Cellular Biology

1. Cellular Biology School of Life Sciences Shaanxi Normal University 1

2. CHAPTER 10 CELL DIFFERENTIATION AND REGULATION

3. I. Fertilization and embryogenesis The structures of sperm and ovum: Animal spermatocyte can be differentiated as four spermatids by meiosis, and the spermatid can be further differentiated as sperm by spermiogenesis. Matured sperm looks like a tadpole composed of head and tail. Two important structures are included in head: nucleus and acrosome. Acrosome is a big lysosome actually and contains many types of hydrolases inside. When an ovum is fertilized, the acrosome releases out a lot of acrosomal enzymes to digest ovum membrane to have sperm entered ovum. The sperm tail is also called as flagellum that is the moving structure for sperm. The tail can be described as 4 regions: neck, middle (mid-piece), trunk (tail), and terminal (end-piece). The neck is very short and contains two centrioles overlapped. The sperms of some animals have no tail.

4. A model fig of sperm

5. The section slide of sperm of some duck. L: vertical section of head. R: cross section. A: acrosome; AS: acrosome spine; N: nucleus

6. Zona pellucida Yolk Cortex Embryonic cells Cortical granules Ovum cell Plasma Corona radiata and cumulus cell layer Egg of bird Ovum of mammalian

7. Fertilization: Fertilization procedures: Capacitation of sperm Acrosome reaction Cortical reaction Formation of original nucleus Fusion During the capacitation, the activity of sperm is obviously increased by the reactions as the follows: 1. Ca+ channel is activated; 2. Both consumption of oxygen and glycolysis are increased significantly, and the value of pH is raided up; 3. The activation of adenylate cyclase results in the intracellular cAMP concentration increased, and protein kinase A activated; 4. Acrosomal enzymes are activated.

8. Sperms

9. Sperms are competing each other to bind and enter the ovum

10. Only one sperm that is most strong and fast ovum receives usually

11. Only one sperm can enter the ovum usually

13. The early events of fertilization (From Janice P. Evans 2002) a:Acrosome reaction b: Cortical reaction c: Formation of original nucleus d: Fusion

14. The fertilized ovum will move to uterus, implant and develop there.

15. The capacitated sperm can pass through the corona radiata and cumulus cell layer of ovocyte, and reach at zona pellucida via its powerful catalytic activity of hyaluronidase from plasma PH-20 of sperm. The sperm can recognize the zona pellucida of ovocyte by the ZP binding protein located on its acrosome, then, the acrosomal reaction is started. During this reaction, the hydrolases are released out from acrosome and the zona pellucida is degenerated to form a channel as an entrance for sperm. When the sperm enters the ovum, it cause the depolarization of ovum cell membrane, then, via a serial complicated reactions, the fertilization membrane (the sclerosed or hardened zona pellucida) is formed in the perivitelline space (between the ovum cell membrane and zona pellucida) where the sperm will be fused to ovum. Sperm can excite the dormant ovum to finish meiosis and release polar bodies. The nuclei of sperm and ovum are called as male pronucleus and female pronucleus at this time. The both pronuclei move to the center of the ovum and touch each other to be fused as a diploidfertilized ovum with their chromosomes mixed. The fertilized ovum can be called as zygote.

16. Usually, one ovum can be combined with one sperm only. If more than one sperms penetrate into an ovum, many polar sites and spindles will be formed and the fertilized ovum will be proliferated abnormally that causes the death of the embryo usually. But, in case of monozygotic twins (uniovular twins, monovular twins) or multifetuses, the combination of one ovum and more than one sperms can be developed normally, but it is very rarely to be seen. In many cases of multiple infants delivery, it is the combination of multiple ova vs multiple sperms. After fertilization, the fertilized ovum inhibit the penetration of other sperms by two ways as the follows: 1. The membrane of fertilized ovum is depolarized; 2. The cortical reaction can damage sperm receptor and inhibit the formation of fertilization membrane. In some insects, molluscs, chondrichthyes, birds, amphibians, and reptilians, the fertilization can be carried out with the combination of one ovum vs multiple sperms. But, only one male pronucleus can be fused to female pronucleus, and other sperm nuclei will be degenerated. The polyspermous fertilization described as above is called as physiologic polyspermous fertilization. During the fertilization, the nucleus, centromere, and mitochondria of sperm are exported into ovum. But, only the maternal mitochondria can survive in fertilized ovum. That is why mitochondrion follows matrilinear inheritance way.

