General Biology Laboratory Bio 112
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General Biology Laboratory BIO 112
Morehouse College
Laboratory Study 4
Animal Development Objectives 1. 2. 3. 4.
Observe egg activation and fertilization in sea urchins Observe early cleavage stages in sea urchins Compare the early development of deuterostomes to protostomes Review prepared slides of frog and chicken development
Introduction The development of each individual in sexually reproducing organisms starts with the development of gametes, eggs, and sperms. The less motile gametes is the egg (ovum). A female is defined by its production of eggs. The egg originates from a population of mitotically multiplying primordial germ cells. A member of this population ceases multiplication and starts to grow and is now called an oocyte. Informally, it is called an egg, a term commonly, but incorrectly, applied from this stage until early cleavage. During this growth, the oocyte remains a diploid (2N) cell, meaning that the nucleus contains two sets of homologous chromosomes. The growth of the egg results in the accumulation of materials that are the products of synthetic activity under the control of its own DNA and materials synthesized in other tissues and transported to the egg. Many of these imported materials are macromolecules that not only serve as building blocks for embryonic development, but also carry information critically important in embryonic development. These molecules are not deposited evenly throughout the egg. Instead, they are found in cellular organelles and cytosol distributed in a characteristic pattern in different parts of the egg. Fertilization cannot be completed until the egg undergoes the divisions of meiosis which reduce the chromosomes in the nucleus to one set (1N). Meiosis results in one copy of each gene, on one chromosome from each homologous pair, in the mature egg. The mature egg is a haploid cell. The meiosis that yields a mature egg involves unequal divisions of cytoplasm, in both divisions, so the cell that becomes the egg gets the majority of the cytoplasm. The other three cells that get only enough cytoplasm to surround their nucleus are called polar bodies. The polar bodies soon die and the one large remaining cell, the mature egg, is called an ovum. The more motile gamete is the sperm (spermatozoan) and the male is defined by the production of these gametes. Sperms also arise from mitotically multiplying dipoid primordial germ cells. However, these cells do not grow and proceed directly to meiosis. Meiosis reduces the chromosomes to one set in each of the four product cells, and the cytoplasmic divisions are equal between all four cells. The haploid products of meiosis in the male differentiate by producing structures needed to move them to and into an ovum. These differentiated motile cells are called spermatozoa. In some species, including humans, the differentiated spermatozoa has a head region containing the nucleus and structures for penetrating the ovum, a mid-piece region that carries the 1
General Biology Laboratory BIO 112
Morehouse College
machinery to transform chemical energy to kinetic energy, and a tail region that has a flagellar structure for propulsion. In many species, this differentiation of spermatozoa does not occur and even amoeboid sperms are possible. Fertilization is the process in which the egg and sperm unite to form a zygote and the haploid nuclei of each gamete contributes one set of chromosomes to make a dipoid nucleus. The zygote undergoes mitotic divisions, cleavages, and the resulting multicellular embryo undergoes a series of developmental events. Development consists of three processes: differentiation, growth and translocation. Differentiation is the structural and functional modification of a cell to a specialized function. A more sophisticated definition would describe a change in the functional characteristics of cells and the sequence of gene activation and/or inactivation that leads to a change in functional characteristics. In most, but not all cases, differentiated cells cannot reacquire their original totipotency (potential of an unspecialized cell to express all of its genes and become specialized to any cell type in the body). Growth is an increase in mass or volume resulting from cell division and cell expansion. If molecules simply increase in number, the cell volume must increase and the cell grows (expands). If molecules accumulate in a defined part of the cell, this accumulation may change the characteristics of the cell. Thus, growth at one level of organization (molecular) can result in differentiation at another level of organization (cellular). Can you think of an example of growth at the cellular level that results in differentiation at the tissue or organ level? Translocation is the movement of materials from one location to another (note that the movement of a piece of a chromosome, and solute movement in plants are very specific uses of this term). The following are some examples of translocation: Proteins produced at the ribosomes are moved to other locations for storage, use, or release outside the cell. Cells or tissues formed at one location may be relocated to another location in the body. The translocation of molecules can result in cell differentiation and translocation of cells can cause tissue or organ differentiation. As you observe developmental changes in the animal specimens, try to relate the events to the processes of growth, differentiation and translocation. The fertilized egg, the zygote, and its daughter cells (blastomeres) undergo repeated mitotic divisions and pass on to each generation of blastomeres an exact copy of their entire genetic material. However, these blastomeres also receive different portions of the cytoplasmic constituents which were compartmentalized during the growth of the unfertilized ovum. Carrying identical genetic information, but differing in the composition of their cytoplasm and being in different locations in an embryo results in the blastomeres undergoing developmental changes. Different location in an embryo results in different levels of access to nutrients and gas exchange which can influence developmental processes. Cells become specialized to ultimately become, for example, liver cells, nerve cells or muscle cell. Each specialized cell has a specific function and a
