Developmental Biology - Essay Example

The Annelid is a triplobalstic protostome with characteristic metamerisation (segmentation) that is found across the whole body except for the procephalon (head) and the 'foot'. Each of these segments is a repeated unit containing the peripheral neurons, the segmental ganglions, excretory, locomotory and respiratory organs. The only organ system not repeated is the digestive system. During development of the embryo, a spiral holoblastic cleavage pattern is seen. In the first round of cleavage 4 blastomeres are produced called A, B, C, and D. During the second round of cleavage unequal division takes place which produces two types of blastomeres, the larger ones are called macromeres while the smaller ones are called micromeres. After certain number of divisions the macromeres give rise to a pair of blastomeres termed as teloblasts M, N, Q and O/P. It is from these teloblasts that the segments of the annelid are derived in the adult. To draw our fate map we used alkaline phosphatase to help in tracing out the cell lineage of many of the adult structures. Alkaline phosphatase being naturally present in a cell and was an ideal candidate as a marker to trace out the fate map. The blastomeres were injected with high concentrations of alkaline phosphatase and then allowed to develop into their corresponding adult structures. The annelid was then bathed with a colourless substrate which coloured on reacting with alkaline phosphatase. Using this we were able to show that the blastomere A, B & C most probably develops into the ectoderm. The blastomere D though proved to be the most vital with most of the endoderm and mesoderm structures being derived from it. We also show the individual fates of each of the teloblasts and by alternating the time of injecting alkaline phosphatase we were able to deduce many vital facts about metamerisation. From our study we came to the conclusion that most probably the segments are produced from the posterior to the anterior direction and that segmentation begins at a very early stage of asymmetric cell division of the teloblasts. Introduction: The phylum Annelid consists of many species that range from the humble earthworm all the way to the maligned leech, but one common feature that binds them all is their segmented body. This segmentation is termed as Metamerism and each ring like segment is called a metamere. In fact the word Annelid is derived from the Latin word Annelis meaning rings. The Annelid body can be further bisected into two equal halves implying that the body architecture is bilaterally symmetrical. Annelids are also triplobalstic i.e. the embryo has 3 distinct germ layers namely the mesoderm, endoderm and ectoderm. It is from these 3 distinct germ layers that all the organ systems arise. The ectoderm that forms the outer layer of the embryo produces the epidermis and the nervous system, the endoderm which forms the inner most layer of the embryo gives rise to the digestive system and its related organs and sandwiched in between the two layers is the mesoderm which produces the circulatory system, muscles and connective tissue. As the yolk in the Annelid embryo is equally spaced out a Holoblastic cleavage pattern is seen and thus the cleavage furrow extends through the entire egg. The cleavage pattern seen in Annelids is a Spiral Cleavage which is also seen in other organisms such as sea urchins. During the first of cleavage, the embryo is divided into 4 cells each of which is called a blastomere and are labelled as A, B, C and D. All the blastomeres are of equal size except for Blastomere D which is slightly larger than the rest. The second round of cleavage produces an unequal division of 4 large blastomeres called Macromeres and 4 smaller blastomeres called Micromeres. The macromeres are labelled as A1, B1, C1, and D1 and the micromeres are labelled as a, b, c, and d. The macromeres keep on dividing to produce more macromeres and micromeres and eventually the entire organism is formed. Of special note is the D1 blastomere which further divides to form blastomeres DM and DNOPQ. Blastomere DM divides to form blastomeres M1 and M2 while the blastomere DNOPQ divides to form two blastomeres NOPQ1 and NOPQ2. Each blastomere NOPQ divides to from blastomere N and OPQ and then each blastomere OPQ divides to form blastomere OP and Q. The blastomere OP divides to form two O/P blastomeres. The final result of these cleavages is two sets of five large blastomeres, designated M1 & M2, N, O/P and Q. These blastomeres are termed as teloblasts. Once the teloblasts are formed, each cell initiates a series of unequal divisions that produces a column of small cells called a germinal bandlet. These columns merge to finally form the germinal band that will give rise to segmented part of the embryo. As a developmental biologist we would like to be able to study all the paths that an organism takes to develop from a single cell to a whole organism. Questions such as does the location of the cell matter during development in deciding its fate Or is its location not important at all and its more about the genealogical line of descent from the egg Or is it in fact really a little bit of both To be able to answer these questions developmental biologists came up with a new field of embryology where they would trace the cell lineage i.e. follow individual cells to see what they develop into. By collating all these cell lineages of different cells during different time points in the development of an organism we can create a fate map. A Fate Map basically tells