Sunil Nandi

Somatic sex identity is cell autonomous in the chicken.

In the mammalian model of sex determination, embryos are considered to be sexually indifferent until the transient action of a sex-determining gene initiates gonadal differentiation. Although this model is thought to apply to all vertebrates, this has yet to be established. Here we have examined three lateral gynandromorph chickens (a rare, naturally occurring phenomenon in which one side of the animal appears male and the other female) to investigate the sex-determining mechanism in birds. These studies demonstrated that gynandromorph birds are genuine male:female chimaeras, and indicated that male and female avian somatic cells may have an inherent sex identity. To test this hypothesis, we transplanted presumptive mesoderm between embryos of reciprocal sexes to generate embryos containing male:female chimaeric gonads. In contrast to the outcome for mammalian mixed-sex chimaeras, in chicken mixed-sex chimaeras the donor cells were excluded from the functional structures of the host gonad. In an example where female tissue was transplanted into a male host, donor cells contributing to the developing testis retained a female identity and expressed a marker of female function. Our study demonstrates that avian somatic cells possess an inherent sex identity and that, in birds, sexual differentiation is substantively cell autonomous.

Evidence for avian cell autonomous sex identity (CASI) and implications for the sex-determination process?

For the majority of animals, males and females are obviously different in terms of appearance, behaviour and physiology, and until recently, these differences were considered to be the result of hormone actions. However, there is now considerable evidence that the development of some sexually dimorphic structures/behaviours is a function of properties inherent to male and female cells (hormone independent). The relative contribution of hormones and cellular identity to the development of the phenotype is not clear and is likely to vary from species to species. The study of gynandromorph birds and chimeric embryos has greatly assisted efforts to distinguish between the effects of hormones and inherent cellular factors on phenotype. It is now clear that in birds, male/female differences are not primarily the result of hormone action and that male and female somatic cells possess a cell autonomous sex identity (CASI). Here, we review evidence for CASI in birds and discuss the implications for the process of sex determination.

Gonadal Asymmetry and Sex Determination in Birds

Although vertebrates display a superficial bilateral symmetry, most internal organs develop and locate with a consistent left:right asymmetry. There is still considerable debate as to when this process actually begins, but it seems that, at least for some species, the initial steps occur at a very early stage of development. In recent years, a number of model systems, including the chick embryo, have been utilised to increase our understanding of the molecular basis of this complex developmental process. While the basic elements of asymmetry are clearly conserved in chick development, the chick embryo also exhibits an additional unusual asymmetry in terms of the development of the gonads. In the female chick embryo, only 1 gonad and accessory structures fully develop, with the result that the adult hen has only 1 ovary and a single oviduct - both on the left side. With a small number of exceptions, this is a consistent feature of avian development. Here, we describe the morphological development and molecular basis of this unusual asymmetry, consider the implications for avian sex determination, and discuss the possible biological reasons why many birds have adopted a single-ovary system.

Efficient TALEN-mediated gene targeting of chicken primordial germ cells

In this work we use TALE nucleases (TALENs) to target a reporter construct to the DDX4 (vasa) locus in chicken primordial germ cells (PGCs). Vasa is a key germ cell determinant in many animal species and is posited to control avian germ cell formation. We show that TALENs mediate homology-directed repair of the DDX4 locus on the Z sex chromosome at high (8.1%) efficiencies. Large genetic deletions of 30 kb encompassing the entire DDX4 locus were also created using a single TALEN pair. The targeted PGCs were germline competent and were used to produce DDX4 null offspring. In DDX4 knockout chickens, PGCs are initially formed but are lost during meiosis in the developing ovary, leading to adult female sterility. TALEN-mediated gene targeting in avian PGCs is therefore an efficient process. Summary: TALE nucleases are used to target the DDX4 (vasa) locus in chicken primordial germ cells and generate DDX4 knockouts, which provide insights into DDX4 function in early chick development.

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.

Cryopreservation of specialized chicken lines using cultured primordial germ cells

Biosecurity and sustainability in poultry production requires reliable germplasm conservation. Germplasm conservation in poultry is more challenging in comparison to other livestock species. Embryo cryopreservation is not feasible for egg-laying animals, and chicken semen conservation has variable success for different chicken breeds. A potential solution is the cryopreservation of the committed diploid stem cell precursors to the gametes, the primordial germ cells (PGCs). Primordial germ cells are the lineage-restricted cells found at early embryonic stages in birds and form the sperm and eggs. We demonstrate here, using flocks of partially inbred, lower-fertility, major histocompatibility complex- (MHC-) restricted lines of chicken, that we can easily derive and cryopreserve a sufficient number of independent lines of male and female PGCs that would be sufficient to reconstitute a poultry breed. We demonstrate that germ-line transmission can be attained from these PGCs using a commercial layer line of chickens as a surrogate host. This research is a major step in developing and demonstrating that cryopreserved PGCs could be used for the biobanking of specialized flocks of birds used in research settings. The prospective application of this technology to poultry production will further increase sustainability to meet current and future production needs.

