Real-Time Sexing of Chicken Embryos and Compatibility with in ovo Protocols.

The chicken embryo is an established model system for studying early vertebrate development. One of the major advantages of this model is the facility to perform manipulations in ovo and then continue incubation and observe the effects on embryonic development. However, in common with other vertebrate models, there is a tendency to disregard the sex of the experimental chicken embryos, and this can lead to erroneous conclusions, a lack of reproducibility, and wasted efforts. That this neglect is untenable is emphasised by the recent demonstration that avian cells and tissues have an inherent sex identity and that male and female tissues respond differently to the same stimulus. These sexually dimorphic characteristics dictate that analyses and manipulations involving chicken embryos should always be performed using tissues/embryos of known sex. Current sexing protocols are unsuitable in many instances because of the time constraints imposed by most in ovo procedures. To address this lack, we have developed a real-time chicken sexing assay that is compatible with in ovo manipulations, reduces the number of embryos required, and conserves resources.

Cell-autonomous sex differences in gene expression in chicken bone marrow-derived macrophages.

We have identified differences in gene expression in macrophages grown from the bone marrow of male and female chickens in recombinant chicken M-CSF (CSF1). Cells were profiled with or without treatment with bacterial LPS for 24 h. Approximately 600 transcripts were induced by prolonged LPS stimulation to an equal extent in the male and female macrophages. Many transcripts encoded on the Z chromosome were expressed ∼1.6-fold higher in males, reflecting a lack of dosage compensation in the homogametic sex. A smaller set of W chromosome-specific genes was expressed only in females. LPS signaling in mammals is associated with induction of type 1 IFN-responsive genes. Unexpectedly, because IFNs are encoded on the Z chromosome of chickens, unstimulated macrophages from the female birds expressed a set of known IFN-inducible genes at much higher levels than male cells under the same conditions. To confirm that these differences were not the consequence of the actions of gonadal hormones, we induced gonadal sex reversal to alter the hormonal environment of the developing chick and analyzed macrophages cultured from male, female, and female sex-reversed embryos. Gonadal sex reversal did not alter the sexually dimorphic expression of either sex-linked or IFN-responsive genes. We suggest that female birds compensate for the reduced dose of inducible IFN with a higher basal set point of IFN-responsive genes.

The expression and activity of ß-catenin in the thalamus and its projections to the cerebral cortex in the mouse embryo.

BACKGROUND: The mammalian thalamus relays sensory information from the periphery to the cerebral cortex for cognitive processing via the thalamocortical tract. The thalamocortical tract forms during embryonic development controlled by mechanisms that are not fully understood. ß-catenin is a nuclear and cytosolic protein that transduces signals from secreted signaling molecules to regulate both cell motility via the cytoskeleton and gene expression in the nucleus. In this study we tested whether ß-catenin is likely to play a role in thalamocortical connectivity by examining its expression and activity in developing thalamic neurons and their axons. RESULTS: At embryonic day (E)15.5, the time when thalamocortical axonal projections are forming, we found that the thalamus is a site of particularly high ß-catenin mRNA and protein expression. As well as being expressed at high levels in thalamic cell bodies, ß-catenin protein is enriched in the axons and growth cones of thalamic axons and its growth cone concentration is sensitive to Netrin-1. Using mice carrying the ß-catenin reporter BAT-gal we find high levels of reporter activity in the thalamus. Further, Netrin-1 induces BAT-gal reporter expression and upregulates levels of endogenous transcripts encoding ß-actin and L1 proteins in cultured thalamic cells. We found that ß-catenin mRNA is enriched in thalamic axons and its 3'UTR is phylogenetically conserved and is able to direct heterologous mRNAs along the thalamic axon, where they can be translated. CONCLUSION: We provide evidence that ß-catenin protein is likely to be an important player in thalamocortcial development. It is abundant both in the nucleus and in the growth cones of post-mitotic thalamic cells during the development of thalamocortical connectivity and ß-catenin mRNA is targeted to thalamic axons and growth cones where it could potentially be translated. ß-catenin is involved in transducing the Netrin-1 signal to thalamic cells suggesting a mechanism by which Netrin-1 guides thalamocortical development.

The chicken type III GnRH receptor homologue is predominantly expressed in the pituitary, and exhibits similar ligand selectivity to the type I receptor.

Two GnRH isoforms (cGnRH-I and GnRH-II) and two GnRH receptor subtypes (cGnRH-R-I and cGnRH-R-III) occur in chickens. Differential roles for these molecules in regulating gonadotrophin secretion or other functions are unclear. To investigate this we cloned cGnRH-R-III from a broiler chicken and compared its structure, expression and pharmacological properties with cGnRH-R-I. The broiler cGnRH-R-III cDNA was 100% identical to the sequence reported in the red jungle fowl and white leghorn breed. Pituitary cGnRH-R-III mRNA was approximately 1400-fold more abundant than cGnRH-R-I mRNA. Northern analysis indicated a single cGnRH-R-III transcript. A pronounced sex and age difference existed, with higher pituitary transcript levels in sexually mature females versus juvenile females. In contrast, higher expression levels occurred in juvenile males versus sexually mature males. Functional studies in COS-7 cells indicated that cGnRH-R-III has a higher binding affinity for GnRH-II than cGnRH-I (K(d): 0.57 vs 19.8 nM) with more potent stimulation of inositol phosphate production (ED(50): 0.8 vs 4.38 nM). Similar results were found for cGnRH-R-I, (K(d): 0.51 vs 10.8 nM) and (ED(50): 0.7 vs 2.8 nM). The initial rate of internalisation was faster for cGnRH-R-III than cGnRH-R-I (26 vs 15.8%/min). Effects of GnRH antagonists were compared at the two receptors. Antagonist #27 distinguished between cGnRH-R-I and cGnRH-R-III (IC(50): 2.3 vs 351 nM). These results suggest that cGnRH-R-III is probably the major mediator of pituitary gonadotroph function, that antagonist #27 may allow delineation of receptor subtype function in vitro and in vivo and that tissue-specific recruitment of cGnRH-R isoforms has occurred during evolution.

Identification of disease markers by differential display: prion disease.

In order to identify molecular markers of prion disease in peripheral tissues, we used the differential display reverse-transcriptase polymerase chain reaction (DDRT-PCR) procedure to compare gene expression in spleens of infected and uninfected mice. In this study, we identified a novel erythroid-specific gene that was differentially expressed as a result of prion infection. We were able to demonstrate that a decrease in the expression levels of this transcript in hematopoietic tissues was a common feature of prion diseases. Our findings suggest a previously unknown role for the blood erythroid lineage in the development of prion diseases and should provide a new focus for research into diagnostic and therapeutic strategies.