SUMMARY Inner ear hair cell differentiation requires Atoh1 function, while Eya1, Six1 and Sox2 are coexpressed in sensory progenitors and mutations in these genes cause sensorineural hearing loss. However, how these genes are linked functionally and the transcriptional networks controlling hair cell induction remain unclear. Here, we show that Eya1/Six1 are necessary for hair cell development and their coexpression in mouse cochlear explants is sufficient to induce hair cell fate in the nonsensory epithelium expressing low level Sox2 by activating not only Atoh1-dependent but also -independent pathways and that both pathways induce Pou4f3 to promote hair cell differentiation. Sox2 cooperates with Eya1/Six1 to synergistically activate Atoh1 transcription via direct binding to the conserved Sox- and Six-binding sites in Atoh1 enhancers and these proteins physically interact. Our findings demonstrate that direct and cooperative interactions between the Sox2, Six1 and Eya1 proteins coordinate Atoh1 expression to specify hair cell fate.

SUMMARYInner ear neurogenesis depends upon the function of the proneural basic helix-loop-helix (bHLH) transcription factors NEUROG1 and NEUROD1. However, the transcriptional regulation of these factors is unknown. Here, using loss-and gain-of-function models, we show that EYA1 and SIX1 are crucial otic neuronal determination factors upstream of NEUROG1 and NEUROD1. Overexpression of both Eya1 and Six1 is sufficient to convert non-neuronal epithelial cells within the otocyst and cochlea as well as the 3T3 fibroblast cells into neurons. Strikingly, all the ectopic neurons express not only Neurog1 and Neurod1 but also mature neuronal markers such as neurofilament, indicating that Eya1 and Six1 function upstream of, and in the same pathway as, Neurog1 and Neurod1 to not only induce neuronal fate but also regulate their differentiation. We demonstrate that EYA1 and SIX1 interact directly with the SWI/SNF chromatin-remodeling subunits BRG1 and BAF170 to drive neurogenesis cooperatively in 3T3 cells and cochlear nonsensory epithelial cells, and that SOX2 cooperates with these factors to mediate neuronal differentiation. Importantly, we show that the ATPase BRG1 activity is required for not only EYA1-and SIX1-induced ectopic neurogenesis but also normal neurogenesis in the otocyst. These findings indicate that EYA1 and SIX1 are key transcription factors in initiating the neuronal developmental program, probably by recruiting and interacting with the SWI/SNF chromatinremodeling complex to specifically mediate Neurog1 and Neurod1 transcription. Moreover, many genes have been shown to require SWI/SNF complexes for activation in yeast, fruit flies and mammals (Armstrong et al., 2002;Krebs et al., 2000;Liu et al., 2001;Ng et al., 2002;Sudarsanam et al., 2000). Recently, the ATP-dependent chromatin-remodeling enzyme CHD7 has been shown to regulate neurogenesis in the inner ear (Hurd et al., 2010). However, whether the SWI/SNF complexes play a role in mammalian inner ear neurogenesis and whether they interact with other transcription factors to regulate the transcriptional activities of Neurog1 and Neurod1 are not understood. The murine eyes absent (Eya) and homeobox Six gene families, homologous to Drosophila eyes absent and sine oculis, respectively, play essential roles for inner ear development. Haploinsufficiency for human EYA1 or SIX1 leads to branchio-oto-renal syndrome (Abdelhak et al., 1997;Ruf et al., 2004), and genetic deletion of either gene in mice results in early arrest of inner ear development at the otocyst stage (Xu et al., 1999;Zheng et al., 2003;Zou et al., 2006). We have shown that Six1 functions downstream of and interacts genetically with Eya1 during inner ear development (Xu et al., 1999;Zheng et al., 2003;Zou et al., 2006), and their gene products participate in protein-protein interaction (Buller et al., 2001). In Eya1-or Six1-null mutants, neurogenesis is initiated normally but the neuroblast cells fail to form a morphologically detectable ganglion owing to abnormal apoptosis (Zou et al., 2004), ind...

In all jaw-bearing vertebrates, three-dimensional mobility relies on segregated, separately innervated epaxial and hypaxial skeletal muscles. In amniotes, these muscles form from the morphologically continuous dermomyotome and myotome, whose epaxial-hypaxial subdivision and hence the formation of distinct epaxial-hypaxial muscles is not understood. Here we show that En1 expression labels a central subdomain of the avian dermomyotome, medially abutting the expression domain of the lead-lateral or hypaxial marker Sim1. En1 expression is maintained when cells from the En1-positive dermomyotome enter the myotome and dermatome, thereby superimposing the En1-Sim1 expression boundary onto the developing musculature and dermis. En1 cells originate from the dorsomedial edge of the somite. Their development is under positive control by notochord and floor plate (Shh), dorsal neural tube (Wnt1) and surface ectoderm (Wnt1-like signalling activity) but negatively regulated by the lateral plate mesoderm (BMP4). This dependence on epaxial signals and suppression by hypaxial signals places En1 into the epaxial somitic programme. Consequently, the En1-Sim1 expression boundary marks the epaxial-hypaxial dermomyotomal or myotomal boundary. In cell aggregation assays, En1- and Sim1-expressing cells sort out, suggesting that the En1-Sim1 expression boundary may represent a true compartment boundary, foreshadowing the epaxial-hypaxial segregation of muscle.