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.

01-P012 The role of micro-RNAs in the development of the chick gonads

causative gene. We have identified a 187 kb microdeletion on chromosome 15q11.2 which narrows down the candidate region to a cluster of non-coding small nucleolar RNAs (snoRNAs), the SNORD116. The deletion results in a loss of expression of the SNORD116 cluster in the patient. The snoRNAs in the adjacent SNORD115 cluster, and the neighbouring genes UBE3A, MAGEL and NECDIN are expressed. Knockout mouse models of Snord116 show features of PWS, thus supporting a role of SNORD116 in the aetiology of PWS. However, nothing is known about the physiological role of the SNORD116 snoRNA cluster and how their lack of expression could cause the multiple symptoms of PWS. SnoRNAs are small non-coding RNAs that are found in the nucleolus,which guide modification of ribosomal RNA via sequence homology to the rRNA target. However, the SNORD116 snoRNAs do not show any sequence homology to any known rRNA or mRNA target. They are so-called ‘orphan’ snoRNAs. To date, it is unknown with which proteins or other RNAs SNORD116 interacts. To identity binding partners of SNORD116, we performed a StreptoTag binding assay.

15-P020 Cell autonomous sexual development in birds

Our analyses of early neurogenesis in several representatives of chelicerates (e.g. spiders) and myriapods (e.g. millipedes) have revealed that the genetic network involved in recruitment and specification of neural precursors is conserved in all euarthropod groups. However, the expression pattern and function of these genes is adapted to the distinct morphology of neural precursor formation in each group. We observed several molecular and morphological characters in the developing central and peripheral nervous system of chelicerates and myriapods that cannot be found in equivalent form in insects and crustaceans. It is possible that these characters are shared derived characters (synapomorphies) of myriapods and chelicerates, providing the first morphological support for a clade uniting these two groups. However, they could also represent ancestral characters (symplesiomorphies) retained in myriapods and chelicerates and lost in the more derived insects and crustaceans. We have therefore analysed neurogenesis in a representative of an outgroup to the euarthropods, the onychophoran Euperipatoides kanangrensis. We have identified Notch and Delta homologues in Euperipatoides kanangrensis. These genes are involved in specification of neural stem cells in insects. We show that they are expressed in a distinct pattern which is neither comparable to insects/crustaceans nor to chelicerates/myriapods. Furthermore, we have analysed the morphology of neural precursor formation by F-actin staining and light microscopic sections. The data suggest that at least some of the characters shared by chelicerates and myriapods are synapomorphies

09-P056 Identification and characterisation of chicken DMRT1 transcript isoforms

In birds, the mechanism of sex determination is unknown: it is uncertain whether the female-specific W-chromosome encodes an ovary-determining gene analogous to the mammalian testisdetermining gene, or whether testis determination is dependent on a double dose of a Z-chromosome gene(s) that escapes inactivation. In addition, birds do not carry a homologue to the Sry gene and the identity of the proposed sex determining gene is unknown. Currently, the gene considered the strongest avian candidate for a ‘classical’ testis-determining gene is DMRT1 (doublesex and Mab-3-related transcription factor 1). DMRT1 is conserved across vertebrates and is expressed in the developing gonads of mammals, birds, reptiles and fish, with higher expression in male than in female gonads. Chicken DMRT1 is located on the Z-chromosome and is expressed at higher levels in male (ZZ) gonads than in female (ZW) gonads, both at and after the suspected point of sex determination (day 5.5/stage 28). Recently we have identified six different transcript isoforms of DMRT1 in the chicken. Sequence analysis of these cDMRT1 cDNAs revealed that they encode a combination of DM domain (a conserved zinc-finger-like DNA-binding domain) and/or a SP domain (conserved domain with unknown function). The identification of five different 5UTR sequences indicates five different transcription initiation sites. PolyA Northern analysis shows that one transcript is expressed at higher levels in male embryonic gonad than in female embryonic gonad from day 5 to 9. Currently, a transgenic chicken with cDMRT1 has been generated and further analysis is underway.