(EN)Special Embryology

Embryology II Development of the respiratory system By the end of the 4th week, from the ventral wall of the foregut, the respiratory diverticulum appears; more or less in front of the 4-6th pharyngeal arches. The laryngotracheal groove, becomes a channel and protrudes as a bud, gemma pulmonarie (buds of the bronchi). The epithelium of the inner side of the larynx, trachea and bronchi has an endodermis origin. The cartilaginous part, the muscular and the connective tissues arrive from the splanchnic mesoderm situated around the foregut. The pulmonary bud has, initially, an open communication with the foregut; but as it expands caudally, two longitudinal tracheo-esophageal septa appear so the foregut divides into an anterior part (trachea bud) and the posterior esophageal primorus (5 mm through the esophageal septum).

The lung buds develop towards the lateral sides of the foregut at the beginning of the 5th week; then enlarge to make the right and the left bronchi. The left bronchi divides in 2 parts and the main right bronchi splits in 3 parts. These broncho-pulmonary buds will divide further in a dihcotomic manner and extend into the body of the embryo in what we call pericardia-peritoneal channels, which are pretty narrow and will be filled slowly. In the end, they will separate and form the primitive pleural cavities (the visceral and the parietal pleura will develop from the mesoderm). Slowly the segmental bronchi will appear and give the final segments of the lung; and from here until the 6th month of development, there will appear around 17 generations of subdivisions of the bronchial tree. After birth, there will appear up to 6 more divisions.

The maturation of the lungs will occur in several periods of time: A. Pseudoglandular period W5 – W16 (divisions until bronchioles terminales) B. Canalicular period W16 – W26 in which bronchioles terminales split in bronchioles respiratory and then alveolar ducts C. Saccus terminalis W26 – term (primitive alveoles pulmonares are in contact with the primitive capillaries) D. Alveolar period in which the pulmonary alveoli develop their specific epithelium and strengthen the contact with the capillaries.

During the last 2 months of the prenatal life and in the first several years after birth, the number of alveoli increase steadily. The internal lining of the cells of the inner side of the alveoli: • Type 1 of epithelium cell become thiner and thiner also the capillaries are going inside the alveoli at the same time realizing in the end a blood to air barrier. • Mature alveoli contain type 2 alveolar epithelial cells, which start to develop at the end of the 7th month. They are important because they produce the surfactant which is a fluid very rich in phospholipids and its role is to reduce the surface tension at the level of the air-alveolar interface. As we approach birth, the surfactant increase. The lungs normally filled with liquid, but at this time they are preparing for air. Embryology of the heart

Starting with the 3rd week of development, the nutritional requirements are no longer satisfied only by diffusion. A cardiovascular system starts developing. Blood islands of the heart start to appear in the mesoderm into an area situated anterior to tubus neuralis, lama precordalis and anterior to the oropharyngeal membrane. In this so called cardiogenic area, 2 angioblastic cords will appear and will transform into 2 tubes since the embryo is folding in a cranial-caudal manner, but also laterally. These 2 two tubes are fusing together, generating one single endocardial tube. From the same mesoderm, the primordium myocardiale, also known as ”cardiac jelly”, will form.

Around these structures, the primordium epicardiale, from the mesothelial cells of venous sinus, will form here. The developing part of the tube, bulges more and more into the pericardial cavity, being initially attached to the dorsal side of this cavity by a fold; dorsal mesocardium, which will disappear creating sinus transversus of the pericardium. Towards what we mentioned until now; D21, further on towards D23, the primordium of the heart evolves to cor tubulare simplex; which presents 4 dilations: 1. Sinus venosus – where the veins of the embryo are draining (inferiorly) 2. Atrium primitivum – small and above the venous sinus. Atrioventricular junction – which is pretty narrow and followed by: 3. Ventriculus primitivum – the most dilated part at this stage. Sulcus bulboventricularis – continuated by: 4. Bulbus primitivum from which goes truncus arteriosus towards the embryo.

The heart tube continues to elongate and will start to bend. (The pericardial growth is slower). From this moment, by starting bending, the so called cor tubulare simplex transforms to cor sigmoideum. The caudal part, sinus venosus and the atrium, go to the left posterior and superior, meanwhile the superior part, the ventricles and bulbus, go to the right, anterior and inferior, creating the cardiac loop. This cardiac loop will be completed by D28. The primordium bulbus and the primordium ventricle are communicating through the ostium bulboventricularis, ”Haller narrowing”.

Sinus venosus The part that bends and rotates the most, reaches in the end a posteriorsuperior position. It presents a transversal part, which receives three major veins: - Common cardinal vein - Umbilical vein - Vitelline vein It also presents 2 lateral sides named cornu dextrum et sinistrum, which are the real recipients of the veins. The sinus venosus starts draining the veins blood in the middle of the 4th week; but there are going to be a few changes. The communication between the sinus and the atrium, is called sinoatrial junction.

