A single dose of AC102 restores hearing in a guinea pig model of noise-induced hearing loss to almost prenoise levels.

Although sudden sensorineural hearing loss (SSNHL) is a serious condition, there are currently no approved drugs for its treatment. Nevertheless, there is a growing understanding that the cochlear pathologies that underlie SSNHL include apoptotic death of sensory outer hair cells (OHCs) as well as loss of ribbon synapses connecting sensory inner hair cells (IHCs) and neurites of the auditory nerve, designated synaptopathy. Noise-induced hearing loss (NIHL) is a common subtype of SSNHL and is widely used to model hearing loss preclinically. Here, we demonstrate that a single interventive application of a small pyridoindole molecule (AC102) into the middle ear restored auditory function almost to prenoise levels in a guinea pig model of NIHL. AC102 prevented noise-triggered loss of OHCs and reduced IHC synaptopathy suggesting a role of AC102 in reconnecting auditory neurons to their sensory target cells. Notably, AC102 exerted its therapeutic properties over a wide frequency range. Such strong improvements in hearing have not previously been demonstrated for other therapeutic agents. In vitro experiments of a neuronal damage model revealed that AC102 protected cells from apoptosis and promoted neurite growth. These effects may be explained by increased production of adenosine triphosphate, indicating improved mitochondrial function, and reduced levels of reactive-oxygen species which prevents the apoptotic processes responsible for OHC death. This action profile of AC102 might be causal for the observed hearing recovery in in vivo models.

Sphingosine 1-phosphate receptor signaling regulates proper embryonic vascular patterning.

Sphingosine 1-phosphate (S1P) binds G-protein-coupled receptors (S1P(1-5)) to regulate a multitude of physiological effects, especially those in the vascular and immune systems. S1P receptors in the vascular system have been characterized primarily in mammals. Here, we report that the S1P receptors and metabolic enzymes are conserved in the genome of zebrafish Danio rerio. Bioinformatic analysis identified seven S1P receptor-like sequences in the zebrafish genome, including duplicated orthologs of receptors 3 and 5. Sphingolipidomic analysis detected erythrocyte and plasma S1P as well as high plasma ceramides and sphingosine. Morpholino-mediated knockdown of s1pr1 causes global and pericardial edema, loss of blood circulation, and vascular defects characterized by both reduced vascularization in intersegmental vessels, decreased proliferation of intersegmental and axial vessels, and hypersprouting in the caudal vein plexus. The s1pr2 gene was previously characterized as a regulator of cell migration and heart development, but its role in angiogenesis is not known. However, when expression of both s1pr1 and s1pr2 is suppressed, severely reduced vascular development of the intersegmental vessels was observed with doses of the s1pr1 morpholino that alone did not cause any discernible vascular defects, suggesting that s1pr1 and s1pr2 function cooperatively to regulate vascular development in zebrafish. Similarly, the S1P transporter, spns2, also cooperated with s1pr1. We propose that extracellular S1P acts through vascular S1P receptors to regulate vascular development.

'In parallel' interconnectivity of the dorsal longitudinal anastomotic vessels requires both VEGF signaling and circulatory flow.

Blood vessels deliver oxygen, nutrients, hormones and immunity factors throughout the body. To perform these vital functions, vascular cords branch, lumenize and interconnect. Yet, little is known about the cellular, molecular and physiological mechanisms that control how circulatory networks form and interconnect. Specifically, how circulatory networks merge by interconnecting 'in parallel' along their boundaries remains unexplored. To examine this process we studied the formation and functional maturation of the plexus that forms between the dorsal longitudinal anastomotic vessels (DLAVs) in the zebrafish. We find that the migration and proliferation of endothelial cells within the DLAVs and their segmental (Se) vessel precursors drives DLAV plexus formation. Remarkably, the presence of Se vessels containing only endothelial cells of the arterial lineage is sufficient for DLAV plexus morphogenesis, suggesting that endothelial cells from the venous lineage make a dispensable or null contribution to this process. The discovery of a circuit that integrates the inputs of circulatory flow and vascular endothelial growth factor (VEGF) signaling to modulate aortic arch angiogenesis, together with the expression of components of this circuit in the trunk vasculature, prompted us to investigate the role of these inputs and their relationship during DLAV plexus formation. We find that circulatory flow and VEGF signaling make additive contributions to DLAV plexus morphogenesis, rather than acting as essential inputs with equivalent contributions as they do during aortic arch angiogenesis. Our observations underscore the existence of context-dependent differences in the integration of physiological stimuli and signaling cascades during vascular development.

The novel transmembrane protein Tmem2 is essential for coordination of myocardial and endocardial morphogenesis.

