Zhou-Feng Chen

Molecular Distinction and Angiogenic Interaction between Embryonic Arteries and Veins Revealed by ephrin-B2 and Its Receptor Eph-B4

The vertebrate circulatory system is composed of arteries and veins. The functional and pathological differences between these vessels have been assumed to reflect physiological differences such as oxygenation and blood pressure. Here we show that ephrin-B2, an Eph family transmembrane ligand, marks arterial but not venous endothelial cells from the onset of angiogenesis. Conversely, Eph-B4, a receptor for ephrin-B2, marks veins but not arteries. ephrin-B2 knockout mice display defects in angiogenesis by both arteries and veins in the capillary networks of the head and yolk sac as well as in myocardial trabeculation. These results provide evidence that differences between arteries and veins are in part genetically determined and suggest that reciprocal signaling between these two types of vessels is crucial for morphogenesis of the capillary beds.

1,25-Dihydroxyvitamin D3 is a negative endocrine regulator of the renin-angiotensin system

Inappropriate activation of the renin-angiotensin system, which plays a central role in the regulation of blood pressure, electrolyte, and volume homeostasis, may represent a major risk factor for hypertension, heart attack, and stroke. Mounting evidence from clinical studies has demonstrated an inverse relationship between circulating vitamin D levels and the blood pressure and/or plasma renin activity, but the mechanism is not understood. We show here that renin expression and plasma angiotensin II production were increased severalfold in vitamin D receptor-null (VDR-null) mice, leading to hypertension, cardiac hypertrophy, and increased water intake. However, the salt- and volume-sensing mechanisms that control renin synthesis are still intact in the mutant mice. In wild-type mice, inhibition of 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] synthesis also led to an increase in renin expression, whereas 1,25(OH)(2)D(3) injection led to renin suppression. We found that vitamin D regulation of renin expression was independent of calcium metabolism and that 1,25(OH)(2)D(3) markedly suppressed renin transcription by a VDR-mediated mechanism in cell cultures. Hence, 1,25(OH)(2)D(3) is a novel negative endocrine regulator of the renin-angiotensin system. Its apparent critical role in electrolytes, volume, and blood pressure homeostasis suggests that vitamin D analogues could help prevent or ameliorate hypertension.

neurogenin1 Is Essential for the Determination of Neuronal Precursors for Proximal Cranial Sensory Ganglia

The NEUROGENINS (NGNs) are neural-specific basic helix-loop-helix (bHLH) transcription factors. Mouse embryos lacking ngn1 fail to generate the proximal subset of cranial sensory neurons. ngn1 is required for the activation of a cascade of downstream bHLH factors, including NeuroD, MATH3, and NSCL1. ngn1 is expressed by placodal ectodermal cells and acts prior to neuroblast delamination. Moreover, NGN1 positively regulates the Delta homolog DLL1 and can be negatively regulated by Notch signaling. Thus, ngn1 functions similarly to the proneural genes in Drosophila. However, the initial pattern of ngn1 expression appears to be Notch independent. Taken together with the fact that ectopic ngn1 expression can convert ectodermal cells to neurons in Xenopus (Ma et al., 1996), these data and those of Fode et al. (1998 [this issue of Neuron]) identify ngns as vertebrate neuronal determination genes, analogous to myoD and myf5 in myogenesis.

Symmetrical Mutant Phenotypes of the Receptor EphB4 and Its Specific Transmembrane Ligand ephrin-B2 in Cardiovascular Development

Ephrin-B2 is a transmembrane ligand that is specifically expressed on arteries but not veins and that is essential for cardiovascular development. However, ephrin-B2 is also expressed in nonvascular tissues and interacts with multiple EphB class receptors expressed in both endothelial and nonendothelial cell types. Thus, the identity of the relevant receptor for ephrin-B2 and the site(s) where these molecules interact to control angiogenesis were not clear. Here we show that EphB4, a specific receptor for ephrin-B2, is exclusively expressed by vascular endothelial cells in embryos and is preferentially expressed on veins. A targeted mutation in EphB4 essentially phenocopies the mutation in ephrin-B2. These data indicate that ephrin-B2-EphB4 interactions are intrinsically required in vascular endothelial cells and are consistent with the idea that they mediate bidirectional signaling essential for angiogenesis.