17. The recognition between sperm and ovum: Heterogenous sperm can not bind to ovum because the ovum binding protein located on the surface of the sperm can not recognize and bind to the sperm receptor located on the membrane of ovocyte. The sperm receptor is located in the zona pellucida, and called as ZP protein. The ovum binding protein recognizes the ZP protein that is from a same species only. There are 3 types of glycoprotein in the mouse zona pellucida: ZP1 (200KD), ZP2 (120KD), and ZP3 (83KD). ZP3 is the first sperm receptor that can start acrosomal reaction. If the sperms are treated with ZP3, the sperms will lose the ability to fertilize ovum. β1，4-galactosyltransferase (GalTase-I) is the protein on sperm surface that can bind to ZP3. GalTase-I can bind to the N-acetylglucosamine at the terminal of ZP3 glucose chain. Experimental data proved that the purified GalTase-I and its antibody can inhibit the combination of sperm and ovum. GalTase-I can activate G protein to start acrosomal reaction. The sperms of GalTase-I gene knockout mice lost the ability to cause acrosomal reaction and pass through zona pellucida. After pass through zona pellucida, the sperm goes into the perivitelline space, and its fertilin (called as ADAM also) recognizes and binds to the integrin located on the membrane of ovocyte. The combination of fertilin and integrin can activate the fusion system for the sperm and ovum to finish the fertilization.

18. ZP3 and GalTase-I (from D. J. Miller 2000)

19. The interaction between sperm and the membrane of ovum (By Janice P. Evans 2002)

20. Ovum cleavage and embryonic development: Fertilization can create a new life with a serial steps of ovum cleavage. A fertilized ovum can be cleaved or developed as an embryo or degenerated. Which final result will be taken is just depended if the fertilized ovum can take nidation (implantation) or not in uterus. Ovum cleavage means a fertilized ovum (Zygote) is cleaved (developed) as new cells to form an embryo. New cleaved cells are called as blastomere. Ovum cleavage is similar to mitosis but without gap phase. So, during the cleavage, the number of cell is increased without cell growth. When the ratio of nucleus and plasma become normal, the cleavage is changed as real mitosis. The way selection of ovum cleavage is depended on yolk quantity and distribution. The meridional cleavage means the cleavage section is parallel with ovum axis. The latitudinal cleavage means the cleavage section is vertical with ovum axis. The cleavage direction is associated with the direction of spindle that is depended on the components of plasma and the signals from environment. The fertilized ovum of amphibian can start cleavage at 2hrs post fertilization. The fertilized ovum of mammalian starts cleavage at one day after fertilization. The cleavage will take 2 cells stage, 4 cells stage, 8 cells stage, 16 cells stage and more stages. But, at 16 cells stage, 1 – 2 cells in center of the cell mass will be developed as embryo, and other cells will be developed as trophocytes to form chorionic membrane.

21. Usually, the embryo of animal is a mass at 64 cells stage called as morula, and a big mass containing a blastocoel in center at 128 cells stage called as blastocyst. Some cells located on surface can move or fold into inside of embryo to form gastrula. The migration of cells to form gastrula is called as gastrulation. The cells remained outside are called as ectoderm, and the cells migrated into inside are called as endoderm and mesoderm. The blastocoel will disappear when the gastrula is formed. Ectoderm will form nerve system, skin, hair, nails, and teeth. Mesoderm will form bone system, muscle system, urogenital system, lymph or lymphoid tissue, connective tissue, and blood system. Endoderm will form pulmonary system, digestion system, liver, and others.

22. From a fertilized ovum (zygote) to an embryo

23. II. The major ways of cell differentiation The cell differentiation is the procedure by that the new generated cells are different from their parental cells in morphology and functions. By differentiation, cell can choose some genes to be expressed, and some genes to be blocked, finally, the expressed structural proteins form different structures and take different functions. So, differentiation is the fundamental bio-protocol to form the complicated and perfect organ system and individual that is a highly orderly and exactly regulated “cell community”. Therefore, differentiation is genes’ differential expression. Cell differentiation is operated with morphogenesis together. Morphogenesis means the procedure by that the appearances of tissues, organ, and individual will be formed by cell proliferation, differentiation, migration, adhesion, apoptosis, and other behaviors. The direction of cell differentiation is determined during the cell development. When the direction is determined irreversibly, the position and future of cell have been determined meantime. So, the procedure above is called as determination. For example, the central cells and peripheral cells of a monrula of mammalian have already got their future development irreversibly, the former will be developed as embryo, and the latter as chorionic layer.

