ABSTRACT
ABSTRACT Objective. The present study aimed to describe in detail the expression patterns of the gene Hey1, an effector of the Notch pathway, during the development of branchial arches and facial prominences. Materials and methods. Fertilized chicken (Gallus gallus) eggs obtained from a local egg farm were incubated at 37.5 -38.5ºC with 70% relative humidity until the embryos reached Hamilton-Hamburger stages HH14 through HH25. Digoxigenin-UTP labeled probes Hey1 were generated from linearized plasmids with either T3 polimerase for in vitro transcription. Whole-mount in situ hybridization was then performed. At least 3 replicates (n=3) were obtained for each stage. To confirm the results observed in whole embryos, sagittal and coronal cryosectioning was performed using a thickness of 10 µm. Results. During developmental stages HH14 and HH18, Hey1 gene expression was localized to the endoderm of branchial pouches. Hey1 gene expression was also observed in the epithelium that covers the maxillary and mandibular prominences during developmental stages HH19 and HH21, as well as in the nasal epithelium between HH19 and HH25. Transcripts were also detected in the epithelium that covers the frontonasal prominence during stage HH21. Conclusions. These expression patterns suggest the participation of this component of the Notch signaling pathway in craniofacial morphogenesis, possibly establishing pharyngeal segmentation patterns during early stages and/or regulating cell proliferation and differentiation during the late stages of facial development.
RESUMEN Objetivo. El presente estudio tuvo como objetivo describir detalladamente los patrones de expresión del gen Hey1, un efector de la vía Notch durante el desarrollo de arcos branquiales y prominencias faciales. Materiales y métodos. Se incubaron huevos fertilizados de pollo (Gallus gallus) obtenidos de una granja local entre 37.5-38.5ºC con humedad relativa del 70% hasta que los embriones alcanzaron los estadios HH14 hasta HH25 de Hamilton-Hamburger. Las sondas Hey1 marcadas con digoxigenina-UTP se generaron a partir de plásmidos linearizados con T3 polimerasa por transcripción in vitro. Luego se realizó hibridación in situ sobre embriones completos. Se obtuvieron al menos 3 repeticiones (n=3) para cada estadio. Para confirmar los resultados observados en embriones completos, se realizaron cortes sagitales y coronales de 10 µm. Resultados. Durante los estadios de desarrollo HH14 y HH18, la expresión del gen Hey1 se localizó en el endodermo de las bolsas branquiales. La expresión génica de Hey1 también se observó en el epitelio que cubre las prominencias maxilares y mandibulares durante las etapas de desarrollo HH19 y HH21, así como en el epitelio nasal entre HH19 y HH25. También se detectaron transcritos de Hey1 en el epitelio que cubre la prominencia frontonasal durante la etapa HH21. Conclusiones. Estos patrones de expresión sugieren la participación de este componente de la vía de señalización Notch en la morfogénesis craneofacial, posiblemente estableciendo patrones de segmentación faríngea durante las primeras etapas y / o regulando la proliferación y diferenciación celular durante las últimas etapas del desarrollo facial.
Subject(s)
Branchial Region , Chick Embryo , ChickensABSTRACT
La via de senalización Notch se caracteriza por mediar la comunicación célula-célula, regulando diferentes procesos celulares como proliferación, apoptosis y definición del destino celular. Esta via ha sido implicada en el desarrollo de estructuras craneofaciales como paladar, diente y bóveda craneal. El objetivo de esta investigación fue identificar los patrones de expresión de los genes componentes de la via Notch, Serrate1 y Notch1, durante el desarrollo del tercio medio facial. Se utilizaron embriones de pollo (Callus gallus) seleccionados de acuerdo a los criterios de Hamilton y Hamburger y sobre los cuales se realizó hibridación in situ con ribosondas marcadas con Digoxigenina (DIG), para luego ser detectadas con anticuerpos Anti-Dig. Los resultados mostraron expresión de los genes evaluados, en las prominencias maxilares (pmx) y frontonasal (pfn) durante el desarrollo del tercio medio facial. Estos resultados sugieren una probable participación de la via Notch a través de estos genes, en los diferentes procesos celulares que determinan la morfogénesis y el desarrollo del tercio medio facial.
The Notch signaling pathway is characterized by mediate cell-cell communication, regulating different cellular processes as proliferation, apoptosis and cell fate definition. This pathway has been implicated in craniofacial structures development as palate, teeth and cranial vault. The objective of this research was to identify the genes expression patterns of some Notch signaling pathway components, Serrate1 and Notch1, during the midface development. It was used chicken embryos (Callus gallus) selected according to Hamilton and Hamburger criteria. We performed in situ hybridization with Digoxigenin (DIG)-labeled riboprobes and detected with the antibody Anti-Dig. The results showed the expression of the evaluated genes in the maxillary (pmx) and frontonasal (pfn) prominences during the midface development. These results suggest a probable involvement of the Notch pathway through these genes in different cellular processes that determine midface morphogenesis and development.
ABSTRACT
The human face develops between the fourth and eighth week after conception. Its development can be traced to five facial primordia appearing around the stomodeum in the fourth week. They are, namely, one frontonasal prominence, two maxillary and two mandibular prominences. Two nasal placodes develop on each side of the lower part of the frontonasal prominence at the end of the fourth week and further develop into the medial and lateral nasal prominences. The early development of the human face is similar to that of other mammals, such as pigs, rats and rabbits; all of which acquire branchial arches in the early stage of development. The rabbit branchial arches develop at the 4-5 mm stage and nearly disappear at the 12-14 mm stage, as the second one grows dorsally and obliterates the cervical sinus. The medial and lateral nasal prominences appear at the 4-5 mm stage. At the 12-14 mm stage the nasal cavities are more extensive than those of a 10 mm pig embryo and are of the same stage of development as a 15 mm pig embryo. Currently, we use 10 and 15 mm pig embryos as models in this study of human facial development. However, in the future, it will be more suitable to use 4-12 mm rabbit embryos because they are obtained more easily.