Twin-Airy Point-Spread Function for Extended-Volume Particle Localization.

The localization of point sources in optical microscopy enables nm-precision imaging of single-molecules and biological dynamics. We report a new method of localization microscopy using twin Airy beams that yields precise 3D localization with the key advantages of extended depth range, higher optical throughput, and potential for imaging higher emitter densities than are possible using other techniques. A precision of better than 30 nm was achieved over a depth range in excess of 7 µm using a 60×, 1.4 NA objective. An illustrative application to extended-depth-range blood-flow imaging in a live zebrafish is also demonstrated.

Defining the molecular pathologies in cloaca malformation: similarities between mouse and human.

Anorectal malformations are congenital anomalies that form a spectrum of disorders, from the most benign type with excellent functional prognosis, to very complex, such as cloaca malformation in females in which the rectum, vagina and urethra fail to develop separately and instead drain via a single common channel into the perineum. The severity of this phenotype suggests that the defect occurs in the early stages of embryonic development of the organs derived from the cloaca. Owing to the inability to directly investigate human embryonic cloaca development, current research has relied on the use of mouse models of anorectal malformations. However, even studies of mouse embryos lack analysis of the earliest stages of cloaca patterning and morphogenesis. Here we compared human and mouse cloaca development and retrospectively identified that early mis-patterning of the embryonic cloaca might underlie the most severe forms of anorectal malformation in humans. In mouse, we identified that defective sonic hedgehog (Shh) signaling results in early dorsal-ventral epithelial abnormalities prior to the reported defects in septation. This is manifested by the absence of Sox2 and aberrant expression of keratins in the embryonic cloaca of Shh knockout mice. Shh knockout embryos additionally develop a hypervascular stroma, which is defective in BMP signaling. These epithelial and stromal defects persist later, creating an indeterminate epithelium with molecular alterations in the common channel. We then used these animals to perform a broad comparison with patients with mild-to-severe forms of anorectal malformations including cloaca malformation. We found striking parallels with the Shh mouse model, including nearly identical defective molecular identity of the epithelium and surrounding stroma. Our work strongly suggests that early embryonic cloacal epithelial differentiation defects might be the underlying cause of severe forms of anorectal malformations in humans. Moreover, deranged Shh and BMP signaling is correlated with severe anorectal malformations in both mouse and humans.

Sustained Bmp signaling is essential for cloaca development in zebrafish.

Bone morphogenetic protein (Bmp) signaling has long been known to be important for the early development of the ventral mesoderm, including blood, vasculature and kidney cells. Although Bmp genes are continually expressed in the ventral cells throughout gastrulation and somitogenesis, previous studies in zebrafish have not addressed how the role of Bmp signaling changes over time to regulate ventral mesoderm development. Here, we describe the use of a transgenic inducible dominant-negative Bmp receptor line to examine the temporal roles of Bmp signaling in ventral mesoderm patterning. Surprisingly, we find that Bmp signaling from the mid-gastrula stage through early somitogenesis is important for excluding blood and vascular precursors from the extreme ventral mesoderm, and we show that this domain is normally required for development of the cloaca (the common gut and urogenital opening). Using a novel assay for cloacal function, we find that larvae with reduced mid-gastrula Bmp signaling cannot properly excrete waste. We show that the cloacal defects result from alterations in the morphogenesis of the cloaca and from changes in the expression of genes marking the excretory system. Finally, we show that HrT, a T-box transcription factor, is a Bmp-regulated gene that has an essential function in cloacal development. We conclude that sustained Bmp signaling plays an important role in specification of the zebrafish cloaca by maintaining the fate of extreme ventral cells during the course of gastrulation and early somitogenesis. Furthermore, our data suggest that alterations in Bmp signaling are one possible cause of anorectal malformations during human embryogenesis.

Further observations on the sensitive innervation of some bird's proctodeum.

