A Bird's-Eye View of Endangered Species Conservation: Avian Genomics and Stem Cell Approaches for Green Peafowl (Pavo muticus).

Aves ranks among the top two classes for the highest number of endangered and extinct species in the kingdom Animalia. Notably, the IUCN Red List classified the green peafowl as endangered. This highlights promising strategies using genetics and reproductive technologies for avian wildlife conservation. These platforms provide the capacity to predict population trends and enable the practical breeding of such species. The conservation of endangered avian species is facilitated through the application of genomic data storage and analysis. Storing the sequence is a form of biobanking. An analysis of sequence can identify genetically distinct individuals for breeding. Here, we reviewed avian genomics and stem cell approaches which not only offer hope for saving endangered species, such as the green peafowl but also for other birds threatened with extinction.

Dosage compensation of Z sex chromosome genes in avian fibroblast cells.

In birds, sex is genetically determined; however, the molecular mechanism is not well-understood. The avian Z sex chromosome (chrZ) lacks whole chromosome inactivation, in contrast to the mammalian chrX. To investigate chrZ dosage compensation and its role in sex specification, we use a highly quantitative method and analyze transcriptional activities of male and female fibroblast cells from seven bird species. Our data indicate that three fourths of chrZ genes are strictly compensated across Aves, similar to mammalian chrX. We also present a complete list of non-compensated chrZ genes and identify Ribosomal Protein S6 (RPS6) as a conserved sex-dimorphic gene in birds.

Avian Embryonic Culture: A Perspective of In Ovo to Ex Ovo and In Vitro Studies.

The avian embryos growing outside the natural eggshell (ex ovo) were observed since the early 19th century, and since then chick embryonic structures have revealed reaching an in-depth view of external and internal anatomy, enabling us to understand conserved vertebrate development. However, the internal environment within an eggshell (in ovo) would still be the ideal place to perform various experiments to understand the nature of avian development and to apply other biotechnology techniques. With the advent of genetic manipulation and cell culture techniques, avian embryonic parts were dissected for explant culture to eventually generate expandable cell lines (in vitro cell culture). The expansion of embryonic cells allowed us to unravel the transcriptional network for understanding pluripotency and differentiation mechanism in the embryos and in combination with stem cell technology facilitated the applications of avian culture to the next levels in transgenesis and wildlife conservation. In this review, we provide a panoramic view of the relationship among different cultivation platforms from in ovo studies to ex ovo as well as in vitro culture of cell lines with recent advances in the stem cell fields.

In vitro study of morphological changes of the cultured otocyst isolated from the chick embryo / Estudio in vitro de los cambios morfológicos del otocisto cultivado aislado del embrión de pollo

The aim of this study was to observe morphological changes of the cultured otocysts isolated from various stages of the chick embryo. Isolated otocysts were dissected from embryonic day, E2.5-4.5 of incubation (HH stage 16-26) according to stages of developing inner ear. Morphology of the chick otocyst exhibited an ovoid shape. The width and height of the otocyst were 0.2 mm and 0.3 mm, respectively. Elongation of a tube-like structure, the endolymphatic duct, was found at the dorsal aspect of the otocyst. The cultured otocyst is lined by the otic epithelium and surrounding periotic mesenchymal cells started to migrate outwards the lateral aspect of such epithelium. Notably, the acoustic-vestibular ganglion (AVG) was observed at the ventrolateral aspect of the otocyst. Appearance of AVG in vitro can be applied for studying chemical-induced ototoxicity and sensorineural hearing loss. It was concluded that the organ-cultured otocyst of the chick embryo could be used as a model to study sensory organ development of avian inner ear.

