Embryonic samplesSample acquisition

In this study, two human embryonic samples were used: one at CS 14 (TOP 174, 4-mm crown-rump length (CRL)) and the other at CS 18 (TOP 318, 15-mm CRL), corresponding to gestational ages of 7 weeks and 8.5 weeks, respectively. These specific samples were included due to their availability from the Dutch Fetal Biobank [14]. The specimens were donated with parental written informed consent after removal of an ectopic pregnancy. Ethical approval of the Dutch Fetal Biobank was granted by the Medical Ethical Committee (METC) of the Amsterdam University Medical Center, location AMC, Amsterdam, The Netherlands (METC 2016_285).

Embryo sample preparation and scanning protocol (Fig. 1)Fig. 1

Graphical visualization of the workflow of data acquisition, demonstrating each step of the process: obtaining, fixation/storage, staining, micro-computed tomography scanning, segmentation, 3-dimensional reconstruction

After surgical resection of the fallopian tube, samples were fixed in 4% paraformaldehyde solution for 48 h, and then stored at 4 °C in a 0.2% paraformaldehyde solution. A diffusible iodine-based contrast-enhanced computed tomography (diceCT) staining technique for imaging of the human embryonic soft tissues was used [13, 14], prior to exposure of the embryo to the micro-CT scan. Staining was performed by submerging the sample for 5 days in a 3.75% buffered Lugol’s solution [20]. Apart from its general contrast-enhancement of tissues, this solution typically enhances the visibility of blood and vessels on micro-CT scans due to its high affinity with blood. Micro-CT scans were performed using a UniTOM XL micro-CT unit (TESCAN XRE Demo Lab, Ghent, Belgium) providing images with a voxel size for samples 1 (CS 14) and 2 (CS 18) of 2.5 μm (scan settings 80 kV, 15 W and scan time 7 h 42 min) and 9.0 μm (scan settings 100 kV, 15 W and scan time 2 h 16 min), respectively.

Data visualization

The software package Amira 3-D (version 2021.2, Thermo Fisher Scientific, MA, USA) was used for the creation of 3-D reconstructions of the intra- and extracranial vascular system [21]. Micro-CT data sets from both human embryonic samples (CS 14 and 18) were used for annotation and segmentation. The reconstructions were mostly manually segmented with a Bamboo tablet and stylus (http://www.wacom.com). After generation of the 3-D models, surfaces were smoothed, without loss of essential details. Labels were assigned to the different anatomical structures after the 3-D reconstructions were completed (Fig. 1). Segmentation and labeling were accomplished by two trained dentistry students, under the supervision of the first author.

Supplementary 3-D-PDFs containing the 3-D reconstructions of both stages were added to the online version of this article and can be viewed with a recent version of Adobe Reader (free-ware available at http://www.adobe.com).

Terminology Weeks/days

Gestational age, calculated from the first day of the last menstrual period, is commonly used to denote the clinical age of an embryo or fetus, while post-conception age refers to the number of days elapsed since fertilization. Throughout our study, we will refer to “days” or “weeks” as gestational age.

CS

The Carnegie stages, proposed by Streeter in 1942 and later revised by O’Rahilly and Müller in 1987 and 2010 [22], are based on the observed external features of developing human embryos in the first 60 days of their development post-fertilization.

Background

The development of the craniofacial arterial system begins with the genesis of five pairs of primitive pharyngeal arch arteries, which undergo significant modifications throughout development [9, 15, 23,24,25,26,27]. The fundamental organization of the pharyngeal regions arises with the emergence of pharyngeal pouches, marking a crucial event in the development of the pharyngeal arches [23, 24]. Each pharyngeal arch contains various embryonic cell types, including ectoderm (external), endoderm (internal), and a mesenchymal filling of the neural crest with a central core of mesoderm [23]. These distinct embryonic cell populations contribute to different components of the arch: the ectoderm forms the epidermis and sensory neurons of the epibranchial ganglia; the endoderm gives rise to the epithelial lining of the pharynx, taste buds, thyroid, parathyroid, and thymus; the neural crest in this layer forms the skeletal and connective tissues of the arches; and the mesoderm contributes to the musculature and endothelial cells [23, 28].

During CS 11 to 20, the intracranial vascular system, including the circle of Willis, is formed. This process has been thoroughly studied and documented in the literature [16, 26, 27, 29], particularly due to its clinical significance and the association between congenital arterial malformations in intracranial vessels and the risk of cerebrovascular pathology later in life [30, 31].

Older pioneers in vascular development, as noted by Bertulli and Robert [15], have focused more on the formation of the extracranial arterial system. Notably, several studies by Streeter, Congdon, and Padget [16, 32,33,34] utilized the Carnegie embryo collection to outline the foundational principles of craniofacial vascular system formation. Additionally, significant contributions have been made by Altmann and Lasjaunias [35,36,37], who provided research on arterial variants, comparative anatomy, neurovascular anatomy, and embryology.

The development of the extracranial arterial system commences at CS 8, with the production of mesodermal substrate during the process of gastrulation. Subsequently, various modification and formation processes occur throughout embryonic and fetal development, ultimately culminating in the formation of the extracranial vascular system. Table 1 provides a schematic overview of these developmental stages.

Table 1 Overview showing developmental progression of the extracranial arterial system across Carnegie stages 7 until 23. From each stage, specific developmental processes are described, providing a comprehensive overview of this complex process

Each of the pharyngeal arteries will differentiate, modulate, and finally participate in the development of specific parts of the extracranial vascular system and vascularize the orofacial and extracranial regions. The bilateral present external carotid artery (ECA) will be the basis of the extracranial vessels. Each of these vessels will find their origin in one of four different pharyngeal arches (1st–4th), with most of them deriving from the 2nd pharyngeal arch (see Fig. 2 with corresponding Table 2 for an overview of ECA branches and the pharyngeal artery they derived from).

Fig. 2

Graphical comprehensive overview of all arterial branches, their derivatives, and the corresponding pharyngeal arches of which they originate from, detailing the oxygenated blood supply to the orofacial and extracranial regions: 1st arch derivatives (dark red), 2nd arch artery derivatives (orange), 3rd arch artery derivatives (green), 4th arch derivatives (blue). CCA common carotid artery, ECA external carotid artery, ICA internal carotid artery (bright red). Details of each of these arteries are provided in Table 2 (Figure adapted from Cartens et al. and Thieme/Prometheus 2010 [38, 39])

Table 2 A comprehensive overview of all arterial branches, their derivatives, and the corresponding pharyngeal arches they originate from, detailing the oxygenated blood supply to the orofacial and extracranial regions, which corresponds to Fig. 2