Occipital foramina development involves localised regulation of mesenchyme proliferation and is independent of apoptosis

Abstract

Cranial foramina are holes within the skull, formed during development, allowing entry and exit of blood vessels and nerves. Once formed they must remain open, due to the vital structures they contain, i.e. optic nerves, jugular vein, carotid artery, and other cranial nerves and blood vessels. Understanding cranial foramina development is essential as cranial malformations lead to the stenosis or complete closure of these structures, resulting in blindness, deafness, facial paralysis, raised intracranial pressure and lethality. Here we focus on describing early events in the formation of the jugular, carotid and hypoglossal cranial foramina that form in the mesoderm-derived, endochondral occipital bones at the base of the embryonic chick skull. Whole-mount skeletal staining of skulls indicates the appearance of these foramina from HH32/D7.5 onwards. Haematoxylin & eosin staining of sections shows that the intimately associated mesenchyme, neighbouring the contents of these cranial foramina, is initially very dense and gradually becomes sparser as development proceeds. Histological examination also revealed that these foramina initially contain relatively large-diameter nerves, which later become refined, and are closely associated with the blood vessel, which they also innervate within the confines of the foramina. Interestingly cranial foramina in the base of the skull contain blood vessels lacking smooth muscle actin, which suggests these blood vessels belong to glomus body structures within the foramina. The blood vessel shape also appears to dictate the overall shape of the resulting foramina. We initially hypothesised that cranial foramina development could involve targeted proliferation and local apoptosis to cause 'mesenchymal clearing' and the creation of cavities in a mechanism similar to joint cavitation. We find that this is not the case, and propose that a mechanism reliant upon local nerve/blood vessel-derived restriction of ossification may contribute to foramina formation during cranial development.

Keywords: blood vessel; chick; cranial; embryonic; foramina; glomus body; nerve; skeletal development; skull.

Fig 1

Diagrams of the chicken skull adapted with permission from Couly et al. (1993) showing the mesoderm-derived bones of the skull (blue) and the neural crest-derived bones of the skull (red). (A) The base of the chicken skull. (B) A side view of the chicken skull.

Fig 2

Whole-mount appearance of cranial foramina in the embryonic chick skull HH32–HH47 (embryonic D7.5–D21). Alcian blue- and alizarin red-stained whole-mount embryonic chick skulls are shown in (A and B) and (D and E). Pre-hatching embryonic D21/HH47 chick skulls are shown in (F and G). A schematic of the base of the chick skull is shown in (C) to illustrate foramina locations. The jugular foramina (jf) are the largest and most prominent cranial foramina in the base of the skull (A, D, F, G). The hypoglossal foramina (hf) can be seen either side of the midline (A and B, D–G) and the carotid foramina (cf) can be seen lateral to the jugular foramina (A). The black dotted line depicts the midline location. of, optic foramen; oaf, ophthalmic artery foramina.

Fig 3

H&E histological staining of jugular and hypoglossal foramina in section. A diagram of the base of the chick skull is shown in (D) to depict foramina and blood vessel content locations. The black dotted line in (A, B, D and E) depicts the midline location. At early stages of cranial foramina development (HH27–HH29/D5–D6), the foramina mesenchyme (fm) is densely packed around the jugular (jbv) and hypoglossal blood vessels (hbv) (A–C). However, by HH34 (D8) the foramina mesenchyme becomes much more sparsely distributed within the developing jugular (jf), carotid (cbv) and hypoglossal foramina (hf) (E–G) and continues to become even more sparsely distributed as development proceeds (I–L). Note that at HH35/D9 a distinct perichondrium (pc) has formed (I + J) to define the foramina. The black dotted lines in (F and G) outline the putative perichondrium before HH35/D9 when it is clearly defined. Quantification of foramina mesenchyme density (mean grey scale refers to the sparseness of the foramina mesenchyme) can be seen in Fig.3H. ov, otic vesicle; NT, neural tube.

Fig 4

Graphs of diameters of cranial foramina during chick embryo development. (a) The diameters of the jugular foramina, (b) the hypoglossal foramina and (c) the carotid foramina diameters. The error bars represent the mean diameters of the jugular, hypoglossal and carotid foramina + the standard error of these mean values. Capped lines with * indicate significant differences.