Somitic and head mesoderm contribute to cartilage and bone and deliver the entire skeletal musculature. Studies on avian somite patterning and cell differentiation led to the view that these processes depend solely on cues from surrounding tissues. However, evidence is accumulating that some developmental decisions depend on information within the somitic tissue itself. Moreover, recent studies established that head and somitic mesoderm, though delivering the same tissue types, are set up to follow their own, distinct developmental programmes. With a particular focus on the chicken embryo, we review the current understanding of how extrinsic signalling, operating in a framework of intrinsically regulated constraints, controls paraxial mesoderm patterning and cell differentiation.

No abstract

The cerebellum is derived from the anterior-most segment of the embryonic hindbrain, rhombomere 1 (r1). Previous studies have shown that the early development and patterning of r1 requires fibroblast growth factor (FGF) signaling. However, many of the developmental processes that shape cerebellar morphogenesis take place later in embryonic development and during the first 2 weeks of postnatal life in the mouse. Here, we present a more comprehensive analysis of the expression patterns of genes encoding FGF receptors and secreted FGF ligands during these later stages of cerebellar development. We show that these genes are expressed in multiple cell types in the developing cerebellum, in an astonishing array of distinct patterns. These data suggest that FGF signaling functions throughout cerebellar development to regulate many processes that shape the formation of a functional cerebellum.

Trunk skeletal muscles are segregated into dorsomedial epaxial and ventrolateral hypaxial muscles, separated by a myoseptum. In amniotes, they are generated from a transient structure, the dermomyotome, which lays down muscle, namely the myotome underneath. However, the dermomyotome and myotome are dorsoventrally continuous, with no morphologically defined epaxial-hypaxial boundary. The transcription factors En1 and Sim1 have been shown to molecularly subdivide the amniote dermomyotome, with En1 labeling the epaxial dermomyotome and Sim1 the hypaxial counterpart. Here, we demonstrate that En1 and Sim1 expression persists in cells leaving the dermomyotome, superimposing the expression boundary onto muscle and skin. En1-expressing cells colonize the myotome initially from the rostral and caudal lips, and slightly later, directly from the de-epithelializing dermomyotomal center. En1 expression in the myotome is concomitant with the appearance of Fgfr4/Pax7-expressing mitotically active myoblasts. This finding suggests that Fgfr4 ϩ /Pax7 ϩ /En1 ϩ cells carry their expression with them when entering the myotome. Furthermore, it suggests that the epaxial-hypaxial boundary of the myotome is established through the late arising, mitotically active myoblasts.

The purpose of this study was to examine the effects of self-reported perceived stress at work and home on the levels, variation and co-variation of ambulatory blood pressure (BP), pulse rate (PR) and urinary catecholamine, cortisol, and aldosterone excretion measured at work, home and during sleep in women employed outside the home. The subjects of the study were 134 women (mean age 34.4 +/- 9.6 years, range 18 to 64 years) who were employed in managerial, technical or clerical positions at the same work place. Perceived stress at work and home was self-reported on a scale from 0 (low) to 10 (high). BP, PR and the urinary rates of excretion of epinephrine, norepinephrine, cortisol and aldosterone were averaged in the daily work environment from 11 AM to 3 PM, in the daily home environment from approximately 6 PM to 10 PM, and during sleep from approximately 10 PM to 6 AM the following morning. The results showed that systolic and diastolic BP (SBP and DBP) and the rates of urinary catecholamine, cortisol, and aldosterone excretion measured in the work environment were significantly higher than corresponding measurements taken in the home environment. SBP measured at work was also positively correlated with the difference in perceived stress between work and home (p < 0.05). PR (p < 0.001) and the rate of urinary norepinephrine excretion (p < 0.05) measured in the home environment were positively correlated with stress at home. When the subjects were divided into groups based on whether the work or home environment was perceived to be most stressful, women reporting greater stress at work (n=85) had higher work SBP (p < 0.005), work DBP (p < 0.05), and sleep SBP (p < 0.005) than women who perceived the home environment to be more stressful (n=34). There were no differences in the urinary hormonal excretion rates between these perceived-stress groups. Among women with greater perceived stress at home, the home-stress score was positively correlated with sleep SBP level (r = 0.310, p < 0.05), its variation (SD of sleep SBP: r = 0.402, p < 0.01) and home pulse rate ( r= 0.414, p < 0.01). These findings suggest that among employed women, work stress may increase ambulatory BP levels throughout the day, while home stress may induce additional sympathetic activation at home. In addition, they also show that among employed women who perceive greater stress at home than at work, higher home stress levels may also elevate sleep BP levels.