Expanding understanding of chick embryo's nervous system development at HH22-HH41 embryonic stages using X-ray microcomputed tomography

Abstract

Assessing the embryotoxicity and teratogenicity of various substances and processes is crucial due to their complexity and resource intensity. The chicken embryo (CE) serves an ideal model for simulating the first months of mammalian embryonic development. This makes the CE a reliable model for testing teratogenic effects, particularly in relation to the nervous system (NS), which experiences developmental abnormalities second in frequency only to cardiovascular teratogenic disorders. Microcomputed tomography (μCT) is a promising method for studying these processes. The advantages of μCT include relatively high research speed, diagnostic accuracy, high resolution and the ability to visualize the entire internal 3D structure of an object while preserving for other types of research. At the same time, there are practically no available databases of normative μCT data, both qualitative and quantitative, which would act as a starting point for screening detection of abnormalities in the development of the NS. In this study, we present a simple method for obtaining very detailed quantitative sets of 2D and 3D μCT data of NS structures of the CE (Gallus Gallus domesticus) at HH22-HH41 embryonic stages with contrasting by 1% phosphotungstic acid. The results of μCT demonstrate the exact boundaries, high general and differentiated contrast of the main and specific structures of the NS of CE, which are quantitatively and qualitatively similar to results of histological analysis. Calculations of the X-ray density and volume of the main structures of the NS at constant exponential growth are presented. In addition to the increase in linear dimensions, significant changes in the structures of various parts of the brain were identified and visualized during the CE development at HH22 to HH41 embryonic stages. The data presented establish the first methodology for obtaining normative data, including subtle localized differences in the NS in CE embryogenesis. The data obtained open up new opportunities for modern embryology, teratology, pharmacology and toxicology.

Fig 1. Dynamics of the visualized volume of nervous system of chick embryo at HH22-HH41 embryonic stages, n = 5.

Fig 2

Representative cross-sectional images of the head region of a chick embryo at day 4 (embryonic stages HH22-24), counterstained with 1% PTA for 24 h at 40°C (A): coronal (X-Z), transaxial (X-Y) and sagittal (Z-Y) planes, histological section with hematoxylin-eosin staining at magnification ×18.0 (B), still image embryo under the stereoscope (C) and isosurface 3D renderings of the head region (D). The following structures are marked on the images: 1 –telencephalon (lat. telencephalon); 2 –diencephalon (lat. diencephalon); 3 –midbrain (lat. mesencephalon); 4 –hindbrain (lat. rhombencephalon); 5 –spinal cord (lat. medulla spinalis); 6 –pineal gland (lat. corpus pineale); 7 –pituitary gland (lat. hypophysis); 8 –eyes (lat. oculus); 9 –chiasma and optic nerves (lat. chiasma opticum).

Fig 3

Representative cross-sectional images of the head region of a chick embryo at day 6 (embryonic stages HH28-29), counterstained with 1% PTA for 24 h at 40°C (A): coronal (X-Z), transaxial (X-Y) and sagittal (Z-Y) planes, histological sagittal section with hematoxylin-eosin staining at magnification ×13.0 (B), still image embryo under the stereoscope (C) and isosurface 3D renderings of the head region (D). The following structures are marked on the images: 1 –telencephalon (lat. telencephalon); 2 –diencephalon (lat. diencephalon); 3 –midbrain (lat. mesencephalon); 4 –hindbrain (lat. rhombencephalon); 5 –medulla oblongata (lat. myelencephalon); 6 –spinal cord (lat. medulla spinalis); 7 –pineal gland (lat. corpus pineale); 8 –pituitary gland (lat. hypophysis); 9 –eyes (lat. oculus); 10 –chiasma and optic nerves (lat. chiasma opticum).

Fig 4

Representative cross-sectional images of the head region of a chick embryo at day 8 (embryonic stages HH33-34), counterstained with 1% PTA for 24 h at 40°C (A): coronal (X-Z), transaxial (X-Y) and sagittal (Z-Y) planes, histological sagittal section with hematoxylin-eosin staining at magnification ×13.0 (B) and still image embryo under the stereoscope (C) and isosurface 3D renderings of the head region (D). The following structures are marked on the images: 1 –telencephalon (lat. telencephalon); 2 –diencephalon (lat. diencephalon); 3 –midbrain (lat. mesencephalon); 4 –hindbrain (lat. rhombencephalon); 5 –medulla oblongata (lat. myelencephalon); 6 –spinal cord (lat. medulla spinalis); 7 –pineal gland (lat. corpus pineale); 8 –pituitary gland (lat. hypophysis); 9 –eyes (lat. oculus); 10 –chiasma and optic nerves (lat. chiasma opticum).