24. Different animal species has different determination time point. For mammalian embryo, each cell at 8 cells stage or even 16 cells stage can be developed as an individual. The mechanism of cell differentiation is very complicated. Briefly, a cell determination is depended the internal features of cell and the environment in that the cell exists. The former is associated with asymmetric division, and the latter means the cell receives the external signals and responses to them. Asymmetric division Animal fertilized ovum is not a homogeneous in structures. The nucleus is located at a site closed to membrane, and the centroles are formed in this site. We call this site as northern polaroranimal polar, and the opposite site as southern polar or plant polar. The distribution of proteins and mRNA is not homogeneous also. There are 20,000 – 50,000 types of mRNA in animal ovum to the differentiation and development of the fertilized ovum. The homogeneity makes ovum cleavage asymmetrically, that means each new cell will get different genetic legacy or inheritance from their parental cell. During the embryonic development, asymmetric division is carried out very often.

25. Pathways of induction Induction means that a cell’s development direction can be induced by adjacent or distant cells. We call this induction as embryonic induction, and the cells from that the inducing signals are released out as inductor or inducer. We call a same type of cells that can response to signals as morphogenetic field. There are many morphogenetic fields in an embryo. Other inducing pathways include cascade signaling, gradient signaling, antagonistic signaling, combinatorial signaling, and lateral signaling. Some induction signals

26. The pathways of induction signals (From Thomas Edlund and Thomas M. Jessell 1999)

27. Cascade signaling：Cascade signaling means the cells or tissue that were generated by the primary induction can induct the differentiation for other cells or tissues grade by grade. For example, the original visual cell can induct the ectoderm cells rounding it to be differentiated as the lithocysts for crystalloid lens, and the cells of crystalloid lens can induct the ectoderm cells to be differentiated as cornea. Gradient signaling：The external signals are distributed in a gradient. Each cell has a threshold concentration of signal to response to the signal. The different threshold concentration will cause the different spatiotemporal differentiations. We call the signals in a cell or morphogenetic field that can regulate the cell differentiation as morphogen. Antagonistic signaling：The cell secrets can bind to the receptor or ligand of some signal pathways to block the signaling. Many morphogenetic events are caused by this pathway. Combinatorial signaling：One type of signal can determine a differentiation for a cell, but two types of signals can cause another differentiation way. Lateral signaling：A little bit difference between the signals can be identified. But, this difference can be obviously enlarged by the feedback regulation to determine the differentiation. For example, in fruit fly, Notch signal pathway can activate the lateral inhibition to inhibit the pre-neuron to be differentiated as neuron.

28. It is not true that all differentiation events are caused by signal induction. Some differentiations are associated with the cell internal features. We call this differentiation regulation as autonomous mechanism. For example, the asymmetric division is associated with homogeneity of ovum. The regulation of cell quantity It is easy to answer the question, why human body is large than mouse? You can say because human body contains more cells than mouse body. But, it is not easy to answer these questions: Why there are more cells contained in human body than in mouse body? Why the cell quantity in each type of organ is consistent almost? The regulation of cell quantity is important not only for the tissue or organ structures construction but also for the maintenance of an individual. The regulation of cell quantity depends on cell proliferation and cell death. Enhancement of cell division: Most of cell proliferations are started by the excitation of external signals of the induction ways. So, in a cell community, a cell proliferation should be started or not, that depends on the request or permission from other cells or community. The signals for cell proliferation include cytokines (growth factor), hormone, and extracellular matrix (ECM).

29. 1．Peptide growth factors: Peptide growth factor excites target cells by paracrine way. But, the selfcrine way probably exists among the cells of same type. If you culture one or several cells in a dish, they will not grow or grow very slowly because there is no excitation from other cells. If you culture cells with a medium containing no growth factor inside, the cells will stop to grow at G1/S and turn to G0 phase. Same event can happen in tumor development. Fisher（1967）injected a big number of tumor cells into mouse portal vein and resulted in broad metastasis in liver. The mouse died soon. But, when he injected 50 tumor cells into the mouse portal vein, nothing happened to the mouse. He took a surgery operation to the mouse to check the liver, and no any metastasis could be found in the liver. But, after his performance, the mouse died from tumor metastasis in liver soon because the level of growth factor was raised during the surgery wound healing. The described above indicates that tumor development and metastasis are depended on paracrine way and selfcrine way.