The left horn will degenerate, and from it the oblique vein of ”Marshall” and the sinus coronalis will derive. Due to this, the right horn enlarges its dimension, and at the same time the sinoatrial junction moves its position towards the right side, becoming the sinoatrial orifice which is bordered by the right and the left venous valves.

The right venous valve will disappear in its inferior part. The superior part will remain as septum spurium which will fuse later on with septum secundum. The left venous valve transforms. Its superior part will generate crista terminalis and its inferior part will give the valve for vena cava inferior and the sinus coronalis. Atrium primitivum The atrium primitivum goes through a major transformation by its rotation being placed at the end, between sinus venosus and bulbus cordis, posteriorly, and truncus arteriosus, anteriorly. It will incorporate the sinus venosus so it enlarges its dimensions, but what remains from the original atrium primitivum is only auricula dextra and sinister, which are rough traberculated parts of the atria. The smooth part of the atria will be given into the right side by sinus venosus, which will degenerate sinus venalis cavarum and on the left side by the pulmonary veins. Initially, there is only one pulmonary vein just on the left side of the septum. This vein will connect with the veins coming from the pulmonary buds, having in the end 4 pulmonary veins draining in the left atrium. The septation of the atria

At the end of the 4th week, a circle shaped crest grows from the posterior-superior roof of the common artery and grows towards the A-V channel. It will be called septum primitivum, and the opening in between the lower ring of it and the endocardial cushion of the A-V channel is called ostium primum. Before septum primitivum grows completely in its posterior superior position, some perforations will appear which will fuse together creating ostium secundum. Further on, from the anterior-superior roof of the right atrium, an other fold (septum secundum) appears, which grows towards the A-V channel and towards the posterior side of the atrium; posterior-superiorly fusing with septum spurium. As it grows like this, it will cover slowly what remains free from this growing septum

secundum is called foramen ovale, meanwhile the septum spurium starts to degenerate slowly. The part of it that covers foramen ovale will remain being called valvula of foramen ovalis. Like this the passage in between the 2 atria, will be an oblique communication through fossa ovale and through the ostium secundum. The septation of the ventricles It takes place after the septation of the atria, starting at the end of the 4th week. The myocardium of both sides are growing and slowly create the muscular part of the interventricular septum. From the level of the atrioventricular plane, the membranous part of the septum grows, starting from the midline at the A-V channel (septum intermedium) and grows inferiorly until they will meet. Meanwhile, there is going to be an interventricular communication, communication of ”Panizza”. At the end of the 4th week, until the A-V channel, the so called endocardial cushions appear. First, 1 anterior and 1 posterior, and then 2 smaller laterally. Near the 5th week, the

anterior and posterior cushions almost fuse together. In the end, the lateral ones generate the right and the left A-V channels. The ventricles When the blood starts to come in the cavities, it will start to perforate the tissue creating a space into the tissue right under the A-V plane. From the muscular tissue, which is detached from the wall, slowly the muscular fibers will degenerate and be replaced with dense tissues; superiorly, plus the cordae tendinae and inferiorly they will grow even more deeper into this detaching tissue and create the papillary muscles. Above this structure at the left and right A-V channels, from the dense mesenchymal tissue, the cusps of the A-V valve will appear. The semilunar valve When the aorta and the truncus pulmonaris are almost completely separated, the base of them where the junction of the bulbus is, 3 symmetrical tubercles with an endocardial origin will appear, which will grow into the lumen. The blood inside, by the action of gravity will enlarge the space in between the aorta/truncus pulmonaris and the tubercles creating the sinus of valsalva with the corresponding semilunar valves. The septation of bulbus cordis and bulbus secundum is made by the development of 2 cushions at the level of the junctions. First to the right, superior and the other to the left, inferior. The right grows superiorly and the left grows inferiorly, distally to the right; both by twisting one an other generating 2 aorta-pulmonary crests. These will fuse and create septum aortopulmonare, which will have a spiral disposition in space; and in the end having the adult position of the aorta and pulmonary trunk. At the same time, more or less, the ventricles grow inferiorly. In the end, the aorta from the left ventricle emerge posteriorly to the right of truncus pulmonaris, and then anterior to it; whereas from the right ventricle, truncus pulmonaris emerge anteriorly and to the left towards posterior The development of the conduction system of the heart The development of the conduction system of the heart starts in the caudal part of the left cardiac tube, then moves to the right part of sinus venosus. The cells are migrating near the junction with the future vena cava superior, giving sinoatrial node, then from here, then cells migrate to the anterior-inferior part of the interatrial septum. The cells from the A-V channels are also coming here, creating the A-V node. From here, the cells are migrating through septum intermedium towards the membranous part of the interventricular septum and from there split on to both sides of the muscular part of the interventricular septum going through the myocardium towards the cells into the rete of ”Purkinje”. The development of the arterial system In the development of the blood vessels, 2 processes are involved 1. Vasculogenesis – the vessels appear in coalescence of the angioblasts 2. Angiogenesis – the vessels do develop from other vessels that already exist