Fig 5

Representative cross-sectional images of the head region of a chick embryo at day 11 (embryonic stages HH37), counterstained with 1% PTA for 96 h at 40°C (A): coronal (X-Z), transaxial (X-Y) and sagittal (Z-Y) planes, histological sagittal section with hematoxylin-eosin staining at magnification ×8.5 (B), still image embryo under the stereoscope (C) and isosurface 3D renderings of the head region (D). The following structures are marked on the images: 1 –telencephalon (lat. telencephalon); 2 –diencephalon (lat. diencephalon); 3 –midbrain (lat. mesencephalon); 4 –cerebellum (lat. cerebellum); 5 –fourth ventricle (lat. ventriculus quartus); 6 –pons (lat. pons); 7 –medulla oblongata (lat. myelencephalon); 8 –spinal cord (lat. medulla spinalis); 9 –pineal gland (lat. corpus pineale); 10 –pituitary gland (lat. hypophysis); 11 –eyes (lat. oculus); 12 –chiasma and optic nerves (lat. chiasma opticum).

Fig 6

Representative cross-sectional images of the head region of a chick embryo at day 14 (embryonic stages HH40), counterstained with 1% PTA for 96 h at 40°C (A): coronal (X-Z), transaxial (X-Y) and sagittal (Z-Y) planes, histological sagittal section with hematoxylin-eosin staining at magnification ×8.0 (B), still image embryo under the stereoscope (C) and isosurface 3D renderings of the head region (D). The following structures are marked on the images: 1 –telencephalon (lat. telencephalon); 2 –diencephalon (lat. diencephalon); 3 –midbrain (lat. mesencephalon); 4 –cerebellum (lat. cerebellum); 5 –fourth ventricle (lat. ventriculus quartus); 6 –pons (lat. pons); 7 –medulla oblongata (lat. myelencephalon); 8 –spinal cord (lat. medulla spinalis); 9 –pineal gland (lat. corpus pineale); 10 –pituitary gland (lat. hypophysis); 11 –eyes (lat. oculus); 12 –chiasma and optic nerves (lat. chiasma opticum).

Fig 7

Representative frontal sectional images of the head region of a chick embryo, counterstained with 1% PTA for 24 h at 40°C (A) and histological frontal section with hematoxylin-eosin staining (B, magnification ×18.0) for comparison (day 4, embryonic stages HH22-24). The following structures are marked on the images: Acardv–anterior cardinal vein; Daorta–dorsal aorta; Dien–diencephalon; Giz–gizzard; Ictd–internal carotid artery; Lens–lens; Liver–liver; Mesen–mesencephalon; Noto–notochord; Omesv–omphalomesenteric (vitelline) vein; OptRe–optic recess of 3^rd ventricle; Os–optic stalk; Som–somite; SpC–spinal cord; V3V –ventral third ventricle; Vent–ventricle of heart; Vitr–vitreous humor of eye.

Fig 8

Representative frontal sectional images of the head region of a chick embryo, counterstained with 1% PTA for 24 h at 40°C (A) and histological frontal section with hematoxylin-eosin staining (B, magnification ×17.5) for comparison (day 6, embryonic stages HH28-29). The following structures are marked on the images: 4V –fourth ventricle; Acardv–anterior cardinal vein; Cb–cerebellum; CD–cochlear duct; CGn–cochlear ganglion; DRG–dorsal root ganglion; LRAq–lateral recess of the cerebral aqueduct; Mesen–mesencephalon; Noto–notochord; Rhomb–rhombencephalon; SpC–spinal cord; SpSO–nucleus of trigeminal spinal tr., oral part; Tect–tectum.

Fig 9

Representative frontal sectional images of the head region of a chick embryo, counterstained with 1% PTA for 24 h at 40°C (A) and histological frontal section with hematoxylin-eosin staining (B, magnification ×13.0) for comparison (day 8, embryonic stages HH33-34). The following structures are marked on the images: D3V –dorsal third ventricle; Dien–diencephalon; Mesen–mesencephalon; Optic–optic nerve; Oral–oral cavity; Ppit–posterior lobe of pituitary gland; SGFS–stratum griseum and fibrosum of tectum; SpC–spinal cord; Tect–tectum; V3V –ventral third ventricle.

Fig 10

Representative frontal sectional images of the head region of a chick embryo, counterstained with 1% PTA for 96 h at 40°C (A) and histological frontal section with hematoxylin-eosin staining (B, magnification ×10.0) for comparison (day 11, embryonic stage HH37). The following structures are marked on the images: 4V –fourth ventricle; CCrus–common crus of semicircular duct; Cb–cerebellum; HSCD–horizontal semicircular duct; Mesen–mesencephalon; Periotic–periotic capsule; Pons–pons; PSCD–posterior semicircular duct; Sacc–saccule of inner ear; SGFS–stratum griseum and fibrosum of tectum; SpC–spinal cord; SSCD–superior semicircular duct; Tect–tectum; Utr–utricle of inner ear.

Fig 11

Representative frontal sectional images of the head region of a chick embryo, counterstained with 1% PTA for 96 h at 40°C (A) and histological frontal section with hematoxylin-eosin staining (B, magnification ×10.0) for comparison (day 14, embryonic stage HH41). The following structures are marked on the images: 5Gn–trigeminal ganglion; D3V –dorsal third ventricle; Dien–diencephalon; Eso–esophagus; LRAq–lateral recess of the cerebral aqueduct; LV–lateral ventricle; Mesen–mesencephalon; Telen–telencephalon; Trachea–trachea.