Transcribed by Albert Cheng Craniofaial Lecture 3 Embryology III

3/12/14

Slide 62: Intermediate Mesoderm So lets pick up where we left. We were talking about the different3 germ layers and some of the derivatives of each one of those germ layers. Now we are still in the mesoderm talking about caudamesoderm. We talked abouta little bit of our axial mesoderm, were gonna talk more about it. But now we are into intermediate mesoderm which is the region of the mesoderm that contribute the urogenital system. So its located between the paraxial mesoderm and the lateral plate mesoderm. And thats the region where the primordial germ cells will migrate into eventually. Slide 63: The development of the urinary system and genital system are interconnected anatomically. The mesonephros and the mesonephric duct which are the embryonic kidney, which are viewed here, is closely associated with the developing gonad. This embryonic kidney will eventually degenerate in the adult. It will be replaced by the metaphrenic kidney and metaphrenic duct, which are present here. So both of the those system are very closely associated during the development. Part of the mesonephros, the embryonic kidney and some of the ducts that are associated with itthe kidney will degenerate but some of the ducts will be reused as part of the urogenital system differently in males and females. And you will hear more about that in the context of organs system when you talk about reproductive system. Slide 64: Somite development So the somite is one of the earlyso yea I started a little earlyso the somite is the major derivative of the paraxial mesoderm and those somites are essentially an aggregation of cells that you find on each of the neural tube that will appear as those strucuture here that is more caudal which is known as the presomitic mesoderm. So the presmomitic mesoderm is unsegmented and those aggregration of cells that form the somite will appear progressively as the embryo ages. Slide 65: Somite periodicity So the somite, there is a periodicity in appearance along in the anterior-posterior axis which is a process thats very highly regulated and its species specific. So each species will have a number of somite that are very unique to the species. This process by which the somite are sequentially appearing is regulate by two families of molecules Notch and Wnt signaling pathway. And the number of somite are usually a good indication to estimate the age of an embryo. As you can see here, for example, 28 somites embryo is approximately 28 days of age for this developming embryo Slide 66: Somite Differentiation So the somite will differentiate into a number of components (4): Sclerotome, Myotome, Dermatome, and Syndetome. When the somites are formed, when this aggregation of cell separate from the prisomitic mesoderm, those cells are not committed to a specific

ineage. As the somite mature, in the most anterior part of the embryo where the first somites tend to appear. Those cells will get progressing acquire specific fate as the embryo gets older. Slide 67: So this is the appearance of somites at different time point. It appear as an aggregation of cells without any specific commitment to any specific lineage and then those cellsthis structure will be remodeled very extensively to give rise to different domain (Dermamyotome and Sclerotome). The Dermamyotome is usually more dorsal than the Sclerotome. A litte bit later, the dermamyotome will be separated into dermatome and myotome. The dermatone being more localized in that central aspect of the somite. And then evemtually the dermamyotome and myotome start to be visible more clearly here rather than if the dermatone and here the more ?? ? region of the somite will differentiate to be the Sclerotome. Slide 68: So to summarize the most ventral aspect of somite, once it starts to differentiate, is the Sclerotome which will migrate away from this region of somite and go around the notochord to form the cartilage vertebrae. This region here of the Dermamyotome is call primaxial dermamyotome. Its closer to the neural tube, will give rise to the muscles of the back. The abaxial dermamyotome away from the axis of neural tube. The abaxial dermamyotome will give rise to the muscle of the body wall, limbs, and tongue. Finally this region here in the middle, Dermatome will give rise to the dermis itself. Slide 69: So the 4th segment that I mentioned which is the Syndetome which is the portion of the somites that will give rise to tendon form at the junction here between the Myotome and the Sclerotome. So this is the Syndetome which is the compartment of the somite to give rise to tendon progenitors. Slide 70 So what determines cell fate within the somite. Its all about the location and where the cell type is located with regards to surrounding tissue. So essentially an inductive process. You have these group of cells that are not committed to anything and based on their location with regards to the neural tube, overlying ectoderm, lateral plate mesoderm, the notochordthey will receive different sets of signal that will dictate their fate in the adult. Slide 71 And thats what I was trying to show on this diagram here. The spinal cord, the notochord hereand here you have the somite which has not yet differentiated with the different region here closer to the axis of the neural tube, theres the primaxial muscle and abaxial muscle. Here the dermis in the middle and the Sclerotome here. So it all makes sense when you look at the position of those different tissues with regards to the inducer. So the notochord for example which is located closer to the ventral aspect of the somite will secrete a factor sonic hedgehog whichwill dictate the formationturn on a number of

