No description available.

In most mammals, sexual fate is determining genetically by the presence of the SRY gene which encoded the testis-determining factors on the Y chromosome. Likewise, avian sex is determined genetically. At day 3.5 (stage 22; HH) in chicken embryogenesis, the gonadal primordium begins forming. Thus, to identify the novel sex-determinating genes in early chicken embryos, subtractive cDNA libraries from male-minus-female (M-F) and female-minus-male (F-M) of 3 Dpc. embryos were established. Both collected male and female chicken total mRNAs were purified using Dynabeads. After a blund-end restriction endonuclease Rsa I digestion of cDNA, adaptor ligation for tester cDNA was performed. When first and second cDNA hybridization was finished, those nonredundant cDNA between tester and driver will be amplified by two rounds of PCR. Subsequently, TA-cloning was performed and the cDNA fragments were PCR-amplified using M13 primers. PCR products of Clones were first screened by differential screening hybridization to decrease false positive inserts. Then, gene annotation was carried out by data-mining in public databases, GeneBank (NCBI, USA). Finally, 40 known and 71 novel transcripts of M-F cDNA library, 88 known and 128 novel transcripts of F-M cDNA library were identified. In M-F subtracted library, 4 identified known genes were located on Z sex chromosome such as WD repeat domain 36 (WDR36), PC4 and SFRS1 interacting protein 1 (PSIP1), serum response factor binding protein 1 (SRFBP1) and glycine dehydrogenase (decarboxylating) (GLDC). Another two identified known genes, laminin alpha 1 (LAMA1) and leukocyte cell derived chemotaxin 1 (LECT1) were reported be relate to cell differentiation and development. In F-M subtracted library, only Wpkic-8 was located on W sex chromosome. Other identified genes like slowmo homolog 2 (Drosophila) (SLMO2), collagen, type IV, alpha 1 (COL4A1), anterior gradient 2 homolog (Xenopus laevis), transcript variant 2 (AGR2), solute carrier family 25 (mitochondrial carrier; adenine nucleotide translocator), member 6 (SLC25A6) and prolyl endopeptidase (PREP) were also found expressed higer in human ovary then testis. PREP was proposed that it may play a role in mediating sperm death by regulating the levels of thyrotropin-releasing hormone analogs and in mediating sperm death associated with necrozoospermia. These transcripts located on W or Z sex chromosome identified from subtracted libraries may play an important role in sex determination mechanism.

Hematocrit and hematocrit regulation have the potential to affect developing embryos. To examine the ability of chicken embryos at day 15 to regulate hematocrit, they were subjected to either repeated saline injections (5% of total blood volume) or repeated blood removal (5% of total blood volume). Embryos showed an ability to maintain hematocrit (~20%) despite blood volume increases up to 115% of initial blood volume. Embryos were not able to maintain hematocrit in the face of dramatic blood volume loss. Oxygen consumption of embryos could be affected by their level of hematocrit. To examine this, chicken embryos at day 15, 16, and 17 of incubation were given a high hematocrit (~50-60%) sample of blood (400 μl) to artificially increase the hematocrit of the embryos (~10-12%). Despite the increase in oxygen availability, when monitored over a period of six hours, embryos showed no difference (0.36 ± 0.01 (ml O2 - min-1- egg-1) in metabolism from baseline measurements at day 15, 16 and 17.

To examine if a rhythm can be entrained in either heart rate or oxygen consumption in late stage embryos (days 17-19.5) with light as a zeitgeber, chicken embryos were incubated in complete darkness (D:D) and 12:12 light:dark cycle (L:D). Light had no impact on oxygen consumption (390 µL O2∙min-1∙egg-1) but increased heart rate for non-internally pipped embryos (260 to 270 beats∙min-1 during light cycle). Oxygen consumption increased independent of pipping while heart rate increased (255 to 265 beats∙min-1) in D:D embryos due to pipping. A light-induced rhythm or effect occurred in heart rate but not oxygen consumption, suggesting heart rate and oxygen consumption may be uncoupled.

The chicken ductus arteriosi (DA) are two embryonic blood vessels that shunt blood away from the non-ventilated lungs and towards the body and chorioallantoic membrane. I show that prostaglandins have a diminished role in maintaining chicken DA patency and nitric oxide inhibits oxygen induced contraction of the day 19 proximal DA in a time dependent manner. The pathways governing oxygen induced contraction in the chicken DA are similar to those found in mammals and include contributions from ROS, Kv channels, L-type Ca2+ channels, and the Rho kinase pathway. Longer exposure to high oxygen generates increased oxygen induced constriction of the day 19 DA that may be mediated through the Rho kinase pathway.

