The retrograde IFT dynein is required for normal function of diverse mechanosensory cilia in Drosophila

IntroductionCilia biogenesis relies on intraflagellar transport (IFT), a conserved transport mechanism which functions bi-directionally to bring protein complexes to the growing ciliary tip and recycle signaling and transport proteins between the cilium and cell body. In Drosophila, anterograde IFT is critical for assembly of sensory cilia in the neurons of both chordotonal (ch) organs, which have relatively long ciliary axonemes, and external sensory (es) organs, which have short axonemal segments with microtubules in distal sensory segments forming non-axonemal bundles. We previously isolated the beethoven (btv) mutant in a mutagenesis screen for auditory mutants. Although many btv mutant flies are deaf, some retain a small residual auditory function as determined both by behavior and by auditory electrophysiology.ResultsHere we molecularly characterize the btv gene and demonstrate that it encodes the IFT-associated dynein-2 heavy chain Dync2h1. We also describe morphological changes in Johnston’s organ as flies age to 30 days, and we find that morphological and electrophysiological phenotypes in this ch organ of btv mutants become more severe with age. We show that NompB protein, encoding the conserved IFT88 protein, an IFT complex B component, fails to be cleared from chordotonal cilia in btv mutants, instead accumulating in the distorted cilia. In macrochaete bristles, a class of es organ, btv mutants show a 50% reduction in mechanoreceptor potentials.DiscussionThus, the btv-encoded Dync2h1 functions as the retrograde IFT motor in the assembly of long ciliary axonemes in ch organs and is also important for normal function of the short ciliary axonemes in es organs

Dynamic Range Compression in the Honey Bee Auditory System toward Waggle Dance Sounds

Honey bee foragers use a “waggle dance” to inform nestmates about direction and distance to locations of attractive food. The sound and air flows generated by dancer's wing and abdominal vibrations have been implicated as important cues, but the decoding mechanisms for these dance messages are poorly understood. To understand the neural mechanisms of honey bee dance communication, we analyzed the anatomy of antenna and Johnston's organ (JO) in the pedicel of the antenna, as well as the mechanical and neural response characteristics of antenna and JO to acoustic stimuli, respectively. The honey bee JO consists of about 300–320 scolopidia connected with about 48 cuticular “knobs” around the circumference of the pedicel. Each scolopidium contains bipolar sensory neurons with both type I and II cilia. The mechanical sensitivities of the antennal flagellum are specifically high in response to low but not high intensity stimuli of 265–350 Hz frequencies. The structural characteristics of antenna but not JO neurons seem to be responsible for the non-linear responses of the flagellum in contrast to mosquito and fruit fly. The honey bee flagellum is a sensitive movement detector responding to 20 nm tip displacement, which is comparable to female mosquito. Furthermore, the JO neurons have the ability to preserve both frequency and temporal information of acoustic stimuli including the “waggle dance” sound. Intriguingly, the response of JO neurons was found to be age-dependent, demonstrating that the dance communication is only possible between aged foragers. These results suggest that the matured honey bee antennae and JO neurons are best tuned to detect 250–300 Hz sound generated during “waggle dance” from the distance in a dark hive, and that sufficient responses of the JO neurons are obtained by reducing the mechanical sensitivity of the flagellum in a near-field of dancer. This nonlinear effect brings about dynamic range compression in the honey bee auditory system

Improved Survival, Vascular Differentiation and Wound Healing Potential of Stem Cells Co-Cultured with Endothelial Cells

