in this video we'll be talking about the tetrapod limb development this is a complicated topic to understand but we would make it simple for you stay tuned till the end of this video and this concept would be nailed down so what are tetrapods tetrapods are those animals which has four limbs like mammals birds reptiles Etc now be it the hand of a human leg of a cat uh flipper of a well or the wing of a bat all of that has a basic architecture which is defined by stylopod Zod and autopod stylopod is basically the femur and the humorus the zeugopod comprises the fivia tibia and the radius and alna the autopod comprises the tels and carpal and all these feses so now we understand that there is an intricate architecture in the tetrapod 4 Lim and this beautiful architecture is developmentally sculpted but that's a difficult job to do it right if you can imagine how you can make this kind of intricate architecture can you can you uh conceptualize how you would make it in the development it's hard to understand right but in development it is nicely sculpted but it all starts with the simple structure so we have to understand the embryonic Landmark a little bit let's try to do that so this is the fenial mesoderm this is the anterior lateral plate mesoderm this is the posterior lateral plate mesoderm and just opposite to the posterior plate mesoderm there is a field there is a zone of tissue which is defined as four limb field it is not limb yet but it is destined to be limb in the future so this for liim field which rather looks very simple would eventually create a complicated structure or ecture like this and this is a long journey question is how this kind of complex transformation really happens from the set point to the end point it's a huge Journey so we can clearly understand this field which was defined as a limb field which eventually bulge out form a limb bud and that Lim bird extends grow creates bones muscles Etc underneath to ultimately form the Ford limp isn't it a long and beautiful Journey we would try to appreciate that now limp development is happening along different developmental axis so that is why first we should start understanding the axis to get a better sense of the development here is a proximal to distal axis proximal means that is anything close close to the body and distal means anything away from the body here is the dorsal to ventral axis and here is posterior and anterior axxis dorsal Al means anything which is basically at the back and the belly side is basically ventral so you can see your pump is basically ventral and the outside of your pump is basically dorsal now just like a developed hand or developed leg has its own architecture and developmental axis you would have the similar kind of axis in that precursor of the limb that means the limb bird limbird has its own anatomical and developmental axis so limb bird is the hero of this story because this is the first visible sign of limb development now moral of the story lymph bird would eventually be patterned to create that extremely complicated structure now we have to understand why and how so there are sequential processes first there would be induction of the lymph field then eventually there would be outgrowth of the Lim bird and then the cellet specific ification of limb but would eventually lead to specific regions of the tetrapod limb so now we can clearly appreciate this sequential process now if we zoom in and cut a cross-section of this embryo we can see that there are different zones you have to understand that this Bud would eventually grow Whenever there is a growth you have to understand two mechanisms can operate one is extensive proliferate ation and migration of nearby cells to that region it turns out that underneath the limb bird migration of cells happens from hypaxial myoto bud and lateral myoto lateral plate mesoderm so these Limb and muscle precursor cells populate underneath that limb bud so basic formula of making a hand is like you have to create a muscle you have to create bones eventually this basic archistructure has to be innervated by Blood vessels and nerve so these precursor cells has to be defined and these precursor cells are now coming just underneath the limb Bud now the question is once you have a lot of mesenchimal cells underneath this limb but how do you pattern them how does a mimal cell knows that it has to create a stylopod in future or zop pod in future or let's say autopod in future how does this decision is made and the answer is rather simple because in developmental biology everything happens by instructions from morphogens so is there any rule of course there is a rule question is who sets up this Rule and how this rule is followed up and what happens if this rule is abrogated so if we zoom in we can see the lymph but itself has defined anatomical regions there is some region known as zpa Zone of polarizing activity obviously the name suggests it has some sort of morphogen that polarize things there is Progressive Zone which progresses and grows so it has enough amount of stem cells and ultimately the epical ectodermal Ridge which is a ectodermal epex of the developing lymph butd so each of this region interacts with each other in synchrony and ultimately sculpt the development of the four limb now let us look at three axis of development to understand what morphogenetic actions are happening there so there is proximal to distal axis there is anterior to posterior axis and there is dorsal to ventral axis now in all these axis different morphogen acts in a synchronous pattern so in proximal to distal axis retinoic acid and fgf8 or fiberblast growth factor 8 acts in anterior to posterior axis there is SSH or Sonic Hedgehog gradient in basically dorsal to ventral axis there is wind and BMP gradient now each of these morphogen also interact among themselves these are not isolated events we break it down in an isolated fashion to understand it better but all this is happening in synchrony underlying the lymph development now let us get back to the basics remember how morphogen works there is a cell which would secrete the morphogen cells which are near the vicinity would get most of the morphogen which are away from the Source would get list of the morphogen and that kind of sets up the interpretation differences right so B basically there could be tissue specific competence dynamics of morphogen in terms of time signaling Dynamics and transcription Factor Network which help the cell to interpret the morphogen Dynamics and ultimately that lead to the different fate acquisition exactly the same mathematical formula applies in the tetrapod limb development in a moment we would understand how so first let's talk about the proximal to distal axis remember here retinoic acid and fibroblast growth factor opposing gradient operates in this region and based on the concentration threshold there would be differential gene expression different Hawk genes or homeobox genes would be expressed and each of them would