A gastrin-releasing peptide receptor mediates the itch sensation in the spinal cord

Itching, or pruritus, is defined as an unpleasant cutaneous sensation that serves as a physiological self-protective mechanism to prevent the body from being hurt by harmful external agents. Chronic itch represents a significant clinical problem resulting from renal diseases and liver diseases, as well as several serious skin diseases such as atopic dermatitis. The identity of the itch-specific mediator in the central nervous system, however, remains elusive. Here we describe that the gastrin-releasing peptide receptor (GRPR) plays an important part in mediating itch sensation in the dorsal spinal cord. We found that gastrin-releasing peptide is specifically expressed in a small subset of peptidergic dorsal root ganglion neurons, whereas expression of its receptor GRPR is restricted to lamina I of the dorsal spinal cord. GRPR mutant mice showed comparable thermal, mechanical, inflammatory and neuropathic pain responses relative to wild-type mice. In contrast, induction of scratching behaviour was significantly reduced in GRPR mutant mice in response to pruritogenic stimuli, whereas normal responses were evoked by painful stimuli. Moreover, direct spinal cerebrospinal fluid injection of a GRPR antagonist significantly inhibited scratching behaviour in three independent itch models. These data demonstrate that GRPR is required for mediating the itch sensation rather than pain, at the spinal level. Our results thus indicate that GRPR may represent the first molecule that is dedicated to mediating the itch sensation in the dorsal horn of the spinal cord, and thus may provide a central therapeutic target for antipruritic drug development.

Sensory neuron-specific GPCR Mrgprs are itch receptors mediating chloroquine-induced pruritus.

The cellular and molecular mechanisms mediating histamine-independent itch in primary sensory neurons are largely unknown. Itch induced by chloroquine (CQ) is a common side effect of this widely used antimalarial drug. Here, we show that Mrgprs, a family of G protein-coupled receptors expressed exclusively in peripheral sensory neurons, function as itch receptors. Mice lacking a cluster of Mrgpr genes display significant deficits in itch induced by CQ but not histamine. CQ directly excites sensory neurons in an Mrgpr-dependent manner. CQ specifically activates mouse MrgprA3 and human MrgprX1. Loss- and gain-of-function studies demonstrate that MrgprA3 is required for CQ responsiveness in mice. Furthermore, MrgprA3-expressing neurons respond to histamine and coexpress gastrin-releasing peptide, a peptide involved in itch sensation, and MrgprC11. Activation of these neurons with the MrgprC11-specific agonist BAM8-22 induces itch in wild-type but not mutant mice. Therefore, Mrgprs may provide molecular access to itch-selective neurons and constitute novel targets for itch therapeutics.

Cellular Basis of Itch Sensation

A Separate System for Itch Processing It has been a long-standing question if itch is a subquality of pain involving the same neuronal elements or if distinct, so-called labeled lines exist in the nervous system for both sensations. To address this question directly, Sun et al. (p. 1531, published online 6 August) destroyed neurons in the superficial spinal dorsal horn that express the gastrin-releasing peptide receptor. This receptor is known to be involved in mediating itch but not pain sensations. In various animal models, ablation of gastrin-releasing peptide receptor-expressing spinal dorsal horn neurons reduced itch without changing pain perception. Thus, itch and pain indeed appear to be mediated by distinct labeled lines in the central nervous system. Itch, but not pain sensation, is abolished by selective ablation of a small subpopulation of spinal neurons. Itch and pain are two distinct sensations. Although our previous study suggested that gastrin-releasing peptide receptor (GRPR) is an itch-specific gene in the spinal cord, a long-standing question of whether there are separate neuronal pathways for itch and pain remains unsettled. We selectively ablated lamina I neurons expressing GRPR in the spinal cord of mice. These mice showed profound scratching deficits in response to all of the itching (pruritogenic) stimuli tested, irrespective of their histamine dependence. In contrast, pain behaviors were unaffected. Our data also suggest that GRPR+ neurons are different from the spinothalamic tract neurons that have been the focus of the debate. Together, the present study suggests that GRPR+ neurons constitute a long-sought labeled line for itch sensation in the spinal cord.