2
General Biology Laboratory BIO 112
Morehouse College
complement of proteins and other molecules necessary for that specialized role. This dynamic and complicated process is ultimately described in the “blueprint” information in the DNA of the zygote. However, the expression of the DNA “blueprint” is regulated by the environment, which includes the macromolecules received from the female parent when the ovum was produced. The macromolecules present in the ovum are determined by the genotype of the female parent and the environment in which the ovum was produced. Developmental events occur in a predetermined sequence that build and are influenced by those that preceed them. The control of developmental processes, and ultimately the control of gene expression, is a subject of continuing intense research. Understanding the control of gene expression is not only the key to understanding development but also applies to the studies of cancer and molecular genetic technology. Early Embryonic Development Development can be divided into stages whose major events are described below. These stages apply to all animals. Fertilization: This is the fusion (uniting = syngamy) between an ovum and one spermatozoan which occurs in two steps, egg activation and nuclear fusion (karyogamy). Activation begins when the sperm penetrates the plasma membrane of the ovum. Activation initiates many changes in the ovum such as an increase in metabolic rate, an alteration in the permeability of the plasma membrane, and the lifting of the fertilization membranes. Activation is followed by karyogamy to form the diploid zygote nucleus. Depending on the species, sperm penetration of the egg plasma membrane and the initiation of activation, precedes (and triggers) the first (in Ascaris round worms) or the second (in frogs and humans) meiotic divisions. Karyogamy cannot occur until meiosis is completed so there is always some delay between activation and karyogamy. Cleavage: This is the stage during which mitotic divisions of the zygote produce a clone of cells (blastomeres) which package the heterogeneous cytoplasm of the ovum in separate cells each containing the same DNA information. Cleavage begins shortly after fertilization and no increase in mass (or volume) occurs although the cell number increases. The pattern of cleavage is influenced by the amount and distribution of yolk in the ovum. Yolk is protein and lipid rich material in a mature ovum that is stored food for the developing embryo. When large quantities of yolk are present in a zygote, cytokinesis may not involve the entire zygote cytoplasm (incomplete cleavage). Alternatively, if little yolk is present, cytokinesis involves the entire cytoplasm of the zygote (complete cleavage). Egg Types Alecithal: The cytoplasm is heterogeneous but there is no yolk present. Human eggs are of this type. Most nourishment for the developing embryo is from the mother’s circulation. Cleavage is complete.
3
General Biology Laboratory BIO 112
Morehouse College
Isolecital: Yolk is present in small amounts but is not sufficiently localized to make cell division unequal. Sea urchin eggs are of this type. Cleavage is complete. Centrolecital: Yolk is concentrated in the central region of the cytoplasm in the egg. Insect eggs are of this type. The cleavage divisions initially produce numerous nuclei that share the same cell cytoplasm (syncytium) around the yolk and cytokinesis occurs later. Telolecithal: Yolk is concentrated at one end of the ovum, near the vegetal hemisphere (the region that becomes the ventral side of the animal). The nucleus of the zygote is located in the animal hemisphere. The vegetal hemisphere region is incorporated into the gut of the embryo while the embryo proper develops from cells in the animal hemisphere. Depending on the amount of yolk, cleavage is complete (frogs) or incomplete (birds). Incomplete cleavage results in the yolk filled portion of the egg not being divided in cleavage cells (blastomeres). Types of Cleavage The precise form of early cleavage divisions is not the same in all animals. Systematic variation occurs in the first division of the zygote, whether the first two cells of the embryo are equal or unequal in size, and the relative positions of blastomeres produced in successive cell divisions. There are two major patterns in the relative positions of blastomeres, spiral cleavage (in protosomes) and radial cleavage (in deuterostomes). Spiral cleavage results in new blastomeres produced in the third cell division (going from a 4-cell to an 8-cell embryo) positioned in the cleavage furrows of the previously existing four cells located beneath them. If cleavage is complete, the first division in spiral cleavage is unequal, producing one larger and one smaller cell. In radial cleavage, new blastomeres are from the third mitotic division are positioned directly above the previously existing four cells located beneath them. If cleavage is complete, the first division in radial cleavage produces two cells of equal volume. Blastulation: This is the process in which a ball of cells produced by the early cleavage divisions is transformed to a sphere (sea urchin, human, frog) or sheet (birds) called a blastula with a central (sphere) or underlying (sheet) cavity called a blastocoel. Gastulation: The blastula is transformed in this process which is characterized by cell movements to produce a three layered (triploblastic) embryo. Sheets of cells move from the superficial layer of the blastula into the interior by movements that vary depending on the amount of yolk in the embryo. Typically, movement to the interior of the embryo occurs at a specific location called the blastopore and cessation of cell movement occurs once cells reach specific new locations. The process is complete when the organs of the gastrula are formed. These organs include the archenteron (primitive gut) which fills the old blastocoel and three tissue layers (germ layers): the outer ectoderm, the middle