be responsible to patter certain portion of the limp now this is not this simple there are complex network of uh different transcription factors Regulators in this entire Cascade for the Simplicity of this video we are skipping that in a later video we would try to delve into details okay now let's talk about the anterior now let's talk about the dorso ventral axis in the dorso ventral axis wind BMP acts wind triggers the expression of lmx1b it's a strong dorsalizing factor and lmx1b is selectively expressed in the dorsal side not in the ventral side so lmx1b is responsible or one of the key modulators which pattern the dorsal side and it these kind of understanding came from the mutant studies in the control mouse uh if you cut up cross-section across the Paw you can see this is the dorsal ventral axis there is a paw paw digits and these are the foot pad which are vental structures in lmx1 mutant mice the food Pat is duplic ated as if the dorsal structure become ventralized why is that because the strong determinant of the dorsal fate lmx1b was absent it was mutated this in turns tell us why lmx1b which is under under the influence of wind signaling is really important to pattern the dorsal side now let's talk about the digit identity specification SSH is a strong driving factor that determines this kind of specification two things matter time duration of SSH expression and concentration of the SSH protein that is experienced by a receiver cell so here you can see there are two cells which are destined to become digit 5 cell or digit four cell and two of these cell has different kinetics or different temporal dynamics of the SSH expression digit five cell Express SSH for longer duration compared to digit four cell and that sets up the difference of Auto signaling this leads to the specification of digit four and five so clearly there is a complex set of rules digit four and five get specified based on the time duration of expression digit three is specified based on the SSH time time of expression and also the concentration of SSH that is experienced from The Source digit two is solely specified based on the SSH concentration that is experienced from the posterior side and this digit one specification is completely SSH independent so now you can see how the heterogenity of the rule give rise to different digits in proper place now it's important to note that SSH has a very strong function and it all cames from the chick embryonic exp uh experiment a stage 19 to 23 chick embryo was used in this experiment where the Z pa uh was there in the posterior side and people reason that the SSH is actually the driving factor which is coming out of the zpa and polarize this kind of anterior posterior axis development so in order to prove that they took or culture some fibroblast which is secreting SSH and they implant it to the anterior side just opposing to the zpa and if this signaling Center is duplicated right now the entire body axis gets duplicated and and what they found to their surprise there is a mirror image in terms of the development the limbs exhibit a mirror image digit duplication phenotype so this internally this exactly says that when SSH is ectopically produced then the anterior margin of the limb bird becomes basically uh mirror imaged to the posterior margin so SSH is sufficient for action of the zpa this is the moral of the story now this is a glimpses of Limar development in human you can understand how prolonged and a long process this is it starts at week five and it kind of goes on till week 8 so it's a long process in space and time now let me tell you that underneath these morphogen gradient there are strong correlation of homeobox gene which pattern the skeleton identity now we already know from other developmental biology lectures that homeobox genes kind of Define body segments and these body segments in the hand are actually defined by a rule of homeobox gene expression scientists noticed that along these different region of the uh limb there are differential expression of homeobox genes for example Hawks 9 and 10 are very strongly expressed in the stylopod region Hawks 11 is expressed in the zugo Pod Hawks 12 and 13 is expressed in basically uh near the autopod region so already it looks like there is a communial code and the proof that a communial code exist come from mutant experiments so in a mouse mutant where Hawks 11 and Hawks D Hawks a11 and Hawks d11 are actually mutated in that case the scientist noticed that the zeugopod was totally absent this in turn tells us that there is a rule zeugopod requires Hawk 11 Hawk 11 was now mutated so the rule goes haare and that lead to a problem in the zapot development but look other regions are developed properly so now we can appreciate how intricate this rules are and how that sets in another example comes from humans in human there is basically a disease known as polya where the fingers of the hands fuse so this is known as poly inducti and when these people with poly inducti was sequenced it was found that Hawks the 13 Locus which is responsible for the autopods that was mutated now it tells us that depends on which type of Hawk genes are mutated and what part of the hawk code is jumbled up that lead to different kind of consequences and that that in turns tells us that the hawk code is important for limb development now we are kind of at the end of this video let me tell you this limb Evolution happened long back the fish didn't have any kind of like limb but So eventually there is a missing link between the fish and the amphibians this is basically tict talic this is a fish with wrist and finger even fishes has that so it was thought that when these fish was about to uh move out from the Aquatic environment to uh explore the territorial environment so they developed these kind of fingers and digits like thing which is quite similar to human uh or any kind of like tetrapod Limbs and eventually now in uh in basically tetrapods we have defined sets of uh our limbs right so the basically the decision was set 375 Millions ago and this is a missing link so I hope you enjoyed the video in this video summary I would like to emphasize on two points we understood that this process of limb butd development was quite complicated and this complicated process has to be done or executed by set of defined rules morphogen gradient sets up the rules in three developmental axis along the limb butd we looked at that in quite a lot of details and lastly we also see that there are specific Hawk Gene rules that defines different regions of the limb like Zod stylopod or autopod so basically we can appreciate how precise molecular rules Tunes the development of the Maman tetrapod vertebrate limb development so I hope this was useful if you like this video give it a quick thumbs up don't forget to like share and subscribe you can access our notes and flash cards from our website or from Facebook so please support us using super thanks or pay us via patreon see you in next video