30. 2．Hormone: Hormone stimulation can be considered as distant signaling excitation. Hormone can be distributed at any where in body by the blood circulation system. But, hormone stimulates the specific target cells only. For example, sex hormone can put powerful effect on the sex differentiation. Androgen can promote the development of penis and male germ organs, and estrogen can promote the development of vagina, uterus and other female germ channels or cavities. If the embryonic testes were removed, the embryo will be developed as female individual. 3．Extracellular matrix (ECM): ECM can interact with the integrin on cell surface to activate the focal adhesion kinase (FAK), and by the growth receptor bound protein 2 (Grb2), FAK can start the Ras signal pathway to cause cell proliferation. In vitro, ECM can induct the differentiation direction of stem cells. If the stem cells are cultured on a collagen IV coated dish, the stem cells will be differentiated as epidermal cells. If on a collagen I or laminin coated dish, the stem cells will be differentiated as fibroblasts. If on a collagen II coated dish, the stem cells will be differentiated as chondrocytes.

31. Inhibition of cell division: There are two ways at least to inhibit cell division in embryo: 1. Regulate down the level of stimulation signals. 2. Inhibit cell cycle promoter. During myogenesis, the myostatin, a member of TGF-β super family, is a negative regulator for muscle growth. The myostatin mutated animal can present the double-muscled phenotype. If this gene is knockout, the weight of muscle tissue of the mouse will be increased by 2 – 3 folds. Myostatin inhibits the proliferation of muscle by up-regulating the level of p21, an inhibitor to CDK2, and down-regulating the level of CDK2. The inefficiency of activity of CDK2 causes that the transcription factor, E2F can not be released out from Rb, so, cell can not enter S phase from G1 phase. Myostatin is secreted by muscle cells. The level of myostatin is high in embryo, but low in adult. The excessive expression of myostatin will cause muscular atrophy.

32. Myostatin downregulates the muscle growth (from Heather Arnold, 2001)

33. Apoptosis: Apoptosis: Disorder of programmed cell death (PCD) caused by apoptosis genes and other inducers. PCD: Cell death follows spatiotemporal sequence of cell proliferation regulation. Necrosis: Cell death caused accidentally by external pathogens, stimulations or wounds. Cell death We can regulate apoptosis partially for some special aims. Apoptosis research is very hot for many years. The detail about it will be presented in next chapter.

34. The behaviors of cell Cell behaviors are depended on the external signals and the features of cell. The cell behaviors include directed mitosis, differential growth, apoptosis, migration, differential adhesion, cell contraction, Matrix swelling, gap junction, and fusion. Directed mitosis: External signal can regulate the direction of spindle and have the division directed. The new generated cells can be located on a specific area during the directed mitosis. Directed mitosis is associated with asymmetric division. Differential growth: The original pattern will be changed during the individual development. Different parts will form different organ or tissue. Different growth follows spatiotemporal sequence. Migration: Migration means that a mass of cells migrates to a place from another place. Migration is important in the development of organ and tissue, and wound healing. Migration rate is a useful parameter to the cell mobility or motility.

35. Differential adhesion: Differential adhesion means the cells form junction by the glycoprotein on their surface or ECM temporarily or consistently. The functions of differential adhesion include: (1) the cells in same type or relative type are combined together to form tissue or organ; (2) the spine, folds, cavity, and others will be formed by differential adhesion; (3) adhesion and release are the basic procedures of migration. Contraction: Contraction is driven by myosin and actin. Fusion: Muscle cell is a plasmodium fused by myoblasts. Gap junction: Cell communication can regulates differentiation by gap junction. This is a popular way to differentiation regulation.

36. Behaviors of developing cells (From Salazar-Ciudad, 2003）

37. Myotube is fused by myoblasts (From Harvey Lodish, 1999)

38. The changes of cell structure The changes of chromosome structure The deletion of genes: It means the lost of some parts of chromosome during the cell differentiation in some animals, such as protozoa and insect. The amplification of genes: It means that the copy quantity of some special genes was specifically increased in a cell of some animals, such as in some cells of fruit fly, the DNA was replicated without any cell division, so, the polytene nucleus was formed. The rearrangement of genes: It is a very important way to regulate the gene expressions. For example, 106 – 108 antibodies can be generated in mammalian body, but that does not means there are so many genes for antibodies in body. The variety of antibody is just depended on the rearrangement of antibody gene fragments. The methylation of DNA: The activities of some genes of vertebrates are inactivated by methylating them. Of course, demethylation can activate them. All genes in cell can be sorted as two types: house-keeping genes and luxury genes. The former means some genes are important for cell survival. The latter means some genes are associated with cell differentiation, and expressed specifically in some tissues only. Luxury genes keep demethylated in some tissues and methylated in other tissues. All methylation are almost located on the “C” of 5'-CG-3’, and makes cytimidine becomes methyl cytimidine.