us from which part of the embryo the adult structure arose. To create a fate map we first need to track cells during its development which is easy if the cells were naturally pigmented as is the case of Tunicates, but not all organisms are so accommodating thus the need for markers to help in the mapping. Earlier embryologists used vital dyes, dyes that would stain cells but would not kill them. Now many more different types of markers have been created such as radioactive labels, fluorescent dyes, and genetic markers where an inheritable mutation is induced in the cell and its daughter cells traced on the basis of the mutation. Whichever marker is used though it has to fulfil the following minimum criteria, i.e. it should be stable, it should in no way influence the outcome of the experiment, it should be easily detectable and lastly but most importantly it should be hereditable. To create a fate map a single blastomere or a group of blastomeres are marked at different stages of embryonic development. The blastomeres are then allowed to develop normally and the adult or larvae is studied to see what adult/larval structures they finally develop into. Then another blastomere or a group of blastomeres is chosen and put through a similar process. This process is repeated until all the blastomeres fates have been mapped out and we can finally trace out the entire lineage of the organism and draw its fate map. Along with giving us information about cell lineage this technique also provides developmental biologists with other useful information such as the whether a single blastomere is responsible for a particular structure or are there multiple blastomeres involved in the development process. Other useful information such as whether a particular blastomere will always give rise to the same adult structures in every embryo (lineage restricted) or will they give rise to similar adult structures thus most probably being influenced by events which occur further on in the development process. In our experiment we used Alkaline Phosphatase as a marker to map out the cell lineage of the Annelid. Alkaline phosphatase is a naturally occurring enzyme in the cell which dephoshphorylates proteins. Since it is naturally occurring in the cell we can use this as marker. By injecting high concentrations of alkaline phosphatase we can track the structures the blastomere forms as each dividing cell will also inherit the alkaline phosphatase. At the end of development the embryos were immersed in a colourless substrate which reacts with the alkaline phosphatase to give rise to a coloured precipitate which can be easily visualised under a light microscope. Any structure that is coloured is most probably derived from the cell that was injected with alkaline phosphate earlier, thereby concluding its lineage. The experiments were preformed in 3 stages. In the first stage we wanted to ascertain broadly which of the four blastomeres is responsible for which of the germ layers or were they all playing an equal role. After this we wanted to determine what the fates of the teloblasts were and finally in the third set we injected the teloblasts immediately after asymmetrical division and after a time delay so as to ascertain where and when does segmentation begin. Results: Fates of the blastomeres A, B & C: On injecting any of the 3 blastomeres, the endoderm derived structures such as the gut, mouth and anus were partially stained thus all the endoderm structures are most probably derived from these 3 blastomeres. Of the ectoderm derived structures the sub oesophogeal was partially stained and so was the epidermis of the procephalon and the 'foot'. None of the mesoderm derived structures were stained thereby indicating that these blastomeres are most probably not involved in their development. Fate of the blastomere D: On injecting blastomere D, of the ectoderm derived structures the epidermis was completely stained except for the procephalon and the 'foot' region i.e. the segmental regions only. The segmental ganglia and the peripheral neurons were also completely stained. Of the mesoderm derived structures the muscles was completely stained. None of the endoderm derived structures were stained thereby implying that this blastomere is most probably not involved in development of this germ layer. Fates of the macromeres A1, B1 & C1: On injecting any of the 3 macromeres the endoderm derived structures such as the gut, mouth and anus were partially stained implying that these macromeres most probably develop into all the endoderm structures. Fate of the macromere D1: On injecting the macromere D1 we get the same results as was for injecting blastomere D. Fates of the micromeres a, b, & c: Though we didn't actually inject any of the micromeres to study their fates we can still predict what their fates would be by basically 'subtracting' the results of the fates of the macromeres A1, B1, & C1 from the blastomeres A, B, & C. From this we can say that the micromeres most probably develop into the ectoderm derived structures the sub oesophogeal and the epidermis of the procephalon and the 'foot'. Fate of micromere d: By 'subtracting' the results of the macromere D1 from the blastomere D we notice that the fates of the two are the same and hence we can't conclude the fate of the micromere d. Fate of blastomere DM: On injecting the blastomere DM all the muscles were found to be stained implying that the blastomere DM most probably develops into all the muscles of the annelid. Fate of blastomere DNOPQ: On injecting the blastomere DNOPQ all the segmental