The big vessels of the beginning of development like dorsal aortae or the cardinal veins are developing via vasculogenesis, while the rest develop via angiogenesis; this growth being influenced by some other factors including VEGF (vascular endothelial growth factor). Into the extra embryonal mesenchyme also into the fixation pedicle, starting with the 3rd week, angioblastic cells, which are forming blood islands, appear. Inside of them, small cavities will appear. They will fuse together; and the connection between them will form the rete capilare. Around these cavities, angioblastic cells will appear; which will give the endothelium of the vessels and from here the blood cells will start forming at the level of

the liver, spleen, bone marrow and lymphatics. From truncus arteriosus, which is situated above bulbus cordis, saccus aorticus which is the most distal part of the truncus will derive. During the 4th and the 5th weeks, from the saccus aorticus, 6 pairs of symmetrical aortic arches will appear, for the correspond pharyngeal arches. These arches are going to finish into the dorsal aortae (the aortae remain separated in this region of pharyngeal arches, but inferiorly under the level of the 7th intersegmental artery, these 2 dorsal aortae fuse together forming aorta descendens). From these arches, that are emerging from saccus aorticus, the 5th one forms incompletely or it never forms. From the other arches, different parts of the arterial system will start to appear, but not at the same time, because all the arches do not appear at the same time; and they also disappear at different times.

Until D27 the first aortic arch is gone, what remains from it is arteria maxillaris. The same thing happens with the second arch, but what remains is the stapedius and the hyoid. With further development the aortic arches are losing their arrangement and will give at the end the final arterial pattern of the adult human. From saccus aorticus, truncus pulmonaris and the most proximal part of aorta ascendens will derive, whereas from truncus aorticus the entire aorta ascendens plus the adult aortic arch until and including the origin of arteria communis sinistra plus the truncus brachiocephalicus will derive. From the 3rd aortic arch, arteria carotis communis plus the initial part of arteria carotis interna on both sides will derive. What remains from arteria carotis interna, is developing from the dorsal aorta of their sides. Arteria carotis externa, will develop on both sides as a branch of the 3rd aortic arch. From the 4th aortic arch, on the right side, the first part of arteria subclavia dextra will derive and of the left side, the part of the aortic arch situated in between the origin of arteria carotis communis and arteria subclavia sinistra will derive. The next part of arteria subclavia dextra will be given by the right dorsal aorta and the most distal part of

ACD, as well as ACS are deriving from the 7th intersegmental artery on each side) The 6th aortic arch gives on the right side the first part of the right pulmonary artery and on the left side the first part of the left pulmonary artery, plus on this side, the distal part of the arch remains patent (open) as ductus arteriosus, ”Botalli”, connecting the pulmonary artery with the most proximal point of aorta descendens. Under the level of pharyngeal arches, we have aorta thoracica and abdominalis, which will give the branches that we know in the adult life. These branches are deriving, not directly from aorta, but from the intersegmental arteries which are the primary arteries.

The vitelline arteries are supplying the yolk sac, and in the end they will fuse, generating the arteries for the digestive system. The umbilical arteries, also paired, initially as branches of the dorsal aorta, go to the placenta. In the 4th week, they shift their origin with and the common iliac artery. At the end, after birth their initial part will remain patent as internal iliac artery and superior vascicle artery; but the distal part of them will obliterate, generating lig umbilicale mediale. The development of the venous system Into the 5th week, the major component of the venous system of the embryo consists of 3 pairs of veins which are draining into the sinus venosus A. Cardinal veins – which really are draining the embryoʼs blood; the anterior and posterior cardinal veins unite. B. Umbilical veins – carry oxygenated blood from the placenta, and drain into the sinus venosus C. Vitelline veins – drain blood from the yolk sac to the embryo, confronting with sinus venosus more medially than the umbilical veins. The vitelline veins, before entering sinus venosus, form a network around the duodenum which in the end will generate the hepatic sinusoid having the liver cord and cells around them. The left cord of sinus venosus is regressing so the blood from the left side of the liver is directed towards the right side of it, so that the right vitelline vein will increase its diameter generating at the end, the hepatic cardiac part of vena cava inferior.