<div><p>Zika virus (ZIKV) is a mosquito-borne pathogen that stayed unnoticed for over half a century. Only after the 2015-16 Brazilian outbreak did the severity of the infectious outcome, particularly the Congenital Zika Syndrome, become apparent. ZIKV is associated with severe neurodevelopmental impairments in human fetuses, including microencephaly, ventriculomegaly, retinopathy, and sensorineural hearing loss. Though the pandemic is now under control in the Latin American countries, several tropical countries could still be at risk of widespread infection. This warrants a better understanding of the congenital Zika syndrome; this project attempts to contribute towards this goal.</p><p><br></p><p>Previous reports examining neural progenitor tropism of ZIKV in organoid and animal models did not address whether the virus infects all neural progenitors uniformly. To explore this, ZIKV was injected into the neural tube of 2-day-old chicken embryos, resulting in non-uniform periventricular infection 3 days later. Recurrent foci of intense infection were present at specific signaling centers that influence neuroepithelial patterning at a distance through secretion of morphogens. ZIKV infection reduced transcript levels for 3 morphogens, SHH, BMP7, and FGF8, expressed at the midbrain basal plate, hypothalamic floor plate, and isthmus, respectively. Levels of Patched1, a SHH-pathway downstream gene, were also reduced and a SHH-dependent cell population in the ventral midbrain was shifted in position. Thus, the diminishment of signaling centers through ZIKV-mediated apoptosis may yield broader, non-cell autonomous changes in brain patterning.</p><p><br></p></div><p>Sensorineural hearing loss is a relatively understudied consequence of congenital Zika syndrome, and balance disorders are essentially unreported to date. ZIKV pathogenesis was explored in the developing inner ear using the accessible chicken embryo model system. One goal was to assess the spatiotemporal susceptibility of otic epithelial-derived structures to ZIKV infectivity. Direct injections of the inner ear or the inner ear primordium were performed <i>in ovo</i>with subsequent harvests at 2 to 8 days-post-infection. The degree of infection in sensory/prosensory organs was evaluated histologically to determine the susceptibility of one auditory and five vestibular organs. ZIKV infection of the sensory as well as non-sensory epithelia was observed at most stages of analysis, with no apparent preference for one over the other. The lagena, the ventral most tip of the chicken inner ear, and the endolymphatic sac/duct were least frequently infected. In this report, two novel findings in sequela of ZIKV infection are presented: the vestibular labyrinth can present with stalled canal morphogenesis, and the auditory ganglion can be severely shrunken, perhaps due to an increased cell death upon early ZIKV infection of the inner ear.</p><p><br></p><p>Additional methods of peripheral infection in the chicken embryos were tested to examine ZIKV transmission to the central nervous system: E3 blood vessel, E4 limb bud, and E10 chorioallantoic membrane infections. Although none of these methods resulted in a histologically significant infection of the developing brain 3 to 6 days-post-infection, evidence of ZIKV genome replication and viremia was detected in several tissue types.<br></p>

No description available.

No description available.

Bone morphogenetic proteins (BMPs) are diffusible molecules involved in a variety of cellular interactions during development. In particular, Bmp4 expression accompanies the development of the ear sensory organs during patterning and specification of sensory cell fates, and it has been shown to play a role in inner ear development and morphogenesis. However, there is no understanding of the cellular effects of BMP4 in prosensory progenitors, and about its role in the process of sensory fate specification. The present thesis project was aimed at exploring the effects of BMP-signaling on the development of hair-cells, using the chick inner ear as a model.The specific aims proposed were:1- Analyze the cellular effects caused by addition of BMP4 in a model of isolated chick otic vesicles in culture, measuring parameters of cell proliferation, cell death and sensory cell fate specification.2- Analyze the cellular effects caused by inhibition of BMP4 signaling in a model of isolated chick otic vesicles in culture, measuring parameters of cell proliferation, cell death and sensory cell fate specification.3- Analyze the expression in the innear ear of downstream targets of BMP signalling, in particular, analyse the members of Id gene family.4- Analyze the regulation of Id genes by BMP signalling in the inner ear.5- Analyze the expression of genes involved in the process of terminal differentiation, in particular, Btg1 and Btg2 genes6- Analyze the regulation of Btg1 and Btg2 gene by BMP signalling in the inner ear

The focus of this research was the spontaneous magnetic alignment responses of animals. We show that snapping turtles (Chelydra serpentina) and crayfish (Cambarus sciotensis) spontaneously align their body axes relative to the magnetic field. In snapping turtles, this response is sensitive to low-level radio frequency fields, consistent with a mechanism involving a light-dependent radical pair mechanism. Findings from the turtle experiments also suggest that the Earth's magnetic field plays an important role in encoding spatial information in novel surroundings, and may help to organize multiple locales into a 'mental map' of familiar space. Given the importance of magnetic input in many aspects of spatial behavior, another important finding was that magnetic alignment of yearling turtles was disrupted by high levels of maternally transferred mercury, an industrial waste product found at high levels in some fresh water ecosystems. In crayfish, we investigated the effects of ectosymbionts (Annelida: Branchiobdellida) on magnetic alignment responses. Interestingly, the response of crayfish to magnetic cues parallels the complex symbiotic interaction between crayfish and their ectosymbiotic worms, which changes from mutualistic to parasitic with increasing worm density. Our working hypothesis was that these changes in spatial behavior may increase or decrease contact to other crayfish, and therefore increase or decrease transmission rates. Next, to address the ontogeny of the SMA, we attempted to replicate an earlier study showing a possible magnetic alignment response in chicken embryos. Although chicken embryos did show non-random alignment, we were not able to find a magnetic effect. Alignment is also an important feature of animal constructions and is very likely to have fitness consequences, which we explored in woodpecker cavity alignments in a meta-analysis of available global data. The latitudinal and continental pattern in 23 species of woodpeckers suggests that an alignment response can have the proximate function to regulate microclimate in the cavity and therefore, presumably, optimize incubation temperatures and increase hatching success. Overall, the presented findings show how experimental and observational studies of spontaneous alignment behavior can provide insight into the ecology and sensory biology of a wide range of animals. / Ph. D.