ganglia, the peripheral neurons and the epidermis of the segments were stained implying that the blastomere most probably develops into these structures. Fates of blastomere NOPQ1/NOPQ2: On injecting either the blastomeres NOPQ1/NOPQ2 half the segmental ganglia, the peripheral neurons and the epidermis of the segments were stained implying that the blastomere most probably develops into these structures. Fate of teloblast M1/M2: On injecting either the teloblast M1 or M2 half the muscles of the annelid were stained implying that these blastomeres most probably develop into these structures. Fate of teloblast N On injecting either teloblasts N half the segmental ganglia and the peripheral neurons were stained implying that these blastomeres most probably develop into these structures. Fate of teloblast OPQ On injecting either of the teloblasts OPQ half the peripheral neurons and the half the epidermis of the segments were stained implying that these blastomeres most probably develop into these structures. Fate of teloblast Q On injecting either of the teloblasts Q half the segments epidermis were stained but even these were not completely stained implying that the teloblasts Q alone are not responsible for the development of the epidermis in these regions. Fate of teloblast O/P By 'subtracting' the results of the teloblast Q from the teloblast OPQ we can conclude that these teloblasts most probably develop into segmental ganglion and may also play a role in developing into the epidermis of the segments. A point to note is that on staining either one of the teloblasts led to half their corresponding structures being stained. This leads us to believe that most probably the bilaterally symmetry of the organism is probably laid out after the cleavage of the blastomeres DM and DNOPQ since each of the teloblasts staining results showed in half the annelid and not completely as was seen till blastomere DM and DNOPQ. For the final set of experiments that we preformed we wanted to see when and where segmentation of the annelid begins. In order to visualise this we injected the teloblast after the initiation of the asymmetric cell divisions that give rise to the germinal bandlets. As it is impossible to stage the embryos to the resolution of a particular division, the injections were done either 'early' i.e. shortly after the initiation of asymmetric cell division or then 'late' i.e. after the asymmetric cell divisions was well underway. From this we noted that all the 'early' injections almost 2/3 of the corresponding annelid structures were stained starting from the posterior end. Similarly for all the 'late' injections only 1/3 of the corresponding annelid structures were stained but once again starting from the posterior end of the organism. In both cases the segmentation had not reached the procephalon region which probably means that the segmentation of the annelid most probably begins from the posterior end of the annelid. Thus the 'foot' region would have the youngest segment while the segment near the procephalon would most probably be the oldest. Also when we consider the results from the 'early' injections we notice that the cells were stained 2/3 of the way while those from the 'late' injections covered just 1/3 of the organism. This could probably indicate that very few cells are required for the formation of the germinal bandlets and hence for the formation of the segments as the 'early' phase would mean few divisions and hence fewer cells. Discussion: From our studies we were able to deduce how the development proceeds in the annelid. After the first cleavage division 4 blastomeres were produced. Blastomeres A, B & C are essential in the development of the ectoderm. The blastomere D however undergoes further rounds of cleavage to eventually produce the teloblasts which are essential in the development of the segments of the annelid and all the ectoderm and mesoderm derived structures that are associated with them. The 'early' and 'late' injection experiments helped in elucidating the when and where of segmentation. All the 'early' and 'late' injections showed staining from the posterior end of the annelid moving to the anterior end. In both cases the segmentation had not reached the procephalon region. Thus segmentation begins from the posterior region of the annelid. Another point to note was that the 'early' injections showed more staining as compared to the 'late' injections indicating that most probably very few cells are required for the formation of the germinal bandlets. On comparison of the metamerisation pattern of Drosophila with that of the Annelid we notice many similarities. Segmentation in Drosophila is based on the gradients of morphogens present within the syncitium. Through the formation of gradients a segment is formed and also depending on the gradient the segments fate is decided i.e. whether the segment should form a head region or a tail region or neither. This mechanism could probably also explain the formation of segments in the annelid but it would have to be a simpler one because all the segments in the annelid are the same and the only discerning regions are the procephalon and the 'foot' region. The probable mechanism is that at either of the two extreme ends of the annelid are present high concentration of the morhpogen and in between their concentrations are less. Only in the regions of high concentration morphogens would a head or tail region form while the rest of the segments would remain the same as the concentrations would not be enough to induce head or tail formation. Read More