Skeletal development in blue‐breasted quail embryos

Abstract The blue‐breasted quail (Coturnix chinensis), the smallest species of quail with short generation interval and excellent reproductive performance, is a potential avian research model. A normal series of skeletal development of avian embryos could be served as a reference standard in the fields of developmental biology and teratological testing as well as in the investigation of mutation with skeletal abnormalities and in the study of the molecular mechanisms of skeletal development through genome manipulation. Furthermore, ossification sequence shows a species‐specific pattern and has potential utility in phylogeny. However, data on the skeletal development of blue‐breasted quail embryos are scarce. Here, we established a series of normal stages for the skeletal development of blue‐breasted quail embryos. Cartilage and ossified bones of blue‐breasted quail embryos were stained blue and red with Alcian blue 8GX and Alizarin red S, respectively. The time and order of chondrification and calcification of their skeletons were documented every 24 hr from 3 to 17 days of incubation, and a 15‐stage series of skeletal development was created. Moreover, a comparative study with the Japanese quail (Coturnix japonica) demonstrated that ossification sequence differed significantly between these two species.

the ability to fly. Avian skeletal system is extremely lightweight due to their hollow bones but tough enough to withstand the stresses of flying. One major morphological feature of the avian skeletal system is that some parts of vertebrae are fused into a single ossification.
Hence, birds usually have a smaller number of bones than mammals or reptiles. Most of avian species have the keeled sternum that provides a large surface area for the strong attachment of the breast muscles used in flying or swimming. Birds lack teeth and the heavy jaws to support them, and instead have beaks, which is much more lightweight.
There are two essential processes of skeletogenous during embryonic development, that is, intramembranous ossification and endochondral ossification. Intramembranous ossification produces many of the craniofacial bones directly from mesenchymal tissues.
On the other hands, endochondral ossification is the principle embryonic process of bone formation in which long bones develop by replacing cartilage templates. The replacement from cartilage to bone is tightly coupled with chondrocyte, osteoblast, and vascular differentiation. In birds, ossification spans the latter two-thirds of embryonic development and continued after hatching.
Application of avian embryos in teratological tests has been proposed as one of the alternative methods (Hashizume et al., 1992).
Thalidomide was widely prescribed to pregnant women as a sedative but was found to be teratogenic, causing multiple birth defects, for example, malformation of limbs and defects of ears, eyes, heart, kidney, and other internal organs (Knobloch & Rüther, 2008).
Chicken embryos have been used as a well-established model system for studying the molecular mechanisms of thalidomide teratogenicity (Debock & Peters, 1963;Knobloch, Shaughnessy, & Rüther, 2007;Therapontos, Erskine, Gardner, Figg, & Vargesson, 2009). Thalidomide initiates its teratogenic effects by binding to cereblon and inhibiting the associated ubiquitin ligase activity in limbs of chicken embryos (Ito et al., 2010). The list of the steps comprising normal skeletal development of blue-breasted quail embryos is required when this species is used to teratogenic tests.
Moreover, skeletogenous list of the blue-breasted quail would be useful in identification of mutations with skeletal defects and for manipulation of their genome for studying molecular mechanisms of skeletal development in future.
The purpose of this study is to document the normal staging table of skeletal development in blue-breasted quail embryos. Thereafter, ossification sequence of blue-breasted quail embryos was compared with that of Japanese quail embryos.

| Birds and eggs
Fertilized eggs of wild-type blue-breasted quail (Tsudzuki, 1994) were collected within 6 hr after laying and stored at 15°C for ~5 days. They were then incubated at 37.7 ± 0.2°C and a relative humidity of 70% while being tilted at a 90° angle once per hour (MIC-14C; M's Factory, Nagoya, Japan). Embryos were collected every 24 hr from 3 to 17 days of the incubation. Newly hatched chicks were sacrificed by CO 2 inhalation. Embryos were staged according to developmental series reported by Nakamura, Nakane, and Tsudzuki (NNT stages; in press