From the anastamosis around duodenum, the portal vein will form; and from the distal part of the right vitelline vein, vena mesenterica superior will form. The left vitelline vein will disappear. The umbilical veins are situated initially, on each side of the liver, but they start to connect slowly with the hepatic sinusoid. Slowly the proximal part of both umbilical veins will disappear, so the distal part of the left vitelline vein, will carry the oxygenated blood from the placenta to the liver; but a direct communication will appear with the hepatic-cardiac channel, ductus venosus, which will bypass the liver. After birth, vena umbilicalis sinistra and ductus venosus, will obliterate becoming lig teres hepatis and lig venosum hepatis. The cardinal veins are draining the main part of the embryo. - Anterior ones – drain the cephalic part - Posterior – drain the rest of the body Joining together in vena cava inferior and ductus venosus, starting with the 5th week until the 7th, some additional systems will appear: 1. Subcardinal veins – draining the kidneys 2. Sacrocardinal veins – draining the lower extremities 3. Supracardinal veins – daring the body wall of the embryo, taking over the function of the posterior cardinal veins. The anastamosis between the subcardinal veins, will give rise to the renal vein and from the left subcardinal vein, only the left gonadal vein will remain. Due to this, the right subcardinal vein will enlarge and become the renal segment of vena cava inferior. The anastamosis between the sacrocardinal veins, will generate vena iliaca communis sinistra at the same time as the right sacrocardinal vein will increase its diameter generating the infrarenal segment of vena cava inferior. Due to the fact that the posterior cardinal veins are disappearing, the supracardinal veins are becoming more important, generating the intercostal veins, 4 – 11, which eventually will drain in venae azygos et hemiazygos. The anastamosis between the anterior cardinal veins (draining the head), will generate v brachiocephalica sinistra, meanwhile the right anterior cardinal vein and the right common cardinal vein are enlarging and forming vena cava superior. The distal part of the anterior cardinal veins are generating v jugularis interna. The left superior intercostal vein is deriving from the terminal part of the left posterior cardinal vein. The pharyngeal arches, clefts and pouches The most typical feature in head/neck development are pharyngeal arches which appear in W4-5 of development. In the beginning these arches consists in bars of mesenchymal tissue separated by clefts and will penetrate into the surrounding mesenchyme but do not communicate with the external clefts.

Each pharyngeal arch consists of an inner part of mesenchymal tissue which is covered by ectoderm and on the inner side covered by endoderm. In the mesenchyme, there are cells which migrated from the neural crest, which will generate some of the face skeleton. The mesodermal component, generates muscles and into the arch a nerve for the muscles and an artery to supply them.

The 1st pharyngeal arch has a dorsal part called maxillary process a ventral part called mandibular process. Inside the mandibular process, there is the cartilage of Meckel which will generate the incus and malleus (two internal ear bones). The mesenchyme surrounding will generate the mandible. From the maxillary process; the maxilla, os zygomaticus and a part of the temporal bone will appear. The muscles that appear are: muscles for mastication (maseter), m pyteroidea, temporal part of m oligasticus, m mylohyoideus, m tensori tympani and m tensor palatini. The nerves arising are: n mandibularis (principal), n maxillaris (branches) and olfactory (branches). Ligaments deriving are lig sphenimandibularis and some of lig malleus. The 2nd pharyngeal arch presents the cartilage of Reichert, which will generate the stapedius process, the stylohyoid ligament and cornu minor and the upper part of the hyoid bone. The deriving muscles are: m stapedius, m stylohyoideus, posterior m hypogasticus, m auricularis and muscles of mimick.

The nerve that innervates the muscles is the facial nerve From the cartilage of the 3rd pharyngeal arch appear: cornu major and lower part of hyoid bone, m stylopharyngeus and n glossopharyngeus. The 5th does not really exist, but the 4th/6th cartilaginous component gives: cartilago thyroideus, cricoideus, corniculata, cuneiforme and arytenoideus. The muscles deriving from the 4th arch are: m cricothyroideus, m levator palatini, mm constrictor of the pharynx. The nervous innervation: n laryngeus superior. Muscles deriving from the 6th arch; intrinsic muscles of the larynx except m cricothyroideus. Innervation is made by nervus laryngeus recurrens (nervus vagus). The embryo has 4 pairs of pharyngeal pouches. The 5th one being small. The epithelial endodermal coating is going to generate different parts of the structures of the head such as:

1st pharyngeal pouch Recessus tubo tympanicus (comes close to the first pharyngeal cleft, which generates meatus acusticus internus). Zenkerʼs diverticulum (pharyngeal pouch) generates the middle ear cavity – proximal part: tuba auditiva. Later on in the tympanic cavity, the tympanic membrane will be generated. 2nd pharyngeal pouch Buds will receive some mesodermal tissue forming tonsilla palatina + fossa tonsillaris rd 3 pharyngeal pouch

At the distal extremity, will have a dorsal + ventral wing (also 4th). In W5, the distal epithelium will generate glandula parathyroidea inferior and the ventral one will generate the thymus. th 4 pharyngeal pouch Glandula parathyroid superior (on its dorsal part) + last branchial body (ventrally) which will be incorporated in glandula thyroidea and will generate the ”C” or parafollicular cells which secrete the hormone calcitonin.