The AA. studied the autonomic and sensitive somatic innervation of some female bird's proctodeum, through the properly modified Ruffini's gold chloride method. The vegetative component was constituted by ganglion cells of different size, isolated or grouped to form ganglia, found along the course of nerve trunks or in the concurrent point of different nerve bundles. The sensitive somatic innervation was represented by free and encapsulated endings differently distributed in the thickness of the wall. The former were composed of thin networks, while the latter, located more frequently in the muscular tunica and in the subadventitial connective, were composed of encapsulated receptors classified as Pacini, Pacini-like and Herbst corpuscles. The morphology of these receptors was described and hypotheses were brought up about their probable functional role. The AA, also found, even if very rarely, helicoidal collagen fibres around nerve fascicles.

Suprimento arterial da cloaca e corpo vascular paracloacal da codorna Nothura maculosa (Temminck, 1815) / The arterial suply to the cloaca and paracloacal vascular body of Nothura maculosa (Temminck, 1815)

A cloaca da codorna Nothura maculosa (Temminck, 1815) é irrigada por ramos da artéria sacral mediana, principalmente as artérias pudendas internas, as quais säo também responsáveis pela formaçäo dos corpos vasculares paracloacais. No lado esquerdo, a artéria pudenda interna, após formar o corpo vascular paracloacal, continua-se como artéria fálica, que se distribui no fálus intromitente desta espécie, através de dois ramos principais. Os corpos vasculares paracloacais apresentam cavidades linfáticas contínuas com as cavidades linfáticas do fálus, sugerindo sua participaçäo no mecanismo de ereçäo

The mechanism of the ejection of a frothy fluid from the cloaca in the male turkey.

The mechanism of the ejection of a frothy fluid from the "tissue at the vicinity of the papilla" (TVP) of the ductus deferens and triangular fold (TF) of the cloaca of the male turkey during natural copulation was investigated. Injection of Evans blue (T-1824) solution into the wing vein of the intact male led to the ejection, immediately after injection, of frothy fluid colored blue. Ligation of the A. pudenda interna decreased the volume of fluid to a negligible amount, and removal of the paracloacal vascular body (Corpus vasculare paracloacale) completely suppressed the tumescence of the copulatory organ and the flow of the fluid (lymph-like fluid). Evans blue solution, introduced into the A. pudenda interna of dissected cloacal specimens, exuded out from the TVP but not from the lymphatic folds (plicae lymphaticae). Injection of dye solution into the paracloacal vascular body elicited only the erection of the phallus, and none flowed out from the surface of the lymphatic folds. Also, no ejection of the injected dye solution into the A. pudenda interna was found in the dorsal wall of the cloaca, particularly from the TF. The results suggest that lymph generated in the paracloacal vascular body caused only the tumescence of the copulatory organ and a lymph generated in the TVP exuded through the surface epithelium. In addition, a foam (froth) is produced in the TF of the middorsal proctodeum during mounting behavior which appears to be added to the aforementioned lymph from the TVP at copulation.

On the structure of the vascular body in the domestic fowl.

The microscopic structure of the vascular body has been described. Three cell types have been identified. The arrangement of blood and lymph vessels has been demonstrated by means of injection techniques, and these vessels have been investigated at an ultrastructural level. The function of the vascular body is discussed.

Studies on the accessory reproductive organs in the drake. 2. Macroscopic and microscopic observatorys on the cloaca of the drake with special reference to the ejaculatory groove region.

The anatomical structure of the ejaculatory groove region (EGR) of the drake was investigated macro- and microscopically in connection with its function. The EGR covers a part of the urodeum and the second fold of the cloaca. The EGR is unique to the males and is characterized by red colored appearance and less smooth surface of the mucosa. The mucosa of EGR forms folds giving less smooth surface to it and is lined with psuedostratified columnar epithelium. A vascular layer which contains many capillaries and lymphocytes and gives red color to the mucosa lies just beneath the epithelium. The EGR develops at puberty together with the developments of the testis and the penis. Blood supply and the arrangement of the lymphatic sinuses of EGR were described and their functions were discussed.