In this study, we developed a methodology to improve the survival, vascular differentiation and regenerative potential of umbilical cord blood (UCB)-derived hematopoietic stem cells (CD34+ cells), by co-culturing the stem cells in a 3D fibrin gel with CD34+-derived endothelial cells (ECs). ECs differentiated from CD34+ cells appear to have superior angiogenic properties to fully differentiated ECs, such as human umbilical vein endothelial cells (HUVECs). Our results indicate that the pro-survival effect of CD34+-derived ECs on CD34+ cells is mediated, at least in part, by bioactive factors released from ECs. This effect likely involves the secretion of novel cytokines, including interleukin-17 (IL-17) and interleukin-10 (IL-10), and the activation of the ERK 1/2 pathway in CD34+ cells. We also show that the endothelial differentiation of CD34+ cells in co-culture with CD34+-derived ECs is mediated by a combination of soluble and insoluble factors. The regenerative potential of this co-culture system was demonstrated in a chronic wound diabetic animal model. The co-transplantation of CD34+ cells with CD34+-derived ECs improved the wound healing relatively to controls, by decreasing the inflammatory reaction and increasing the neovascularization of the wound

Myosin VIIA, Important for Human Auditory Function, Is Necessary for Drosophila Auditory Organ Development

BACKGROUND: Myosin VIIA (MyoVIIA) is an unconventional myosin necessary for vertebrate audition [1]-[5]. Human auditory transduction occurs in sensory hair cells with a staircase-like arrangement of apical protrusions called stereocilia. In these hair cells, MyoVIIA maintains stereocilia organization [6]. Severe mutations in the Drosophila MyoVIIA orthologue, crinkled (ck), are semi-lethal [7] and lead to deafness by disrupting antennal auditory organ (Johnston's Organ, JO) organization [8]. ck/MyoVIIA mutations result in apical detachment of auditory transduction units (scolopidia) from the cuticle that transmits antennal vibrations as mechanical stimuli to JO. PRINCIPAL FINDINGS: Using flies expressing GFP-tagged NompA, a protein required for auditory organ organization in Drosophila, we examined the role of ck/MyoVIIA in JO development and maintenance through confocal microscopy and extracellular electrophysiology. Here we show that ck/MyoVIIA is necessary early in the developing antenna for initial apical attachment of the scolopidia to the articulating joint. ck/MyoVIIA is also necessary to maintain scolopidial attachment throughout adulthood. Moreover, in the adult JO, ck/MyoVIIA genetically interacts with the non-muscle myosin II (through its regulatory light chain protein and the myosin binding subunit of myosin II phosphatase). Such genetic interactions have not previously been observed in scolopidia. These factors are therefore candidates for modulating MyoVIIA activity in vertebrates. CONCLUSIONS: Our findings indicate that MyoVIIA plays evolutionarily conserved roles in auditory organ development and maintenance in invertebrates and vertebrates, enhancing our understanding of auditory organ development and function, as well as providing significant clues for future research

Bone Marrow-Derived Progenitor Cells Augment Venous Remodeling in a Mouse Dorsal Skinfold Chamber Model

The delivery of bone marrow-derived cells (BMDCs) has been widely used to stimulate angiogenesis and arteriogenesis. We identified a progenitor-enriched subpopulation of BMDCs that is able to augment venular remodeling, a generally unexplored area in microvascular research. Two populations of BMDCs, whole bone marrow (WBM) and Lin−/Sca-1+ progenitor cells, were encapsulated in sodium alginate and delivered to a mouse dorsal skinfold chamber model. Upon observation that encapsulated Sca-1+ progenitor cells enhance venular remodeling, the cells and tissue were analyzed on structural and molecular levels. Venule walls were thickened and contained more nuclei after Sca-1+ progenitor cell delivery. In addition, progenitors expressed mRNA transcript levels of chemokine (C-X-C motif) ligand 2 (CXCL2) and interferon gamma (IFNγ) that are over 5-fold higher compared to WBM. Tissues that received progenitors expressed significantly higher protein levels of vascular endothelial growth factor (VEGF), monocyte chemotactic protein-1 (MCP-1), and platelet derived growth factor-BB (PDGF-BB) compared to tissues that received an alginate control construct. Nine days following cell delivery, tissue from progenitor recipients contained 39% more CD45+ leukocytes, suggesting that these cells may enhance venular remodeling through the modulation of the local immune environment. Results show that different BMDC populations elicit different microvascular responses. In this model, Sca-1+ progenitor cell-derived CXCL2 and IFNγ may mediate venule enlargement via modulation of the local inflammatory environment