Into W5, they are present but only the 1st one contributes to the final embryonic structure. The dorsal part penetrates into the underlying mesentin generating part of meatus acusticus internus and part of membrana tympanica. As the mesenchymal tissue of the 2nd arch grows rapidly; the 2nd arch will overlap the 3rd/4th arches resulting in the epicardial ridge and therefor the 2nd, 3rd + 4th clefts lose contact with the exterior. They fuse and form the cervical sinus which will disappear in the end. The development of the face At the end of W4, proeminencia facialis consists of several components situated around stomodeum (primitive oral cavity). These prominences derive mainly from the 1st and 2nd pharyngeal arches and consist of 2 maxillary prominences situated laterally to the stomodeum, 2 mandibular prominences situated inferiorly and proeminencia frontonasalis situated above stomodeum. On both sides of proeminencia frontonasalis the ectoderm becomes thicker generating placoda nasalis or olfactoria. During W5, the placoda invaginates, generating the nasal pits (around them the nasal prominence which has a lateral and medial part). The placoda nasalis will split inferiorly, having a horse shoe shape with an inside groove, while the maxillary process grows – compressing the rudial part of proeminencia nasalis towards the midline, resulting in the fusion the medial parts. Thus will appear the upper lip, meanwhile the two mandibular processes will fuse – generating the lower lip/mandible.

Initially the maxillary + lateral parts of the nasal prominence are separated by the nasolacrimal groove which will give ductus nasolacrimalis. The nose will be formed by the frontal prominence which will give the bridge (dorsum nasii). As a result of the medial growing of the maxillary prominence, the two medial-nasal prominence are fusing both at a superficial level and a deep level; creating the inter-maxillary segment which generates several components:

A. Philtrum of upper lip B. Upper part of arcus dentalis (4 midline incisives) C. Palatinal component (forms the anterior triangular palate) The secondary palatum derives from 2 components which belong to the maxillary prominence being named palatine shelves. They appear in W6 and are oriented one to the other on each side of the tongue and then fuse in W7. Oral cavity The oral cavity derives from endo-/ectoderm. The primary intestine does not communicate in the first phase with stomodeum, being separated by a double membrane. At the level of the ectoderm there is a pouch of Rathke that will migrate to the brain and will generate the anterior lobe of hypophysis. The double membrane will disappear at the end of W3 resulting in a direct communication between intestine and stomodeum. Nasal cavity The nasal cavity in the first part of development (during W6) consists of the nasal pits which will become deeper and deeper. At first, the nasal pits are separated from the primitive oral cavity by membrana oronasalis, which also presents some openings: primitive choanae situated on each side of the midline, posterior to the primary palatum. Posteriorly as the secondary palatum develops, the choanae become permanent and the lateral sides will develop as paranasal sinus (frontal, maxilla). These sinuses reach maximum size in puberty and generate the final shape of the face. Development of the digestive system Due to the folding of the embryo on the lateral sides and cranial-caudal, a part of the cavitas vitellina (coated with endoderm) will be incorporated into the embryo forming the primitive gut. During itʼs entire development until the end, the amnionic and vitelline cavities will remain outside of the embryo. The primitive gut, which is a tube with broth its endings closed in, is split in: - Anterior part – foregut, hindgut - Mid gut – temporarily connected with the vitelline cavity through ductus vitellinus Further on, we split the parts of the gut in 4 components: 1) Pharyngeal gut: - Starts at membrana oropharyngealis - Ends at the level of the respiratory diverticulum 2) Foregut: - Starts at the end of the pharyngeal tube - Ends at the level of the liver bud 3) Mid gut: - Starts after the liver bud