4
General Biology Laboratory BIO 112
Morehouse College
mesoderm, and the inner endoderm. All future organs develop from these three germ layers. The ectoderm is the source of structures that have the most direct contact the with external environment (skin, nervous system). Muscles, bone, and blood are all derived of mesoderm, and digestive structures are all derived of the endoderm. The site of the blastopore is the location of the mouth in organisms with spiral cleavage. Animals with spiral cleavage are protostomes (first mouth). The site of the blastopore is the location of the anus in animals with radial cleavage and the mouth forms at the opposite end of the embryo. Animals with radial cleavage are deuterostomes (second mouth). Morphogenesis: The formation of the characteristic structures and organs in an animal following gastrulation is the process of morphogenesis. This is a series of stages that varies between animal groups but results in the development of external and internal structures that will be found in the mature animal. Translocations of cells and sheets of cells dominate these developmental stages but growth and differentiation are also occurring. Methods and Materials You will be performing sea urchin fertilizations under the microscope so you can observed the fertilization process first-hand and see the first few cleavages occur. Prepared slides and preserved embryos of will be available to observe and compare the developmental stages in mollusks (protostomes), as well as sea urchins, frogs, and birds (deuterostomes). Bring your lecture textbook to your laboratory class. Acknowledgements This study was reprinted with revision from H. Ikuma, P. Harley and C. Yocum (1985) Animal Development, in Biology 105 Course Pack, Division of Biological Sciences, University of Michigan, Ann Arbor. Revisions made 2/2005 by L. Blumer
5
Morehouse College
Laboratory Study 4
Animal Development Objectives 1. 2. 3. 4.
Observe egg activation and fertilization in sea urchins Observe early cleavage stages in sea urchins Compare the early development of deuterostomes to protostomes Review prepared slides of frog and chicken development
Introduction The development of each individual in sexually reproducing organisms starts with the development of gametes, eggs, and sperms. The less motile gametes is the egg (ovum). A female is defined by its production of eggs. The egg originates from a population of mitotically multiplying primordial germ cells. A member of this population ceases multiplication and starts to grow and is now called an oocyte. Informally, it is called an egg, a term commonly, but incorrectly, applied from this stage until early cleavage. During this growth, the oocyte remains a diploid (2N) cell, meaning that the nucleus contains two sets of homologous chromosomes. The growth of the egg results in the accumulation of materials that are the products of synthetic activity under the control of its own DNA and materials synthesized in other tissues and transported to the egg. Many of these imported materials are macromolecules that not only serve as building blocks for embryonic development, but also carry information critically important in embryonic development. These molecules are not deposited evenly throughout the egg. Instead, they are found in cellular organelles and cytosol distributed in a characteristic pattern in different parts of the egg. Fertilization cannot be completed until the egg undergoes the divisions of meiosis which reduce the chromosomes in the nucleus to one set (1N). Meiosis results in one copy of each gene, on one chromosome from each homologous pair, in the mature egg. The mature egg is a haploid cell. The meiosis that yields a mature egg involves unequal divisions of cytoplasm, in both divisions, so the cell that becomes the egg gets the majority of the cytoplasm. The other three cells that get only enough cytoplasm to surround their nucleus are called polar bodies. The polar bodies soon die and the one large remaining cell, the mature egg, is called an ovum. The more motile gamete is the sperm (spermatozoan) and the male is defined by the production of these gametes. Sperms also arise from mitotically multiplying dipoid primordial germ cells. However, these cells do not grow and proceed directly to meiosis. Meiosis reduces the chromosomes to one set in each of the four product cells, and the cytoplasmic divisions are equal between all four cells. The haploid products of meiosis in the male differentiate by producing structures needed to move them to and into an ovum. These differentiated motile cells are called spermatozoa. In some species, including humans, the differentiated spermatozoa has a head region containing the nucleus and structures for penetrating the ovum, a mid-piece region that carries the 1
General Biology Laboratory BIO 112
Morehouse College