| Double staining of the embryonic skeleton
Blue-breasted quail embryo skeletons were double-stained with Alcian Blue 8GX S (Wako Pure Chemical Industries Ltd., Osaka, Japan) and Alizarin Red S (Wako Pure Chemical Industries Ltd.) for cartilage and ossified bones, respectively. Staining was carried out according to Nakane and Tsudzuki (1999), with minor modifications as described below.
For 3-to 6-day embryos (NNT stages 19-25), they were fixed and stained for 18 hr in a 4:1 mixture of 95% v/v ethanol and acetic acid containing 0.015% Alcian Blue 8GX. Fixed samples were dehydrated three times in 95% v/v ethanol for 3 days then macerated for 3 hr in 1% w/v KOH. After clearing in a glycerol/H 2 O concentration series (25%, 50%, and 75%) for 7 days at each concentration, stained samples were stored in 100% glycerol.
For 7-and 8-day embryos (NNT stages 27 and 29), they were fixed, stained, and dehydrated according to the same procedure as written for 3-to 6-day embryos. After staining of the cartilage for 24 hr, skin and viscera were removed. Samples were stained and macerated for 6 hr in 0.002% Alizarin Red S/1% KOH. Finally, they were cleared and stored in the same procedure described for 3-to 6-day embryos.
For 9-to 11-day embryos (NNT stages 31-33), all procedures are the same as described for 7-and 8-day embryos except for staining period. Ossified bones were stained for 24 hr.
For 12-to 16-day embryos and newly hatched chicks (NNT stages 34-39), skin, and viscera were removed. Subsequent fixation, staining, dehydration, clearing, and storing were performed according to the same procedure as written for 7-and 8-day embryos.
Ossified bones were stained for 2 days.

| Photography and measurement
Chondrification was visualized with a blue color from Alcian Blue 8GX S staining. Calcification was visualized with a red color from Alizarin Red S staining. They were observed under a stereomicroscope (S9i, Leica Microsystems, Tokyo, Japan). Calcific region was identified as the region where stained with only red in color as described elsewhere (Nakane & Tsudzuki, 1999). On and after 7 days of incubation, the lengths of calcific region as well as full lengths of the humerus, ulna, femur, and tibia were measured using a micrometer as described (Nakane & Tsudzuki, 1999).
The percentages of calcific region (length of the red-stained region/full length of the humerus, ulna, femur, and cervical vertebrae; means ± SD) were also calculated as described (Nakane & Tsudzuki, 1999).

| RE SULTS
The sequential development of blue-breasted quail embryo skeletons is shown in Figure 1. The skeleton was divided into the skull, vertebrae, ribs and sternum, and fore-and hindlimbs. The transition from chondrification to ossification in each part of the skeleton is summa- Vertebrae. All the centrums were chondrified as blue staining appeared in the coccygeal region. Bilateral blue staining on each centrum appeared in the bases of the vertebral arches from the cervical to lumbosacral regions.
Forelimb. The humerus was stained blue.
Hindlimb. The femur, tibia, and fibula were stained blue.
Vertebrae. The bases of the arches in the coccygeal vertebrae were bilaterally stained blue.
Ribs and sternum. Blue staining appeared in the proximal portion from the first to the seventh vertebral ribs.
Forelimb. The scapula, coracoid, radius, ulna, and the second and third metacarpals were stained blue.
Hindlimb. The ilium and the first-to-fourth metatarsals were stained blue 3.4 | Stage IV: 6-day embryos (NNT stage 25; Figure 1d) Skull. The parasphenoid and articular cartilage were stained blue.
Vertebrae. All vertebral arches were bow shaped.
Ribs and sternum. The first-to-seventh vertebral ribs lengthened.
Forelimb. Blue staining was present in the first metacarpal. The first phalanges of the second and third digits were stained blue.
The first phalanx of the second toe and the first and second phalanges of the third and fourth toes were stained blue. Vertebrae. Henceforth, the cervical, thoracic, lumbosacral, and coccygeal vertebrae were divided into the upper, medial, and lower regions. The bilateral ribs on the ventral side of the medial-to-lower cervical vertebrae were stained blue.