- Ends at the junction in between the right 2/3 with the left 1/3 of the mesocolon transversum 4) Hind gut - Starts with the left 1/3 of mesocolon transversum - Ends at membrana cloacalis The endoderm which coats this tube, will generate the specific cells of the glands (liver and pancreas), meanwhile the connective tissues of these glands come from mesoderm (also muscles, connective tissue and the peritoneal components of the digestive system arise from the mesoderm). Oesophagus At the end of W4, the respiratory bud (diverticulum) appears on the ventral wall of the foregut. Along with it, a septum develops between the trachea and the oesophagus (which develops fro the posterior side of the same tube). Slowly the will be separated, generating anteriorly the respiratory primordium and posteriorly the oesophagus primordium. Due to the fact that the heart + lungs will descend at this level towards the thorax, the oesophagus will follow them, getting longer and longer until it reaches the abdominal cavity. Like a fusiform dilation of the same foregut (also in W4), the stomach appears. Stomach The stomach has specific development in shape and position due to a different development of its parts rotation; 1. 90° clockwise rotation, on the longitudinal axis - Left side becomes anterior - Right side becomes posterior During this rotation the initial posterior wall grows faster than the initial anterior wall; so at last the left curvature will be a bigger wall. 2. On the anterior-posterior axis (the stomach rotates at the same time as the longitudinal rotation) it rotates towards the left side so the esophagealstomach junction + fundus gastrica go to the left and inferiorly, and the pyloro-duodenum go to the right and superiorly: reaching its final position. Since the stomach is attached to the dorsal body wall by the dorsal mesogastrium and to ventral body wall by the ventral mesogastrium the rotation results in the pulling of the dorsal mesogastrium to the left creating a space behind the stomach; bursa omentalis. In the same way the ventral mesogastrium will be pulled to the right. Spleen The primordium spleen appears in W5 as a mesodermal proliferation situated in between the 2 components of the mesogastrium. When this rotates, it increases the length so the part in between the spleen and the midline will fuse with the peritoneum of the posterior wall. The spleen will still remain in the peritoneal cavity as the last component of the dorsal mesogastrium. A part of the pancreas will also develop in the dorsal mesogastrium from a position situated posterior to the spleen. Since this part of the dorsal mesogastrium will fuse during the rotation with the peritoneum of the posterior abdominal wall (as

mentioned before); this part of the pancreas becomes situated in a retroperitoneal position although it started its development intra-peritoneum. Also this dorsal mesogastrium will elongate inferiorly, as a double layered structure, over cool transversum and the small intestines creating omentum major. From the ventral mesogastrium the omentum minor and lig falciforme develop. Duodenum The last part of the foregut and the 1st part of the midgut will generate the duodenum. The junction of them being distal to the liver bud. As the stomach rotates, the duodenum also moves towards the right side and gets a C-shape. In its concavity, it has the anterior primordium of the pancreas. As a part of the liver develops in front of them, they will be compressed against the posterior abdominal wall so the right posterior surface of the meso-duodenum will fuse with the posterior peritoneum of the abdominal wall; and like this the duodenum and the head of the pancreas will reach a retroperitoneal position except the 1st part of the duodenum which is intraperitoneal. Liver The liver bud appears in W3 at the distal end of the foregut, but superior to the duodenum and the anterior part of the pancreas. Its cells penetrate in septum transversum (which is a mesodermal plate separating the pericardial cavity from the vitelline one). As the cells are penetrating the septum, their connection with the foregut will become thinner and in the end forming canalis choledocus; connecting the liver with the duodenum. In the anterior region of the liver bud, from where canalis choledocus connects with it, an other bud will grow forming ductus cysticus + vesica biliaris. When the cells of the liver bud invade the septum transversum entirely, the organ starts to grow towards the abdomen in front of the duodenum and the pancreas. The mesoderm on the surface of the liver, will generate the visceral peritoneum except on area nuda, which is in contact with the septum transversum. In W10, the liver weights 10% of the embryo and from W12 the bile secretion starts towards the duodenum. Due to the change of the duodenumʼs position the entrance of ductus choledocus inside shifts from anterior to posterior, reaching its final position. Pancreas Its originally from 2 buds; anterior and posterior. The posterior one situated in the dorsal mesogastrium and the ventral one near the opening of ductus choledocus. With the rotation of the duodenum, the anterior bud will go with it reaching in the end a position near and inferior the dorsal bud. At last, they will fuse resulting in processus uncinatus (from the anterior one) and the rest of the pancreas (from the posterior one). The main pancreatic duct is formed by the distal part of the dorsal pancreatic bud united with the entire ventral pancreatic duct. The proximal part of the dorsal pancreatic duct will generate the accessory pancreatic duct of Santorini.