El objetivo de este estudio fue observar los cambios morfológicos de otocistos cultivados aislados en las diversas etapas del desarrollo del embrión de pollo. Otocistos aislados fueron obtenidos de embriones día, E2.5-4.5 de incubación (HH etapa 16-26) de acuerdo a las etapas de desarrollo del oído interno. El otocisto de pollo presentó una morfología ovoide. El ancho y la altura del otocisto fue de 0,2 mm y 0,3 mm, respectivamente. En la cara dorsal del otocisto se visualizó el alargamiento de una estructura similar a un tubo, el conducto endolinfático. El otocisto cultivado está revestido por epitelio ótico y células mesenquimatosas perióticas que comienzan a migrar hacia el exterior de la cara lateral en búsqueda del epitelio. En particular, el ganglio acústico-vestibular (GAV) fue observado en la parte ventrolateral del otocisto. La aparición de GAV in vitro puede ser aplicado para el estudio de la ototoxicidad inducida por productos químicos y la pérdida de audición neurosensorial. Se concluyó que el otocisto cultivado de embrión de pollo podría ser utilizado como un modelo para estudiar el desarrollo de órganos sensoriales del oído interno aviar.

Immunohistochemical localization of estrogen receptor in the embryonic gonad of male quail embryo during gonadal differentiation / Localización inmunohistoquímica del receptor de estrógeno en la gónada embrionaria en el embrión de codorniz macho durante la diferenciación gonadal

In birds, male embryo the gonads develop bilateral testes, in which both left and right sides produce functional spermatozoa, whereas female embryo, only the left gonad develops into a functional ovary. Estrogen plays a key role in avian sex determination in both sexes by binding to the estrogen receptor (ER). Surprisingly, chicken estrogen receptor (cER) mRNA is expressed in both sexes; moreover; its expression is only expressed in the left male gonad. The present study aimed to localize ER protein in the left gonad of male quail embryo using immunohistochemistry. The 8-day-old male quail embryos whose embryonic sex distinguished by gonadal morphology were studied. Histology of the left male gonad displayed thin cortex containing 1 to 2 layers of the germinal epithelium, while testicular cords were observed in the medulla. ER-immunoreactive cells were only found in the germinal epithelium but not in the medulla. Localization of ER was detected in the nucleus and cytoplasm of the germinal epithelial cells. The number of ER-immunoreactive cells counted in upper, lateral, and lower regions of the germinal epithelium was 18.20±1.892, 17.60±1.887, and 16.20±1.290, respectively. This study shows the first evidence for expression of ER protein in the left male gonad of the avian embryo, indicating that ER plays a role in avian gonadal sex differentiation.

En las aves, la gónada embrionaria en los machos se desarrolla bilateralmente, ambos testículos producen espermatozoides funcionales, mientras que en el embrión hembra, sólo la gónada izquierda se convierte en un ovario funcional. El estrógeno juega un papel clave en la determinación del sexo aviar, en ambos sexos, mediante la unión al receptor de estrógeno (RE). Fuertemente los receptores de estrógenos de pollo (cRE) el ARNm se expresan en ambos sexos; además, su expresión sólo se produce en la gónada izquierda del macho. El objetivo fue localizar proteínas del RE en la gónada izquierda de embriones de codorniz macho mediante inmunohistoquímica. Se estudiaron embriones de codorniz machos a los 8 días de edad, cuyo sexo embrionario se distinguió por la morfología de las gónadas. La histología de la gónada izquierda estuvo representada por la corteza delgada que contiene de 1 a 2 capas del epitelio germinal, mientras que se observaron cordones testiculares en la médula. El RE se encontró en células inmunorreactivas del epitelio germinal, pero no en la médula. Se detectó la localización de RE en el núcleo y el citoplasma de las células epiteliales germinales. El número de células RE-inmunorreactivas en las regiones superior, lateral e inferior del epitelio germinal fue de 18,20±1,892, 17,60±1,887 y 16,20±1,290, respectivamente. Este estudio muestra la primera evidencia de expresión de la proteína de RE en la gónada izquierda del embrión aviar macho, lo que indica que el RE desempeña un papel en la diferenciación sexual de la gónada aviar.