Ribs and sternum.
A pair of blue-stained sternal rudiments was observed in the dorsolateral thorax wall. The third-to-sixth sternal ribs were stained blue. The vertebral and sternal ribs were not yet connected.
Forelimb. The proximal surfaces of the clavicles were stained red but no staining was seen in the ventral midline. All phalanges were visible. Hindlimb. The first and second phalanges of the first toe, the second and third phalanges of the second toe, and the third and fourth phalanges of the third and fourth toes were stained blue. The central portions of the femur, tibia, fibula, and the second-to-fourth metatarsals turned red. Two proximal carpals were stained blue.

Ribs
First vertebral rib Hindlimb. The first metatarsal partially turned red. Red staining was present in the second phalanx of the first toe, the third phalanx of the second toe, the fourth phalanx of the third toe, and the fifth phalanx of the fourth toe. All bones showed complete ossification except for the patella, which was not ossified until hatching.

| Stage
The basioccipital turned completely red.
Vertebrae. The centrums of the medial-to-lower regions of the coccygeal vertebrae partially turned red. The arches of the lower regions of the coccygeal vertebrae also partially turned red.
Ribs and sternum. Red staining appeared in the sternum body.

| General skeletal development of the blue-breasted quail
The timing and order of chondrification and ossification in almost all bones including the skull, vertebrae, ribs and sternum, and fore and hindlimbs were schematically represented in Tables 1-4. The developmental features of the above four skeletal systems were summarized as below.
Vertebrae system revealed the earliest appearance of cartilage among the four skeletal systems. As shown in Table 2, most bones chondrified between 3 and 5 days of incubation . In contrast, they ossified at relatively late stages (11-17 days of incubation; NNT stages 33-39). Ossification progressed from the cervical to coccygeal regions. In the lumbosacral and coccygeal vertebrae, ossification began at the upper region. Chondrification and ossification mostly occurred from the centrum to the vertebral arch.
Ribs and sternum showed the latest appearance of cartilage in the four skeletal systems. As shown in Table 3, most bones chondrified between 5 and 8 days of incubation (NNT stages 23-29).
Ossification of vertebral and sternal ribs started at the medial region and progressed to the proximal and distal region. Sternum manubrium and crest remained cartilaginous at hatching (NNT stage 39).
Fore and hindlimbs revealed the earliest appearance of ossified regions among the four skeletal systems. As shown in Table 4, most bones started ossification between 7 and 10 days of incubation . Chondrification and calcification progressed from proximal to distal bones. The first metacarpus and patella appeared at 6 and 9 days of incubation (NNT stages 25 and 31), respectively, and remained cartilaginous at hatching (NNT stage 39).