machinery to transform chemical energy to kinetic energy, and a tail region that has a flagellar structure for propulsion. In many species, this differentiation of spermatozoa does not occur and even amoeboid sperms are possible. Fertilization is the process in which the egg and sperm unite to form a zygote and the haploid nuclei of each gamete contributes one set of chromosomes to make a dipoid nucleus. The zygote undergoes mitotic divisions, cleavages, and the resulting multicellular embryo undergoes a series of developmental events. Development consists of three processes: differentiation, growth and translocation. Differentiation is the structural and functional modification of a cell to a specialized function. A more sophisticated definition would describe a change in the functional characteristics of cells and the sequence of gene activation and/or inactivation that leads to a change in functional characteristics. In most, but not all cases, differentiated cells cannot reacquire their original totipotency (potential of an unspecialized cell to express all of its genes and become specialized to any cell type in the body). Growth is an increase in mass or volume resulting from cell division and cell expansion. If molecules simply increase in number, the cell volume must increase and the cell grows (expands). If molecules accumulate in a defined part of the cell, this accumulation may change the characteristics of the cell. Thus, growth at one level of organization (molecular) can result in differentiation at another level of organization (cellular). Can you think of an example of growth at the cellular level that results in differentiation at the tissue or organ level? Translocation is the movement of materials from one location to another (note that the movement of a piece of a chromosome, and solute movement in plants are very specific uses of this term). The following are some examples of translocation: Proteins produced at the ribosomes are moved to other locations for storage, use, or release outside the cell. Cells or tissues formed at one location may be relocated to another location in the body. The translocation of molecules can result in cell differentiation and translocation of cells can cause tissue or organ differentiation. As you observe developmental changes in the animal specimens, try to relate the events to the processes of growth, differentiation and translocation. The fertilized egg, the zygote, and its daughter cells (blastomeres) undergo repeated mitotic divisions and pass on to each generation of blastomeres an exact copy of their entire genetic material. However, these blastomeres also receive different portions of the cytoplasmic constituents which were compartmentalized during the growth of the unfertilized ovum. Carrying identical genetic information, but differing in the composition of their cytoplasm and being in different locations in an embryo results in the blastomeres undergoing developmental changes. Different location in an embryo results in different levels of access to nutrients and gas exchange which can influence developmental processes. Cells become specialized to ultimately become, for example, liver cells, nerve cells or muscle cell. Each specialized cell has a specific function and a
2
General Biology Laboratory BIO 112
Morehouse College
complement of proteins and other molecules necessary for that specialized role. This dynamic and complicated process is ultimately described in the “blueprint” information in the DNA of the zygote. However, the expression of the DNA “blueprint” is regulated by the environment, which includes the macromolecules received from the female parent when the ovum was produced. The macromolecules present in the ovum are determined by the genotype of the female parent and the environment in which the ovum was produced. Developmental events occur in a predetermined sequence that build and are influenced by those that preceed them. The control of developmental processes, and ultimately the control of gene expression, is a subject of continuing intense research. Understanding the control of gene expression is not only the key to understanding development but also applies to the studies of cancer and molecular genetic technology. Early Embryonic Development Development can be divided into stages whose major events are described below. These stages apply to all animals. Fertilization: This is the fusion (uniting = syngamy) between an ovum and one spermatozoan which occurs in two steps, egg activation and nuclear fusion (karyogamy). Activation begins when the sperm penetrates the plasma membrane of the ovum. Activation initiates many changes in the ovum such as an increase in metabolic rate, an alteration in the permeability of the plasma membrane, and the lifting of the fertilization membranes. Activation is followed by karyogamy to form the diploid zygote nucleus. Depending on the species, sperm penetration of the egg plasma membrane and the initiation of activation, precedes (and triggers) the first (in Ascaris round worms) or the second (in frogs and humans) meiotic divisions. Karyogamy cannot occur until meiosis is completed so there is always some delay between activation and karyogamy. Cleavage: This is the stage during which mitotic divisions of the zygote produce a clone of cells (blastomeres) which package the heterogeneous cytoplasm of the ovum in separate cells each containing the same DNA information. Cleavage begins shortly after fertilization and no increase in mass (or volume) occurs although the cell number increases. The pattern of cleavage is influenced by the amount and distribution of yolk in the ovum. Yolk is protein and lipid rich material in a mature ovum that is stored food for the developing embryo. When large quantities of yolk are present in a zygote, cytokinesis may not involve the entire zygote cytoplasm (incomplete cleavage). Alternatively, if little yolk is present, cytokinesis involves the entire cytoplasm of the zygote (complete cleavage). Egg Types Alecithal: The cytoplasm is heterogeneous but there is no yolk present. Human eggs are of this type. Most nourishment for the developing embryo is from the mother’s circulation. Cleavage is complete.