transcription factor such as Pax1here those transcription factor are not for you to remember but location mostly. Will turn on Pax1 and turn this group of cell into Sclerotome. So cartilage that will surround the notochord. Another set of signal is coming from the lateral plate mesoderm which is close proximity to the abaxial muscle and that will turn on genes like Pax3 and MyoD and convert those cells to abaxial muscles. Together, there is also signals coming from the epidermis, Wnt specifically which in combination with those two factors will be essential for that process. Regarding the primaxial muscle and the dermis, signals coming from the dorsal aspect of the neural tube are essential (NT3, Wnt1, Wnt3a). The general organization of neural tube, location of notochord, lateral plate mesoderm, ectodermit all makes sense in that respect Slide 72: Derivatives of the Endoderm So thats the deeper of the 3 layer. It will form the digestive tract and respiratory tract, tonsils, thyroid, parathyroid, thymus, liver, pancreas and gallbladder. All the glands that are organs associated with digestive tract are also derived in part the endoderm Slide 73: So the general concept about the development of the organs associated with the gut, endoderm will form the lining or epithelium and the secondary element of the digestive tubes and glands. The mesoderm which is the mesenchyme that surround the tubesurround the tubethink about this gut tube that is surrounded by this mesoderm will be forming the connective tissue and all the smooth muscle essential for peristalsis. The gut associated organs are generated for reciprocal interaction between the endoderm and the surrounding mesoderm and mesenchyme. Well show you some examples regarding liver and pancreas Slide 74: Formation of the gut tube Two process that takes place around this time where thegut is being formed, there is a process of lateral flexionwe already talked about that which is essentially the curvature of the amniotic cavity that will come towards the ventral aspects of the embryo to separate the gut tube from the yolk sac. Concomittant to that there is also longitudinal flexion, which is a flexion of the embryo in the anterior and posterior end that come close together and define more specifically the digestive tract. So the two process are important for the formation of the embryo. The lateral flexion to separate the gut from yolk sac. The longitudinal flexion to bring back together the anterior and posterior ends of the embryo. Slide 75: Subdivisions of the gut tube There are essentially 4 subdivisions (pharyngeal gut, foregut, midgut, hindgut). They all give rise to different structure Slide 76: Regionalization of the gut tube So the pharyngeal gut will give rise to the lung bud which is not part of the digestive system but the respiratory system. The respiratory system is a derivative of the pharyngeal gut. So that will give rise to the lung buds which are the precursor for the lungs and give rise to the pharyngeal pouches which will be essential for forming the pharyngeal glands which is the thyroid, parathyroid and the thymus. Then we have the