Genetic ablation of parathyroid glands reveals another source of parathyroid hormone.

The parathyroid glands are the only known source of circulating parathyroid hormone (PTH), which initiates an endocrine cascade that regulates serum calcium concentration. Glial cells missing2 (Gcm2), a mouse homologue of Drosophila Gcm, is the only transcription factor whose expression is restricted to the parathyroid glands. Here we show that Gcm2-deficient mice lack parathyroid glands and exhibit a biological hypoparathyroidism, identifying Gcm2 as a master regulatory gene of parathyroid gland development. Unlike PTH receptor-deficient mice, however, Gcm2-deficient mice are viable and fertile, and have only a mildly abnormal bone phenotype. Despite their lack of parathyroid glands, Gcm2-deficient mice have PTH serum levels identical to those of wild-type mice, as do parathyroidectomized wild-type animals. Expression and ablation studies identified the thymus, where Gcm1, another Gcm homologue, is expressed, as the additional, downregulatable source of PTH. Thus, Gcm2 deletion uncovers an auxiliary mechanism for the regulation of calcium homeostasis in the absence of parathyroid glands. We propose that this backup mechanism may be a general feature of endocrine regulation.

Genetic enhancement of inflammatory pain by forebrain NR2B overexpression

N-methyl-D-aspartate (NMDA) receptors contribute to many brain functions. We studied the effect of forebrain-targeted overexpression of the NMDA receptor subunit NR2B on the response of mice to tissue injury and inflammation. Transgenic mice exhibited prominent NR2B expression and enhanced NMDA receptor-mediated synaptic responses in two pain-related forebrain areas, the anterior cingulate cortex and insular cortex, but not in the spinal cord. Although transgenic and wild type mice were indistinguishable in tests of acute pain, transgenic mice exhibited enhanced responsiveness to peripheral injection of two inflammatory stimuli, formalin and complete Freunds adjuvant. Genetic modification of forebrain NMDA receptors can therefore influence pain perception, which suggests that forebrain-selective NMDA receptor antagonists, including NR2B-selective agents, may be useful analgesics for persistent pain.

Defects in Breathing and Thermoregulation in Mice with Near-Complete Absence of Central Serotonin Neurons

Serotonergic neurons project widely throughout the CNS and modulate many different brain functions. Particularly important, but controversial, are the contributions of serotonin (5-HT) neurons to respiratory and thermoregulatory control. To better define the roles of 5-HT neurons in breathing and thermoregulation, we took advantage of a unique conditional knock-out mouse in which Lmx1b is genetically deleted in Pet1-expressing cells (Lmx1bf/f/p), resulting in near-complete absence of central 5-HT neurons. Here, we show that the hypercapnic ventilatory response in adult Lmx1bf/f/p mice was decreased by 50% compared with wild-type mice, whereas baseline ventilation and the hypoxic ventilatory response were normal. In addition, Lmx1bf/f/p mice rapidly became hypothermic when exposed to an ambient temperature of 4°C, decreasing core temperature to 30°C within 120 min. This failure of thermoregulation was caused by impaired shivering and nonshivering thermogenesis, whereas thermosensory perception and heat conservation were normal. Finally, intracerebroventricular infusion of 5-HT stimulated baseline ventilation, and rescued the blunted hypercapnic ventilatory response. These data identify a previously unrecognized role of 5-HT neurons in the CO2 chemoreflex, whereby they enhance the response of the rest of the respiratory network to CO2. We conclude that the proper function of the 5-HT system is particularly important under conditions of environmental stress and contributes significantly to the hypercapnic ventilatory response and thermoregulatory cold defense.