A BMP regulatory network controls ectodermal cell fate decisions at the neural plate border.

During ectodermal patterning the neural crest and preplacodal ectoderm are specified in adjacent domains at the neural plate border. BMP signalling is required for specification of both tissues, but how it is spatially and temporally regulated to achieve this is not understood. Here, using a transgenic zebrafish BMP reporter line in conjunction with double-fluorescent in situ hybridisation, we show that, at the beginning of neurulation, the ventral-to-dorsal gradient of BMP activity evolves into two distinct domains at the neural plate border: one coinciding with the neural crest and the other abutting the epidermis. In between is a region devoid of BMP activity, which is specified as the preplacodal ectoderm. We identify the ligands required for these domains of BMP activity. We show that the BMP-interacting protein Crossveinless 2 is expressed in the BMP activity domains and is under the control of BMP signalling. We establish that Crossveinless 2 functions at this time in a positive-feedback loop to locally enhance BMP activity, and show that it is required for neural crest fate. We further demonstrate that the Distal-less transcription factors Dlx3b and Dlx4b, which are expressed in the preplacodal ectoderm, are required for the expression of a cell-autonomous BMP inhibitor, Bambi-b, which can explain the specific absence of BMP activity in the preplacodal ectoderm. Taken together, our data define a BMP regulatory network that controls cell fate decisions at the neural plate border.

The Neuropeptide Galanin Is Required for Homeostatic Rebound Sleep following Increased Neuronal Activity.

Sleep pressure increases during wake and dissipates during sleep, but the molecules and neurons that measure homeostatic sleep pressure remain poorly understood. We present a pharmacological assay in larval zebrafish that generates short-term increases in wakefulness followed by sustained rebound sleep after washout. The intensity of global neuronal activity during drug-induced wakefulness predicted the amount of subsequent rebound sleep. Whole-brain mapping with the neuronal activity marker phosphorylated extracellular signal-regulated kinase (pERK) identified preoptic Galanin (Galn)-expressing neurons as selectively active during rebound sleep, and the relative induction of galn transcripts was predictive of total rebound sleep time. Galn is required for sleep homeostasis, as galn mutants almost completely lacked rebound sleep following both pharmacologically induced neuronal activity and physical sleep deprivation. These results suggest that Galn plays a key role in responding to sleep pressure signals derived from neuronal activity and functions as an output arm of the vertebrate sleep homeostat.

Light-Dependent Regulation of Sleep and Wake States by Prokineticin 2 in Zebrafish.

Light affects sleep and wake behaviors by providing an indirect cue that entrains circadian rhythms and also by inducing a direct and rapid regulation of behavior. While circadian entrainment by light is well characterized at the molecular level, mechanisms that underlie the direct effect of light on behavior are largely unknown. In zebrafish, a diurnal vertebrate, we found that both overexpression and mutation of the neuropeptide prokineticin 2 (Prok2) affect sleep and wake behaviors in a light-dependent but circadian-independent manner. In light, Prok2 overexpression increases sleep and induces expression of galanin (galn), a hypothalamic sleep-inducing peptide. We also found that light-dependent, Prok2-induced sedation requires prokineticin receptor 2 (prokr2) and is strongly suppressed in galn mutants. These results suggest that Prok2 antagonizes the direct wake-promoting effect of light in zebrafish, in part through the induction of galn expression in the hypothalamus.

CPI-17 drives oncogenic Ras signaling in human melanomas via Ezrin-Radixin-Moesin family proteins.

Hyperactive Ras signaling has strong oncogenic effects causing several different forms of cancer. Hyperactivity is frequently induced by mutations within Ras itself, which account for up to 30% of all human cancers. In addition, hyperactive Ras signaling can also be triggered independent of Ras by either mutation or by misexpression of various upstream regulators and immediate downstream effectors. We have previously reported that C-kinase potentiated protein phosphatase-1 inhibitor of 17 kDa (CPI-17) can drive Ras activity and promote tumorigenic transformation by inhibition of the tumor suppressor Merlin. We now describe an additional element of this oncogenic mechanism in the form of the ezrin-radixin-moesin (ERM) protein family, which exhibits opposing roles in Ras activity control. Thus, CPI-17 drives Ras activity and tumorigenesis in a two-fold way; inactivation of the tumor suppressor merlin and activation of the growth promoting ERM family. The in vivo significance of this oncogenic switch is highlighted by demonstrating CPI-17's involvement in human melanoma pathogenesis.

A new class of small molecule inhibitor of BMP signaling.

Growth factor signaling pathways are tightly regulated by phosphorylation and include many important kinase targets of interest for drug discovery. Small molecule inhibitors of the bone morphogenetic protein (BMP) receptor kinase ALK2 (ACVR1) are needed urgently to treat the progressively debilitating musculoskeletal disease fibrodysplasia ossificans progressiva (FOP). Dorsomorphin analogues, first identified in zebrafish, remain the only BMP inhibitor chemotype reported to date. By screening an assay panel of 250 recombinant human kinases we identified a highly selective 2-aminopyridine-based inhibitor K02288 with in vitro activity against ALK2 at low nanomolar concentrations similar to the current lead compound LDN-193189. K02288 specifically inhibited the BMP-induced Smad pathway without affecting TGF-ß signaling and induced dorsalization of zebrafish embryos. Comparison of the crystal structures of ALK2 with K02288 and LDN-193189 revealed additional contacts in the K02288 complex affording improved shape complementarity and identified the exposed phenol group for further optimization of pharmacokinetics. The discovery of a new chemical series provides an independent pharmacological tool to investigate BMP signaling and offers multiple opportunities for pre-clinical development.