3
General Biology Laboratory BIO 112
Morehouse College
Isolecital: Yolk is present in small amounts but is not sufficiently localized to make cell division unequal. Sea urchin eggs are of this type. Cleavage is complete. Centrolecital: Yolk is concentrated in the central region of the cytoplasm in the egg. Insect eggs are of this type. The cleavage divisions initially produce numerous nuclei that share the same cell cytoplasm (syncytium) around the yolk and cytokinesis occurs later. Telolecithal: Yolk is concentrated at one end of the ovum, near the vegetal hemisphere (the region that becomes the ventral side of the animal). The nucleus of the zygote is located in the animal hemisphere. The vegetal hemisphere region is incorporated into the gut of the embryo while the embryo proper develops from cells in the animal hemisphere. Depending on the amount of yolk, cleavage is complete (frogs) or incomplete (birds). Incomplete cleavage results in the yolk filled portion of the egg not being divided in cleavage cells (blastomeres). Types of Cleavage The precise form of early cleavage divisions is not the same in all animals. Systematic variation occurs in the first division of the zygote, whether the first two cells of the embryo are equal or unequal in size, and the relative positions of blastomeres produced in successive cell divisions. There are two major patterns in the relative positions of blastomeres, spiral cleavage (in protosomes) and radial cleavage (in deuterostomes). Spiral cleavage results in new blastomeres produced in the third cell division (going from a 4-cell to an 8-cell embryo) positioned in the cleavage furrows of the previously existing four cells located beneath them. If cleavage is complete, the first division in spiral cleavage is unequal, producing one larger and one smaller cell. In radial cleavage, new blastomeres are from the third mitotic division are positioned directly above the previously existing four cells located beneath them. If cleavage is complete, the first division in radial cleavage produces two cells of equal volume. Blastulation: This is the process in which a ball of cells produced by the early cleavage divisions is transformed to a sphere (sea urchin, human, frog) or sheet (birds) called a blastula with a central (sphere) or underlying (sheet) cavity called a blastocoel. Gastulation: The blastula is transformed in this process which is characterized by cell movements to produce a three layered (triploblastic) embryo. Sheets of cells move from the superficial layer of the blastula into the interior by movements that vary depending on the amount of yolk in the embryo. Typically, movement to the interior of the embryo occurs at a specific location called the blastopore and cessation of cell movement occurs once cells reach specific new locations. The process is complete when the organs of the gastrula are formed. These organs include the archenteron (primitive gut) which fills the old blastocoel and three tissue layers (germ layers): the outer ectoderm, the middle
4
General Biology Laboratory BIO 112
Morehouse College
mesoderm, and the inner endoderm. All future organs develop from these three germ layers. The ectoderm is the source of structures that have the most direct contact the with external environment (skin, nervous system). Muscles, bone, and blood are all derived of mesoderm, and digestive structures are all derived of the endoderm. The site of the blastopore is the location of the mouth in organisms with spiral cleavage. Animals with spiral cleavage are protostomes (first mouth). The site of the blastopore is the location of the anus in animals with radial cleavage and the mouth forms at the opposite end of the embryo. Animals with radial cleavage are deuterostomes (second mouth). Morphogenesis: The formation of the characteristic structures and organs in an animal following gastrulation is the process of morphogenesis. This is a series of stages that varies between animal groups but results in the development of external and internal structures that will be found in the mature animal. Translocations of cells and sheets of cells dominate these developmental stages but growth and differentiation are also occurring. Methods and Materials You will be performing sea urchin fertilizations under the microscope so you can observed the fertilization process first-hand and see the first few cleavages occur. Prepared slides and preserved embryos of will be available to observe and compare the developmental stages in mollusks (protostomes), as well as sea urchins, frogs, and birds (deuterostomes). Bring your lecture textbook to your laboratory class. Acknowledgements This study was reprinted with revision from H. Ikuma, P. Harley and C. Yocum (1985) Animal Development, in Biology 105 Course Pack, Division of Biological Sciences, University of Michigan, Ann Arbor. Revisions made 2/2005 by L. Blumer
5
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