Coordination between adjacent tissues plays a crucial role during the morphogenesis of developing organs. In the embryonic heart, two tissues - the myocardium and the endocardium - are closely juxtaposed throughout their development. Myocardial and endocardial cells originate in neighboring regions of the lateral mesoderm, migrate medially in a synchronized fashion, collaborate to create concentric layers of the heart tube, and communicate during formation of the atrioventricular canal. Here, we identify a novel transmembrane protein, Tmem2, that has important functions during both myocardial and endocardial morphogenesis. We find that the zebrafish mutation frozen ventricle (frv) causes ectopic atrioventricular canal characteristics in the ventricular myocardium and endocardium, indicating a role of frv in the regional restriction of atrioventricular canal differentiation. Furthermore, in maternal-zygotic frv mutants, both myocardial and endocardial cells fail to move to the midline normally, indicating that frv facilitates cardiac fusion. Positional cloning reveals that the frv locus encodes Tmem2, a predicted type II single-pass transmembrane protein. Homologs of Tmem2 are present in all examined vertebrate genomes, but nothing is known about its molecular or cellular function in any context. By employing transgenes to drive tissue-specific expression of tmem2, we find that Tmem2 can function in the endocardium to repress atrioventricular differentiation within the ventricle. Additionally, Tmem2 can function in the myocardium to promote the medial movement of both myocardial and endocardial cells. Together, our data reveal that Tmem2 is an essential mediator of myocardium-endocardium coordination during cardiac morphogenesis.

Diverse functions for the semaphorin receptor PlexinD1 in development and disease.

Plexins are a family of single-pass transmembrane proteins that serve as cell surface receptors for Semaphorins during the embryonic development of animals. Semaphorin-Plexin signaling is critical for many cellular aspects of organogenesis, including cell migration, proliferation and survival. Until recently, little was known about the function of PlexinD1, the sole member of the vertebrate-specific PlexinD (PlxnD1) subfamily. Here we review novel findings about PlxnD1's roles in the development of the cardiovascular, nervous and immune systems and salivary gland branching morphogenesis and discuss new insights concerning the molecular mechanisms of PlxnD1 activity.

GIPC proteins negatively modulate Plexind1 signaling during vascular development.

Semaphorins (SEMAs) and their Plexin (PLXN) receptors are central regulators of metazoan cellular communication. SEMA-PLXND1 signaling plays important roles in cardiovascular, nervous, and immune system development, and cancer biology. However, little is known about the molecular mechanisms that modulate SEMA-PLXND1 signaling. As PLXND1 associates with GIPC family endocytic adaptors, we evaluated the requirement for the molecular determinants of their association and PLXND1's vascular role. Zebrafish that endogenously express a Plxnd1 receptor with a predicted impairment in GIPC binding exhibit low penetrance angiogenesis deficits and antiangiogenic drug hypersensitivity. Moreover, gipc mutant fish show angiogenic impairments that are ameliorated by reducing Plxnd1 signaling. Finally, GIPC depletion potentiates SEMA-PLXND1 signaling in cultured endothelial cells. These findings expand the vascular roles of GIPCs beyond those of the Vascular Endothelial Growth Factor (VEGF)-dependent, proangiogenic GIPC1-Neuropilin 1 complex, recasting GIPCs as negative modulators of antiangiogenic PLXND1 signaling and suggest that PLXND1 trafficking shapes vascular development.

RhoGAP control of pancreas development: putting cells in the right place at the right time.

Recent evidences suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing ß-cells, depend on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium. We recently found that Rho GTPase and its regulator, Stard13 RhoGAP, coordinate morphogenesis with growth in the developing pancreas. Conditional mutation of Stard13 in the mouse pancreas hampers epithelial remodeling and distal tip domain formation, affecting proliferation and expansion of pancreatic progenitors. These defects eventually result in pancreatic hypoplasia at birth. Stard13 acts by regulating Rho signaling spatially and temporally during pancreas development. In line with this, pharmacological activation or inhibition of Rho mimics or rescues, respectively, the defects observed in Stard13-deficient embryos and pancreatic organ cultures. Furthermore, in the absence of Stard13 uninhibited Rho activity affects the actomyosin contractile network, disrupting its apical distribution and hampering coordinated cell-shape changes. These results unveil therefore the crucial role of actin cytoskeletal dynamics during the onset of pancreatic branching morphogenesis. Finally, our findings define a reciprocal interaction between the actin-MAL/SRF and the MAPK signaling to locally regulate progenitor cell proliferation in the pancreas.

Semaphorin-PlexinD1 signaling limits angiogenic potential via the VEGF decoy receptor sFlt1.

Sprouting angiogenesis expands the embryonic vasculature enabling survival and homeostasis. Yet how the angiogenic capacity to form sprouts is allocated among endothelial cells (ECs) to guarantee the reproducible anatomy of stereotypical vascular beds remains unclear. Here we show that Sema-PlxnD1 signaling, previously implicated in sprout guidance, represses angiogenic potential to ensure the proper abundance and stereotypical distribution of the trunk's segmental arteries (SeAs). We find that Sema-PlxnD1 signaling exerts this effect by antagonizing the proangiogenic activity of vascular endothelial growth factor (VEGF). Specifically, Sema-PlxnD1 signaling ensures the proper endothelial abundance of soluble flt1 (sflt1), an alternatively spliced form of the VEGF receptor Flt1 encoding a potent secreted decoy. Hence, Sema-PlxnD1 signaling regulates distinct but related aspects of angiogenesis: the spatial allocation of angiogenic capacity within a primary vessel and sprout guidance.