Transcriptome analysis of chicken ES, blastodermal and germ cells reveals that chick ES cells are equivalent to mouse ES cells rather than EpiSC.

Pluripotent Embryonic Stem cell (ESC) lines can be derived from a variety of sources. Mouse lines derived from the early blastocyst and from primordial germ cells (PGCs) can contribute to all somatic lineages and to the germ line, whereas cells from slightly later embryos (EpiSC) no longer contribute to the germ line. In chick, pluripotent ESCs can be obtained from PGCs and from early blastoderms. Established PGC lines and freshly isolated blastodermal cells (cBC) can contribute to both germinal and somatic lineages but established lines from the former (cESC) can only produce somatic cell types. For this reason, cESCs are often considered to be equivalent to mouse EpiSC. To define these cell types more rigorously, we have performed comparative microarray analysis to describe a transcriptomic profile specific for each cell type. This is validated by real time RT-PCR and in situ hybridisation. We find that both cES and cBC cells express classic pluripotency-related genes (including cPOUV/OCT4, NANOG, SOX2/3, KLF2 and SALL4), whereas expression of DAZL, DND1, DDX4 and PIWIL1 defines a molecular signature for germ cells. Surprisingly, contrary to the prevailing view, our results also suggest that cES cells resemble mouse ES cells more closely than mouse EpiSC.

Microanatomical Study of Embryonic Gonadal Development in Japanese Quail (Coturnix japonica).

Gonadal development of quail embryos was examined histologically using histological and histochemical methods. In the present study, quail embryos were studied at various stages of incubation period based on phases of gonadogenesis. Germ cell migration was observed on day 3-4 but gonadal differentiation and gonadal function were observed on day 6-8 and day 11-14, respectively. During germ cell migration, quail primordial germ cells (qPGCs) were successfully detected in both left and right genital ridges as well as the dorsal mesentery by lectin histochemistry. Unexpectedly, qPGCs-like cells were found next to the neural tube by Mallory-AZAN stain. During gonadal differentiation, embryonic sex can be distinguished histologically since day 8 of incubation. Embryonic testis exhibited a thin cortex, whereas embryonic ovary exhibited a thick cortex. Testicular cord formation was found in the medulla of embryonic testes while the lacunae and fat-laden cells were found in the medulla of embryonic ovary during gonadal function. This is the first report on a comparison of phases of gonadogenesis and histochemical study of quail embryonic gonads in both sexes.

Chick stem cells: current progress and future prospects.

Chick embryonic stem cells (cESCs) can be derived from cells obtained from stage X embryos (blastoderm stage); these have the ability to contribute to all somatic lineages in chimaeras, but not to the germ line. However, lines of stem cells that are able to contribute to the germ line can be established from chick primordial germ cells (cPGCs) and embryonic germ cells (cEGCs). This review provides information on avian stem cells, emphasizing different sources of cells and current methods for derivation and culture of pluripotent cells from chick embryos. We also review technologies for isolation and derivation of chicken germ cells and the production of transgenic birds.

Sexually dimorphic and sex-independent left-right asymmetries in chicken embryonic gonads.

Female birds develop asymmetric gonads: a functional ovary develops on the left, whereas the right gonad regresses. In males, however, testes develop on both sides. We examined the distribution of germ cells using Vasa/Cvh as a marker. Expression is asymmetric in both sexes: at stage 35 the left gonad contains significantly more germ cells than the right. A similar expression pattern is seen for expression of ERNI (Ens1), a gene expressed in chick embryonic stem cells while they self-renew, but downregulated upon differentiation. Other pluripotency-associated markers (PouV/Oct3/4, Nanog and Sox2) also show asymmetric expression (more expressing cells on the left) in both sexes, but this asymmetry is at least partly due to expression in stromal cells of the developing gonad, and the pattern is different for all the genes. Therefore germ cell and pluripotency-associated genes show both sex-dependent and independent left-right asymmetry and a complex pattern of expression.