foregut that will give rise to the stomach and muscle/glands that are associated with digestive tract, pancreas, liver and gallbladder. The midgut will give rise to the primary intestinal loop. Finally the hindgut will give rise to the cloaca and urinary bladder Slide 77: Regionalization of the gut tube Regionalizationso how is this gut regionalize in such a way. There is believe there are a number of transcription factors that are regionally expressed in the endoderm that will specify those different regions. And those different set of transcription factors that are shown here in color coded. Do not remember those transcription factorthats not the purpose here. But know that this is response to gradient of retinoic acid that is coming from the posterior end to anterior end of the embryo that will essentially the intiate the expression of the different transcription factors at different level and determine the fate of the different region of the gut Slide 78: Regionalization of the gut tube In the more posterior region of the embryo, this is an exampleyouve heard of Hox genes which provide the position the entity along the anterior posterior axiswe know that in the most posterior region of the gutsonic hedgehog (SHH) which is expressed in the endoderm will provide signals in the surrounding mesenchymewhich I was talking about this reciprocal interaction between endoderm and surrounding mesochyme/mesoderm. SHH is expressed throughout endoderm will differentially activate different sets of Hox genes in different regions of the posterior part of the digestive tract. And in such a way allow for the initiation of different segment of the digestive tract. So the number of the Hox genes are not important but you heard about Hox genes and they will always do essentially the same functionestablish identity along the AP axis. So the name is Hox is not familiar, its important in that respect Slide 79: Development of the Liver So a few words about the development of some of the organs associated with the digestive tract. The liver develop from a diverticulum of the foregut which is known as the hepatic diverticulum and as this diverticulum extends out from the foregut into the surrounding tissue which will be mesoderm/mesenchyme that is surrounding them from the lateral plate mesoderm. This mesenchyme that is surrounding this diverticulum will initiate the proliferation of the endoderm so that this duct will branch out and eventually develop the glandular epithelium of the liver. So this is the diverticulum here and this is a little time where the liver expands and very rapidly the liver will occupy a large portion of the body cavity. Slide 80: Molecular regulation of liver induction Molecularly, we know how the liver is specified. This is the schematic representation here of an embryo that went through this longitudinal flexingthe head is here and posterior end hereand here were looking at the heart region. Were looking here at the ectodermwere looking here at the notochord and in yellow here is the endoderm in the anterior region. This is the heart region which is derived from the mesoderm. So whats happening and how do we know how this liver is forming only in that location in response to an inhibitory and stimulatory signals coming from the ??? region of the

embryo. So the endodermtypically if you remove a piece of endoderm from the embryo and put it in culture in vitro, it will spontaneously form the liver diverticulum. So the entire endoderm has the ability to form a liver however, there are signals coming from the ectoderm and notochord that block this ability of the endoderm to form liver in any of those regions. The liver will form only in this region in the hepatic field and that is happening in response to stimulatory signals that are coming from the cardiac mesoderm that is right underneath this endoderm region here and signals like FGF2 and BMP will be essential in promoting formation of liver in this region and only in this region. As you know, liver is one of the rare organs that have the ability to regenerate spontaneously when you cut it. To regenerate from other region of the endodermthats another story.you have this strong inhibition coming from those tissuesin that region not only are signals stimulating the formation of hepatic tissue but they have to also remove this repression coming from this endoderm which is probably coming from the SHH signal Slide 81: Development of the Pancreas The pancreas is an outgrowth of the endoderm muscle that is immediately past the stomach and start to be visible around the 4th week. There is two domains of the pancreas (dorsal and ventral) that will eventually come together and typically the secretion of insulin will began around the 5th month of gestation. In contrast to the liver, the notochord promotes pancreatic development by repressing SHH expression in the endoderm. So its a little bit the opposite from what we discuss regarding the liver. Here the notochord repress ability of endoderm to form liver. Here the notochord has a positive signals on this specific region of this endoderm to generate the pancreas Slide 82: Physiological Herniation An important process that takes place during embryogenesis that is associated with development of the gut is what is referred to as physiological herniation. Essentially, the midgut and the loop of the gut (intestinal loop) will enter through the umbilical cord around the 6th week of gestation. So there is a lot going on in the development of the organs in the body cavity around that time. The embryo cannot make up for this increase in size of all these organs so one way the embryo compromises for that is to push the intestinal loop into the umbilical cord to make more space in the body cavity to allow the organs to grow. So this is a temporary thingthe intestinal loop moves into the umbilical cord and around 10th-12th week of gestationprogressively this loop will retracted back into the body cavity. Slide 83: Development of the Respiratory System So the respiratory system is developing as part of the respiratory diverticulum which will eventually branch out to form the lung bud and that will start to form around 4th week of gestation. Im not gonna go into much detail about that because Im gonna give you a lecture in OS about the respiratory system and I will go more in detail about this process. Slide 84: But know the respiratory diverticulum is an outpouching of the foregut that will branch out to give the lung buds.