39. The replacement of isoform: The one of the most popularly happened events in the development is the serial replacements with spatiotemporal sequence. We call this replacement as isoform replacement. It means that during a special stage, a molecule, cell, or organ is replaced by another cell, molecule or organ that is very similar to original one with same function but better to match development needs than original one in a new environment. The unit in the serial replacement is called as isoform. We have a lot of instances for it: the primary embryonic red blood cells are nucleated erythrocyte, they will be replaced by non-nucleated (acaryote) erythrocytes generated by liver. The embryonic hemoglobin molecules will be replaced by the fetus hemoglobin molecules that will be replaced by adult those. The teeth will be replaced by their isoforms when a baby is developed as a boy or girl. The replacement of kidneys by their isoforms is the most complicated isoform replacement during embryonic development.

40. III. The potential differentiation ability of cells Totipotence, pluripotence, and unipotence: A fertilized ovum can be differentiated as any cell, tissue, organ, and individual, so, it is totipotent cell. With the development, cells will lose their potential differentiation ability from totipotent cells to pluripotent cells, finally to unipotent cells (adult cells or somatic cells). For example, pluripotent hematopoietic stem cells can be developed as any blood cells (unipotent cells). Totipotence of fertilized ovum

41. Totipotence of plant cell

42. Matured animal cells are not of totipotence or pluripotence because of plasma, not nucleus. A nucleus contains the repertoire of genome DNA (Complete set of genes) that means a nucleus keeps totipotence. That is why many scientists could clone the animal individuals from a differentiated (adult) cell, such as goat, pig, and others. Of course, a human adult cell should have the totipotence to be developed as a human individual, but, laws do not allow you to do it! For a person, his/her skin, blood cells, and mucosal cells need to be continuously replaced by new cells generated from stem cells. Stem cells are the cells with pluripotence that can be developed as many types of cells, but not as a new individual naturally. Unipotent stem cells are developed from pluripotent cells, and they can be developed as some specific cells only. Unipotent stem cells is also called as progenitor.

43. Potential differentiation ability of stem cell

44. The features of stem cells The features of stem cells are as the follows: ① Keep non-differentiated or low differentiated status in whole life; ② The number and location in body are not variable usually; ③ Be of the recruiting themselves; ④ Can be proliferated unlimitedly; ⑤ Pluripotence; ⑥ Most of stem cells are G0 phase cells; ⑦ Be cleaved by two ways: symmetric division and asymmetric division. Former will form two new stem cells, and latter will form one stem cell and one progenitor. Totipotent stem cells: These cells keep totipotence to form anycells in body, but not form an individual naturally. Pluripotent stem cells: These cells keep pluripotence to form manytypes of cell, or the cells of some tissue type, such as blood cells. Unipotent stem cells: These cells keep unipotence to form one type of cell only, and they are of the limited ability of recruiting themselves.

45. The embryonic stem cells (ESC) By the spatiotemporal sequence, stem cells can be sorted as ESC and adult stem cells. ESC is the totipotent or pluripotent cell isolated from embryonic cell mass or generated by the transplantation of the nucleus of adult cell. ESC can be used to: ①clone animals. The cloning generation of animals by replacing an ovum nucleus by a nucleus of adult cell is difficult. The cloned animals by this high technology are of some genetic deficiencies, such as immunodeficiency. ②generate transgenic animals. ESC is the best vector to this aim because the success rate can be obviously increased for the generation. ③ cell or tissue engineering. ESC can be artificially and directorially differentiated as some special tissue or organ for the clinic needs.

46. Somatic (Adult) cell Ovum or embryonic cell Generating an ESC by nucleus transplantation

47. Regeneration Regeneration means specially that a wounded organ can repair itself by generating the wounded part with same form and function, and generally that a molecule, cell, or organ can be replaced or regenerated. The types of regeneration: Physiological regeneration: It means cell replacement of isoform. For example, 6 million old erythrocytes are replaced by new generated erythrocytes per second in human body. Repairing regeneration: It means the regeneration of wounded organ. Many invertebrates have powerful ability for repairing regeneration. Reconstruction: It means some special regeneration under experimental conditions. Asexual reproduction: Low grade living things, such as some parasites, can take asexual reproduction. There are some interested questions about regeneration as the follows: 1．How is the body informed for that which organ or part was lost or damaged, and how much was lost? In other words, how to regulate the regeneration? 2．Where are the new replaced part from? Is it from stem cells or the remained differentiated cells adjacent the wound? Many experimental results show that the differentiated (adult) cells can be dedifferentiated, migrated, or proliferated for the wound healing. 3．Is it reconstruction or cell proliferation?