From M3 small islands of Langerhans cells start to appear in the pancreas, and from M5 insulin secretion starts. Midgut In W5 the midgut is connected with the posterior abdominal wall by a short mesenterium and communicates with vitelline cavity through the ductus vitellinus. What derives from here in adults, is everything that develops from the duodenum with left 1/3 of colon transversum being characterized by a fast elongation of the gut and its mesenterium, creating the primary intestinal loop. From here the most distal duodenum, jejunum-ileum (from the proximal limb of this loop), the cecum, appendix, colon ascendens and the 2/3 part of colon transversum (from inferior) develop. This first loop increases a lot (especially the superior limb) because the liver also is growing fast into the abdominal cavity, there is no space for it; so stating with W6 the physiological umbilical hernia of the intestinal loops through the umbilical cord into the extra-embryonic cavity. During W10, the herniated loops return into the abdominal cavity. The first that enters is the proximal part of jejunum (situated on the left part of the abdomen) and the laterentering loops are positioned to the right. The cecal bud, is the last part that enters the abdomen and is situated under the liver and afterwards gradually descends to fossa iliaca (the appendix develops from it during this time). In the same time as the growth in length, the primary intestine loop will rotate along its long axis of 270° degrees, and jejunum/ileum will have a coiled disposition. The large intestine (inferior loop) does not participate in this rotation, so it will not be coiled. The rotation becomes during the herniation about 90° and during retraction 180°. The mesocolon ascendens and descendens fuse partially with the posterior abdominal wall/peritoneum – the 2 components will not have a meso in the adult life. Hind gut The hind gut generates the left 3rd of colon transversum, descendens, sigmoideum, rectum + the superior part of canalis analis. Also from its endoderm, parts of vesica urinalis and urethra will develop. The terminal part of the hind gut reaches the region of the cloaca, where canalis anurectalis will develop superior to it and the allantoid will generate the sinus urogenitalis. The cloaca is coated with endoderm, but anteriorly it is in contact with the ectoderm. This boundary will generate membrana cloacalis (endo-ectodermalis). The septum in between the hind gut and the allantoid is given by the mesoderm generating the uro-rectal septum.

As the embryo grows and folds crania-caudally, the tip of the uro-rectal septum becomes closer to the membrana cloacalis (which at the end of W7 will rupture creating the anal opening); here will the urogenital sinus also be. In between them, uro-rectal septum will give the perineum bud; so the upper 2/3 of canalis analis comes from the endoderm of the hind gut and the lower part from the ectoderm situated at this level. Development of the urinary system Starting with W3, 3 urinary systems will appear into a cranial-caudal manner. The first 2 will disappear being quite rudimentary, and the 3rd one being the functional one 1) Pronephros: - Starts to develop in the 3rd week at the cervical region, as up to 10 solid cells which will open into a collective channel which progresses towards the cloaca. - Disappears in W4 2) Mesonephros: - Appears in W4 by deriving (as pronephros) from the intermediate mesoderm. At this level of the upper thorax until the upper lumbar level (L3). From these solid cells, some excretory tubules will appear towards a longitudinal collecting tube called mesonephric or Wolfian duct. The small tubules are growing fast in length and getting an S-shape. At one end they form they form the ”Bowman” capsule; inside of it has a network of capillaries thus forming together the renal capsule. In this manner the mesonephros looks like a large organ at the end of M2, on each side of the midline having medial to it the primordium of the genitals. Together they form a ridge (prominence) named urogenital. Towards the end of M2 the proximal cells and tubules have already disappeared and slowly also the caudal ones will disappear. In females the mesonephric duct will disappear also, but in males it will persist participating in the formation of the genitals. 3) Metanephros: - Appears in W5, being the final kidney. Its excretory unit develops from the mesoderma metanephrica. From the distal part of the mesonephric duct, the primordium ureteric bud will appear, close to the cloacaʼs entrance. This bud will penetrate into the metanephric tissue which stays on the ureteric bud as a cap. The ureteric bud will dilate forming the renal pelvis which first splits in major calyces, then in minor ones in the building of the renal pyramid (with collecting tubules). Also from the ureteric bud the urethras will form. Each new formed collecting tube is covered on its distal end by a cap made of metanephric tissue, the cells of this tissue will form some vesicles which will unite in between the creating a small tube with an S-shape. At one end of this tube a pocket will appear in which the glomerulus will develop. The excretion tubes together, with the glomerulus will make the nephrons or the excretory units. The proximal end of each nephron will form Bowmanʼs capsule (having the glomerulus inside of it) and the distal end will make an open connection with the collector tubes which are coming from the ureteric valve. This distal end of the glomerulus before making the connection, will elongate and form the proximal convoluted tubule, then the ascending and descending links of Henleyʼs loops; the distal convoluted tubule which connects with the collecting tubes. The nephrons develop until birth = 1 million/kidney. The urine will be produced starting with W10. Initially the kidney is situated in the pelvic region, but will start to ascend as the body curvature diminishes and the lumbar and sacral regions are growing fast.