Figure 2

<p>Organization of the honey bee JO. (A) TEM picture of the honey bee JO is shown. The morphology and positions of the cuticular knob (1), epithelial cell (2), quasi-longitudinal section of long sensory processes (3), quasi-transverse section of scolopidia (4), soft chitin (5), pedicel (P), and flagellum (F) are indicated. (B) The summary of the ultrastructural analysis of the honey bee JO is shown. At the joint of the pedicel (P) and flagellum (F), the cuticle is organized in a complex pattern of radial fibrils (horizontal lines), with circular fibrils surrounding the cuticular “knobs” (K) to which the scolopidia are attached. There are approximately 48 knobs evenly distributed around the circumference of the flagellum at its joint with the pedicel. Epithelial cells (EC; blue) exhibit extensive apical microvilli, likely for the copious secretion of cuticle proteins. The epithelial cell cytoplasm is filled with spongiform membranous organelles, likely also reflecting high secretion levels. Each cuticular knob is the attachment site of 3–10 scolopidia. Each scolopidium forms an independent dendritic cap (red), and these are surrounded by cap cells (CC) which enclose electron dense rods (green) that are thick apically but divide more basally into multiple finer rods. The scolopale cell (SC) of each scolopidium forms a spindle-shaped cage of scolopale rods (organe) and encloses an extracellular scolopale space (ss), through which the ciliary outer dendritic segments of three neurons (N) extend. Morphologically, honey bee JO scolopidia are amphinematic, containing cilia of both Types I and II (see classification described in ref. 30). The two Type I cilia are of uniform diameter, contain an axoneme along their entire length, and attach to the basal end of the dendritic cap; the single Type II cilium contains an axonemal segment up to the dendritic cap, then a wider non-axonemal segment with loose microtubules that continues throughout the length of the dendritic cap. Basally, there are accessory cells (AC) of uncertain classification. The structure of the honey bee JO is consistent with a sensory function for flagellar vibration.</p

Figure 6

<p>Intensity characteristics of SEPs in response to acoustic stimuli. (A) Schematic representation of the preparation is shown. Honey bee heads are fixed and exposed from the end of a micropipette tip, and the recording electrode is inserted at the joint between the scape and pedicel (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000234#pone-0000234-g001" target="_blank">Fig. 1C</a>) and the reference electrode penetrates the head cuticle. Sound stimuli are delivered through a Tygon tube ending in a 7 mm opening close to the honey bee. The dashed line indicates the hemispherical zone where full near-field acoustic conditions are maintained. The SEP magnitudes in response to 750 (B and D) and 265 (C and E) Hz tone stimuli are plotted against the flagellum (B and C) and air particle (D and E) displacements. The SEP magnitudes linearly increase with the flagellar tip displacement up to 100 nm. The response of JO neurons can be detected by 20 nm displacement of flagellar tip in response to 750 Hz stimuli. The background SEP magnitude is <0.02. The response of JO neurons saturates by the flagellar tip displacement above 200nm in response to 265 Hz stimuli. (F) Bar graph shows the mean SEP magnitudes in response to 265 Hz acoustic stimuli at 7 mm/s air particle velocity. SEPs were measured before (Control) and after (Lesioned) lesions in the pedicel. Error bars indicate standard error (N = 5 antennae per treatment, where the SEP of each antenna is the averaged response to 10 trials). (G) SEPs were measured before (Control) and after cutting off the distal one-third (1/3 severed) and two-thirds (2/3 severed) segment of the flagellum. Bar graph of the mean SEP magnitudes as above is shown. Error bars indicate standard error.</p

Figure 1

<p>External morphology of the honey bee antenna. The honey bee antenna was examined by scanning electron microscopy with different magnifications (A; x40, B; x150, C; x 300). Two proximal antennal segments (scape and pedicel) and the ten segments of the flagellum are indicated by arrows in A. Arrowhead in C indicates the position of electrode insertion for SEP recordings. The scale of each panel is shown by a white bar.</p