Slide 85: Stages of Pulmonary Development The lung buds will eventually differentiate into a different phasepseudoglandularcanalicularsaccularalveolar which are referring to the shape of the branching of the respiratory bronchioles and bronchi and the whole process takes a long timeas you can see 40 weeks. So its one of the organ system that is matures later in the embryo along with the nervous system Slide 86: 4 week human embryo So this is human embryo in the 4th week. Looking at the head region, you see the branchial arches here in which the neural crest will migrate. See the heart occupy a very important position herevery predominantyou start to see the beginning of the eyeyoull see all the somites here that are forming along this region. Counting the number of somite you can approximately determine the age of this embryo Slide 87: 5 week human embryo So the embryo already went through this flexion. You have development of the limb that starts to be clearly visible. The heart and the liver again occupy a very high, important predominant position in the body cavity. Slide 88: 6 week human embryo This is when the intestinal loop starts to move into the umbilical cord which appears a little larger as a result. The limb continue to grow Slide 89: 8 week human embryo Few weeks later, we see the predominance of the size of head compared to the rest of the body which is a very important characteristic of the developing embryo during those 8 weeks where the head tend to take a very high volume as compared to the rest of the body General Embryology III Slide 2: Growth of the organ is faster than growth of embryo itself so once there is a reasonable ratio between organ and size of embryo then the loop can come back into the body cavity. So here were gonna talk about what happens in the next 8weeks after that. Very briefly, talk about the fetal membrane and placenta. Talked about birth defects and prenatal diagnosis Slide 3: Timeline of Human Development Were gonna cover essentially whats referred to as the fetal period. So youve seen most of the organs are put in place already during this 8 week period. After that, its just a matter of expanding the different organ systems. Slide 4: Development of the Fetus As I was mentioning in the last slide is that this fetal period at the end of the embryonic period, the embryo has decide essentially where the size of the head is half the size of the entire embryo itself. So the fetal period will be characterized by the maturation of the

tissues and the organs that have already been put in place during embryonic period. There will be a rapid growth in the body length and weight. The growth of the head will start to slow down in such a way that progressively after 5 months birth, the ratio of head versus the rest of the body is 1:3 going to 1:4 essentially. Slide 5: 11 week human fetus Some of the most remarkable process that take place during that fetal period, in which the embryo is 5-7 cm in length. There is a swelling of the umbilical cord that is still visible here in this case. Remember this loop that is emanating through the umbilical cord. The digits are not fully formed but already well developed in the forelimb and hindlimb. The skull is relatively irregular so there is still a lot of remodeling and growth taking place. So you dont want the suture of the skull to be established too early so that there is time for growth and expansion. Interestingly, the eyes at this stage here close to the midlineventrally located but they are really close to the midline and they will move more laterally later Slide 6: 12 week human fetus The embryo is 8cm roughly. Start to see the primary ossification center that are established in the long bones of the limb. The external genitalia are visible. The loop has been completely retracted from the umbilical cord and thats when we first start to see signs of muscular activity. Slide 7: 18 week human fetus The outer ear are in their final position which are really lateral as they should be. The limbs continue to grow with the digits more and more formed. You start to see the first hair that start to appear. This is a very smooth appearance of the top of the head as compared to what weve seen earlier where it was more irregular in shape. Slide 8: 28 weeks human fetus Well rounded contour. Most organ systems are functional except the respiratory and nervous system that are the last ones to fully mature. And birth around the 7th month, 90% of survival chancepretty high. Most of the major systems are in place at that time Slide 9: Fetal Membrane & Placenta So the placenta is the primary site for exchange of nutrients and gas between the mother and the fetus. Also the role of protection, nutrition, respiration, excretion and hormone production. Two components to the placenta which is referred to as fetomaternal organfetal component and maternal component. Fetal component known as the chorion is derived from the trophoblast and the extraembryonic mesoderm. Trophoblasts are the cells that segregate from the inner cell mass that are invading the uterine wall during implantation. The maternal component is derived from the uterine endometrium which is the location where the embryo implants. As the fetus grows, there is an increase of needs in nutrients for the fetus and this will be facilitated by increasing the exchange surface between the trophoblast from the fetus and the endometrium from the mother. There will be formation of villi that increase the surface contact between the two.