The definitive kidneys become functional in W12. In between W4-W7 the cloaca divides into urogenital sinus (anteriorly) and canalis analis (posteriorly), having in between them a layer of mesoderm. The tip of this layer which acts like a septum creates the perineum. The urinary bladder is initially continued with the allantoid, but when the lumen obliterates (closes) this allantoid will give the uracha (later – lig umbilicale medianum). The next part of the urogenital sinus is a narrow channel which will give the prostatic and membranous parts of the urethra. The last part of the urogenital sinus will generate the folic part of the urethra. The distal part of the mesonephric duct will generate in males ductus ejaculatorius, and regress in females. The epithelium of the urethra at the end of M3 will generate in males the buds of prostate and in females the buds of urethral and para-urethral glands. Development of the genitals The sex of the embryo is determined by the Y-chromosome which has on its short arm a gene called ”SRY-gene” (sex determining on Y-chromosome). The protein product of this gene generates the testisʼ determining factor. Under its influence, the male development occurs. If absent (XX) the female development occurs. Although the sex of the embryo is determined genetically at the time of fertilization, the gonads do not acquire male/female morphological characteristics until W7. These gonads appear on a ridge medial to the urinary system by a proliferation of the epithelium and a condensation of the underlying mesenchyme. In this ridge, the germ cells are migrating from the epiblast and reach the final position in W6/W7 After their arrival, they form some primitive sex cords which at this time are called together indifferent gonads. Testis The primitive germ cells (in the testis) under the influence of SRY form some medullary chords which towards the hilum of the glands are generating a network of tiny small strands of cells, creating like this rete (network) testis. Around it, there is layer of fibrotic tissue (tunica albuginea). In M4 the testis have a C-shape having inside cells organized in chords also sustentacular (sustaining) cells of ”Sertoli” derived from the surface of the epithelium of the glands. In between these chords of cells, there are interstitial cells of ”Leydig” derived from the original mesenchyme of the gonadal ridge. Starting with W8, these Leydig cells are producing testosterone which influence the further development. These testis chords remain solid (without channel) until puberty when they acquire a lumen forming, then the seminifer tubes. These seminifer tubes will connect with ductus efferens which is the remaining part of the excretory tubules of the

mesonephric system. This ductus efferens will connect in the end with ductus deferens which is the remaining part of ductus mesonephricus. - Ductus mesonephricus –> ductus deferens - Excretory tubules –> ductus efferens Ovaries In females, the primitive sex chords of cells dissociate themselves in clusters of cells occupying the medullar part of the ovary. Slowly they disappear by degenerating, being replaced by the so called vascular stroma. On the surface of the epithelium there is a proliferation, so in W7 some cortical chords will appear on the surface; at this level. In M3 these chords will split in clusters of cells which continue to proliferate, each of the cells being surrounded by a layer of epithelium so together they form the primary follicles. Genital ducts Initially, both male and female have 2 pairs of genital ducts: 1) The mesonephric duct (Wolf) 2) The para-mesonephric duct (Muller) – arises like a longitudinal invagination of the epithelium on the anterior-lateral surface of the uro-genital ridge. Cranially, the Muller duct opens into the abdominal cavity like a funnel-like structure. Distally goes lateral to the Wolfian duct, then crosses it anteriorly and then on the midline comes in contact with the other Muller duct. Initially, the 2 ducts are separated by a septum, but this will disappear so they will fuse together, generating the uterine canal. With the descending of the ovary, the first 2 parts of the Muller ducts will generate the uterine tube and the last part will give the uterus. From the urogenital sinus, near the pelvic bud, the sino-vaginal bulbs will appear, which are generating the vagina (which is complete, having a channel inside) in M5. However, the upper part of the vagina near the uterus, derives from the uterine channel. The lumen of the vagina is separated from the urogenital sinus by a thin plate called hymen. The external genital starts to appear from W3, when the mesenchyme from the region of the cloaca generates 2 folds united cranially into the genital tubercle and divided posteriorly into a urethral- and anal fold. On each side of the urethral fold, a swelling will appear which will generate the scrotum (in males) and the labia (in females).

From the genital tubercle in males, the phallus will appear, having inside a groove which will transform into a channel (urethra). In females, the clitoris will appear from the genital tubercle; but the urethral folds will not fuse, as in males, but will generate the labia minora. Starting with the end of M2, the mesonephros starts to degenerate so its meso disappears also – only its distal part remains and generates the caudal genital ligament. Also from the inferior pole of the genital ligament, there is a mesenchymal condensation called Cubernaculum testis. These structures will be oriented towards the symphysis pubica, a little bit laterally to the inguinal region and they will direct the descend of the testis towards the scrotum by retraction. In W12 the testis reach the inguinal region, then migrate through the inguinal channel and reach the scrotum in W32. This process is influenced by the male hormone. The descend of the ovaries is not so accentuated, so this will stop at the level of the pelvis near tuba ovalica (ovarian tube).