Fig 5

Anti-acetylated tubulin immunohistochemistry shows the appearance and location of the nerves within jugular foramina (jf) at early stages of development in the chick embryo. A diagram of cranial foramina in transverse section is shown in (A) to depict locations of cranial foramina. At HH25/D4 the nerve (arrowheads) associated with the jugular blood vessel (jbv) is large in comparison to the blood vessel and closely associated to it (B and C). By HH27/D5 the nerve has become smaller but is still closely associated to the foramina blood vessel (D and E). NT, neural tube; not, notochord; bv, blood vessel; ov, otic vesicle. Arrowheads show anti-acetylated tubulin staining.

Fig 6

Anti-acetylated tubulin immunohistochemistry shows the appearance and location of the nerves within jugular foramina (jf) at later stages of development in the chick embryo. A diagram of cranial foramina in transverse section is shown in (G) to depict locations of cranial foramina. At HH31/D7 the nerve (nv) is very closely associated with the foramina blood vessels (A–C). The hypoglossal nerve is harder to see so an insert is displayed with the area of the nerve (black box) magnified within. Fluorescent anti-acetylated tubulin immunohistochemistry at D9 is shown in (D), and interestingly nerve staining (arrowheads) is seen within blood vessels of the jugular foramina (jf). This can be seen also at later stages of development (E and F). hf, hypoglossal foramina; cf, carotid foramina; cbv, carotid blood vessel; hbv, hypoglossal blood vessel; jbv, jugular blood vessel; pc, perichondrium; fm, foramina mesenchyme; odc, occipital differentiated cartilage; NT, neural tube. The arrowhead labelled nv show anti-acetylated tubulin staining. (A–C) and (E) are counterstained with alcian blue. (F) is counterstained with nuclear fast red.

Fig 7

Smooth muscle actin immunohistochemistry reveals hypoglossal (hbv), jugular (jbv) and carotid foramina blood vessels (cbv) are not immunoreactive to smooth muscle actin. A diagram of cranial foramina at early stages of development in transverse section is shown in (A). The jugular blood vessel is not immunoreactive to smooth muscle actin compared with other blood vessels (bv) in the surrounding area (B and C). This negative staining of blood vessels inside hypoglossal and jugular cranial foramina continues to be maintained throughout cranial foramina development in the chick embryo (D–G). ov, otic vesicle; cf, carotid foramina; hf, hypoglossal foramina; jf, jugular foramina. (B and G) are counterstained with nuclear fast red. (C–F) are counterstained with alcian blue. Dotted lines in (C and D) indicate foramina perichondrium.

Fig 8

In situ hybridisation of Sox9 during cranial foramina development. A diagram of cranial foramina in transverse section at early stages of development is shown in (A). Sox9 expression in the mesenchyme immediately surrounding the jugular blood vessel (jbv) (outlined in the figure by a black dotted line) at HH21/D3.5 (B), HH24/D4 (C and D) and HH27/D5 (F and G). Refined sox9 expression at HH29/D6 is seen in (H). The black boxed areas in (C) and (F) are the higher magnification area shown in (D) and (G), respectively. oabv, ophthalmic artery blood vessel; ov, otic vesicle; NT, neural tube; zi; zone of chondrogenic inhibition.

Fig 9

PCNA immunohistochemistry. Diagram of cranial foramina in transverse section at late stages of development is shown in (B). PCNA staining (arrowheads) can be seen in and around jugular (jf) (A, E) and hypoglossal foramina (hf) (C and D). The putative jugular and carotid blood vessels at HH27/D5 are outlined by black dots in (A). (F) A graph based on local PCNA-positive cell counts taken at various stages of cranial foramina development in the foramina mesenchyme. Error bars represent the mean number of PCNA-positive cells at each developmental stage + the standard error of each mean value. Capped lines with * indicate significant differences. jbv, jugular blood vessel; hbv, hypoglossal blood vessel; fm, foramina mesenchyme.

Fig 10

TUNEL labelling in cranial foramina development. Diagrams of cranial foramina in transverse section to show locations of foramina at different stages of development are shown in (Ai) and (Aii). TUNEL labelling (arrowheads) can be seen in and around the jugular blood vessel (jbv) is not clearly apparent at early stages of development (B); however, can be seen in other areas of the head (C). Later in development there is some TUNEL labelling in the foramina mesenchyme (D) and the within the walls of the blood vessel (E and F). The black frames in (C) and (F) show the enlarged insets of the positive TUNEL staining. hn, hypoglossal nerve; jbv, jugular blood vessel; 3V, 3rd ventricle; oc, optic chiasm; hbv, hypoglossal blood vessel; fm; foramina mesenchyme; perichondrium (shown with dotted lines). (E) is counterstained with alcian blue.

Fig 11

Schematic to illustrate the cellular appearance of cranial foramina development in the chick embryo, as assessed by H&E staining.