Slide 10: So this is some examples going back to a little bit earlier this morning. We are looking at the embryo at 13 days after implantation. I indicated the trophoblasts make the two regions (syncytiotrophoblast and cytotrophoblast) that start to make those villi into the endometrium from the uterine wall. And thats where here you find the extraembryonic mesoderm here in pink that will follow those villi. You go from primary villi where it is essentially syncytiotrophoblast and cytotrophoblast to a secondary villi where there is in position of mesoderm from the extraembryonic mesoderm between the syncytiotrophoblast and cytotrophoblasts. So if you make a section at this level, you see mesoderm, cytotrophoblast, and syncytiotrophoblast. The tertiary villi which is an example hereyou start to see those capillary that form in those villi that will essential for establishing contact between the two Slide 11: Beginning 2nd month This is an embryo around 2 months of gestation. You see here the artery from the mother and this is all the villi that is emitted by syncytiotrophoblast and cytotrophoblast. You can see initially that those villi appearing throughout the entire circumference of the chorionic cavity. However, they will only be maintained at one pole which is called the embryonic pole where essentially the embryo is directly attached. This is referred to as the embryonic pole and this is ab-embryonic pole (opposite to the embryo). But note there is initially villi that are forming throughout the periphery of the implanted embryo and they will be preferentially maintained in this region at the embryonic pole Slide 12: So this is just a diagram here to show the blood coming from the fetus here that makes those vessels that are actually form in the extraembryonic mesoderm that are establishing contact with the blood from the mother and there will be a passive transfer of nutrients between the two in this setting Slide 13: Relation of fetal membranes to wall of the uterus So I mentioned that initially the villi are forming until the entire periphery of the implanted embryo however, there will eventually only maintained in this region where the embryo is located. So there is 3 components to the uterine wall and endometrium. There is the deciduas basalis here which will be discarded at birth. There is decidua parietalis which is the other component here and then the deciduas capsularis which is this component in which you have initially those villi but eventually those villi at the abembryonic pole are eventually lost. Decidua capsularis will eventually disappear completely. As the embryo grows, the chorion will come in close contact with the growing amniotic cavity and the decidua parietalis and the decidua capsularis will be completely lost. So the placenta is really formation between chorion frondosum, which is the region at the embryonic pole where you have those extensive villi that are forming into the decidua basalis. Here those two regions come in close juxtaposition. So the placenta is really the juxtaposition between chorion frondosum and decidua basilis of the endometrium

Slide 14: Birth Defects Few words about birth defect. They are sometime referred to as congenital malformations or congenital anomaly. They can be structural, behavioral, functionalthey can be metabolic disorders that are discovered at birth. Birth defects are the leading cause of infant mortality. The major anomalies occur in 3% of live-born infants. Major anomalies referring to anomaly that are impairing major organ system. Mino anomalies occur in 15% of newborns. Those are that not life-threatening anomalies (e.g. extra digits). Genetic factors account for approximately 28% of birth defects while environmental factors create 3-4% of birth defects. And vast majority of birth defects are multifactorial inheritance that can be genetic or environmental or a combination of both. A vast majority of birth defects are largely unknown Slide 15: Risk of Birth being Induced So when you look at risk of birth defect which is visualized here. The increase of risk over time. The highest risk of birth defect occur during embryonic period, which makes sensethis is the period where everything is taking placeall the organogenesis taking place. Slide 16: Types of Anomalities Types of anomalities can be disruptions, deformations, and syndrome which is I show you some syndrome alreadylike DiGeorge syndrome which is a combination of anomalies that are occurring together and that have a common cause. Slide 17: Environmental Factors The role of environmental factors has been really considered as an important factor in birth defects only relative recently. May seem like a long time 1941, but 1941 and 1961 where the first true report showing that there was a correlation between birth defect and the use of some agent. In the case of thalidomide, which was a sedative that was given to mother during pregnancy in the 1950sthat had very dramatic effect on the development of the limbs. Took a long time to establish this link between the sedative and the formation of those defect limbs which was very dramatic most of the time and essentially the attachment of hand at the level of the shoulder without any other component of the limb. The key report that really established a link between environmental factor and birth defect. Slide18 & 19: So I put out this paper that is from 2010 that look at the higher incidence of birth defects in the US for a period between 2004 and 2006 that show a list of the birth defect that have a higher incidence. So those are some of the birth defects listed here. Just wanna point out a few. The orofacial defect like cleft palate or cleft lip have a relatively high incidents. Craniofacial defects are a very predominant type of birth defect. Another one is heart defect. So the incidence of heart defects are relatively high. The prevalence here is 16 and when you add up all that, its close to that point also12 or 13 maybe. And those can beyou heard of Tetralogy of Fallot.ventricular septal defect is another very important and common birth defect. Another onethe incidence is not as high but those are associated with defects in the digestive system. So the CNS defectone of the most

