand the function of HCRT neurons and narcolepsy. Loss of HCRT neurons increased the expression of the HCRT receptor (hcrtr). Behavioral assays revealed that HCRT neuron-ablated larvae had normal locomotor activity, but demonstrated an increase in sleep time during the day and an increased number of sleep/wake transitions during both day and night. Mild sleep disturbance reduced sleep and increasedc-fosexpression in HCRT neuron-ablated larvae. Furthermore, ablation of HCRT neurons altered the behavioral response to external stimuli. Exposure to light during the night decreased locomotor activity of wild-type siblings, but induced an opposite response in HCRT neuron-ablated larvae. Sound stimulus during the day reduced the locomotor activity of wild-type sibling larvae, while HCRT neuron-ablated larvae demonstrated a hyposensitive response. This study establishes zebrafish as a model for narcolepsy, and indicating a role of HCRT neurons in regulation of sleep/wake transitions during both day and night. Our results further suggest a key role of HCRT neurons in mediating behavioral state transitions in response to external stimuli.

For most species, light represents the principal environmental signal for entraining the endogenous circadian clock. The zebrafish is a fascinating vertebrate model for studying this process since unlike mammals, direct exposure of most of its tissues to light leads to local clock entrainment. Importantly, light induces the expression of a set of genes including certain clock genes in most zebrafish cell types in vivo and in vitro. However, the mechanism linking light to gene expression remains poorly understood. To elucidate this key mechanism, here we focus on how light regulates transcription of the zebrafish period2 (per2) gene. Using transgenic fish and stably transfected cell line–based assays, we define a Light Responsive Module (LRM) within the per2 promoter. The LRM lies proximal to the transcription start site and is both necessary and sufficient for light-driven gene expression and also for a light-dependent circadian clock regulation. Curiously, the LRM sequence is strongly conserved in other vertebrate per2 genes, even in species lacking directly light-sensitive peripheral clocks. Furthermore, we reveal that the human LRM can substitute for the zebrafish LRM to confer light-regulated transcription in zebrafish cells. The LRM contains E- and D-box elements that are critical for its function. While the E-box directs circadian clock regulation by mediating BMAL/CLOCK activity, the D-box confers light-driven expression. The zebrafish homolog of the thyrotroph embryonic factor binds efficiently to the LRM D-box and transactivates expression. We demonstrate that tef mRNA levels are light inducible and that knock-down of tef expression attenuates light-driven transcription from the per2 promoter in vivo. Together, our results support a model where a light-dependent crosstalk between E- and D-box binding factors is a central determinant of per2 expression. These findings extend the general understanding of the mechanism whereby the clock is entrained by light and how the regulation of clock gene expression by light has evolved in vertebrates. Light is the principal signal used by animals to synchronize their circadian clocks with the day/night environment. Central to this vital property is the ability of light to trigger changes in gene expression. However, we still lack a complete understanding of how this occurs. The zebrafish is particularly interesting in this regard since direct light exposure induces the expression of clock genes in most of its tissues and in turn adjusts the phase of the intrinsic clocks. Here, by studying the promoter of one key light-regulated zebrafish clock gene, per2, we have identified a Light Responsive Module (LRM) that is necessary and sufficient for light controlled expression. Interestingly, the LRM is also highly conserved in the per2 genes of other vertebrates that lack widespread light-sensing tissues. In addition, the human LRM can substitute for its zebrafish counterpart to confer direct light regulation of gene expression in zebrafish cells. The LRM contains E- and D-box enhancers critical for its function. While the E-box is a target of clock regulation, the D-box directs light driven expression. We show that the expression of the D-box binding transcription factor, tef, is itself induced by light and is essential for normal light-induced per2 expression. These results advance our understanding of the mechanisms underlying entrainment by light and how light-regulated clock gene expression has evolved in vertebrates.