| Comparative ossification sequence of bluebreasted and Japanese quail
A comparison of the skeletal development between the bluebreasted quail and the Japanese quail was conducted on the basis of the skeletogenesis list reported in this study. For the purposes of this comparison, the skeletogenous stages of the Japanese quail were consulted (Nakane & Tsudzuki, 1999). The developmental differences and skeletogenous heterochrony between blue-breasted and Japanese quail embryos are presented below.
Skeletogenous heterochrony between these two species was the most pronounced for the skull system. The development times of the occipital bones were highly variable between the two quail species. The basioccipitals of the blue-breasted quail embryos chondrified later than those of the Japanese quail embryos but calcified earlier than them.
Chondrification and calcification of the exoccipital occurred earlier in the blue-breasted quail embryos than in the Japanese quail embryos.
Chondrification of their supraoccipitals was occurred in the same period but calcification was faster in the blue-breasted quail than in the Japanese quail. Chondrification and calcification of the parasphenoid and the basisphenoid were delayed, but orbitosphenoid calcification was accelerated in the blue-breasted quail. The temporal development was almost the same between two quail species but slightly delayed for blue-breasted quail in the part of petrosal. Calcification of the other membranous and mandibular bones occurred at the same time or earlier than it did in the Japanese quail. The frontal, prefrontal, premaxillary, maxillary, quadrate, supra-angular, angular, splenial, and prearticular appeared earlier in the blue-breasted quail than they did in the Japanese quail. The only exception was the dentary. For the hyoid apparatus, the basihyal, urohyal, ceratobranchial, and epibranchial chondrified later in the blue-breasted quail than they did in the Japanese quail. Calcification of the ceratobranchial was delayed in the blue-breasted quail embryos.
In the cervical vertebrae of the blue-breasted quail embryos, cal- upper-to-medullary regions occurred earlier in the blue-breasted quail than the Japanese quail. In contrast, the centrums of the medial-tolower regions calcified later in the blue-breasted quail embryos than they did in the Japanese quail embryos.
The vertebral ribs chondrified earlier in the blue-breasted quail embryos than they did in the Japanese quail embryos. However, they calcified almost simultaneously in both quail species. In contrast, the uncinate processes chondrified at almost the same time in both species but calcified later in the blue-breasted quail than in the Japanese quail. Chondrification and calcification of the sternal ribs were delayed in the blue-breasted quail embryos. The exception was the third sternal rib, which chondrified earlier in the blue-breasted quail.
Fusion of the sternum body pair occurred at 8 days of incubation in the blue-breasted quail (NNT stage29) but at 9 days of incubation in the Japanese quail (ASE stage 36). Calcification of the sternum body and the uncinate process of the sixth vertebral rib occurred in newly hatched blue-breasted quail but not in newly hatched Japanese quail.
The calcification patterns of the terminal phalanges of the second digit in the forelimb and the first-to-fourth toes in the hindlimb were significantly different between the two quail species. In the bluebreasted quail embryos, these phalanges calcified much later than the other phalanges. On the other hand, these phalanges calcified slightly late but did nonetheless occur in Japanese quail embryos. The lag time between the calcification of the first and second (terminal) phalanges of the first toe was 4 days in the blue-breasted quail but only 1 day in the Japanese quail. Calcification of the first metatarsal was delayed in the blue-breasted quail embryos. The chondrification times of all fore and hindlimb bones were almost equal for both quail species. Hogg (1980) reported that the order in which bones ossify during embryogenesis shows a species-specific pattern in birds. Such sequence variability between species as well as lineages provides mounting evidence that adult morphology influences the order of ossification (Maxwell, 2009;Rippel, 1993;Sánchez-Villagra, Goswami, Weisbecker, Mock, & Kuratani, 2008). Moreover, comparative ossification sequence data have been further examined for its potential phylogenetic utility (Maisano, 2002;Maxwell & Larsson, 2009;Sánchez-Villagra, 2002;Schoch, 2006). Therefore, further accumulation of data for ossification sequences of various avian species is expected.

| CON CLUS ION
In this study, a list of normal embryonic skeletogenous development of the blue-breasted quail was established. A comparative study revealed that for all skeletal systems, there was substantial skeletogenous heterochrony between the blue-breasted quail and the Japanese quail. These embryonic skeletogenous stages of the blue-breasted quail could serve as a reference standard in the fields of developmental biology and teratological testing as well as in the investigation of mutation with skeletal defects and in the study of skeletogenous mechanisms through manipulation of their genome.
Moreover, comparative ossification sequence of the blue-breasted quail and Japanese quail described here would provide insight into their phylogenetic relations and evolutionary history.

This work was supported in part by Grant-in-Aids for Scientific
Research from JSPS (KAKENHI; 18K14570 and 18H05551 to Yoshiaki Nakamura and 08558088 to Masaoki Tsudzuki) and funds for the Development of Human Resources in Science and Technology" under MEXT, through the "Home for Innovative Researchers and Academic Knowledge Users (HIRAKU)" consortium to Yoshiaki Nakamura.