common is spina bifida like I indicated which can have different relation that can be life threatening or barely noticeable. There is not as high of occurrence as the craniofacial or heart defects. Then there is some chromosome abnormalities for example here highlighted trisomy 21 or Down syndrome which have relatively high frequency also. Slide 20: Here are some examples of defects. I mentioned this problem with the sedative thalidomide which was resulting when the mother took this sedative during preganancy would lead to very high incidence of newborns that were missing essentially the different components of limb and end up with the most extreme case the hand attached to the shoulder. Cleft lip quite common. Turner syndrome, its a chromosome abnormality where you lack one X chromosome. Treacher Collins Syndrome which I mentioned is a neurocristopathy defect in neural crest. Very extreme case of spina bifida where the spinal cord is dramatically exposed. Tetralogy of fallot which shows the hypertrophy of right ventricle, ventricular septal defect, overriding aorta, and pulmonary stenosis. For cleft palate, there is genetic evidence that some genes that are very essential for the palate to close but there is also if your tongue during development you will see thatif the tongue doesnt come down the palatal shelf that are closing wont be able to close and that could be an environmental factor such as the tongue doesnt come down or remains swollen for an extended period of time. So it can be a combination of all those factors. Some we know, some we dont. Spina bifida can be caused by many different things (genetic factor, environmental factor) Slide 21: Prenatal Diagnosis To finish, just a few words about prenatal diagnosis. So there is a number of procedure that are typically used that you are probably familiar with. So ultrasonogram, which is a non-invasive technique that use high-frequency sound waves which is reflected on the tissue to create images. Some of these images are here. So they can be trans-abdominal like in this casetransvaginal which offer higher image resolution typically and they are usually performed around 18 or 20 weeks. You can assess overgrowth and development and look for birth defect also. This is a sonogram showing you this feetone handthe headIm not sure what that is. Slide 22: Maternal Serum Screening There is a Maternal Serum Screening which is commonly used also to look for concentration of serum alpha-fetoprotein (AFP) which is an indicator ofso if you have abnormally elevated level of AFP that can lead to neural tube defect such as spina bifida. So AFP is normally produced by fetal liver and peaks at 14 weeks, and pass into the maternal circulation via placenta. The AFP concentration increase in the serum during the second trimester and then it goes through a steadily decrease after the 30th week of gestation. So if there is a maintained high level of AFP that could be an indication of potential birth defect. And there is a whole range of serum molecules that can tested in relation to pregnancy. Slide 23: Aminocentesis

Aminocentesis is another one that is commonly used also which consist of removing amniotic fluid from the amniotic cavity. Typically you need between 20 and 30ml of fluid to be able to do an appropriate test which means the procedure cannot be performed before 15-20 weeks when the amniotic cavity has grown enough that you have enough of this fluid to collect. In the fluid, you can look for factors such as AFP and fetal cells that are sometime present there. And you can use those cells to do karyotyping essentially to look for abnormalities in chromosomes. The way you can find defects such as Trisomy 21 or Down syndrome. Also you can perform PCR to look for specific genetic disease. The problem here is that you start with very small amounts of cells from this amniotic fluid so that you have to expand those cells in culture for a few weeks. So you wont be able to get a result as fast as other technique such as Slide 24: Chorionic Villus Sampling which is essentially very similar to the technique I just described but in this case you dont collect amniotic fluidyou directly collect some of the villus tissue that is found in the placenta. So you as a result end up with a larger amount of tissue and you can do all kinds of genetic testing on those cells. So you have large amount of cells you dont need to expand for a period of time and you can have the results very fast in a few days by extracting the DNA and doing PCR for specific genetic markers that can indicate a specific disease.