Notes on Cellular Differentiation | Cell Biology

In this article we will discuss about Cellular Differentiation:- 1. Subject-Matter of Cellular Differentiation 2. Role of Cytoplasm in Cellular Differentiation 3. Homeotic Genes.

Subject-Matter of Cellular Differentiation:

Differentiation is the process by which the genes are preferentially active and the gene products are utilised to bring some phenotypic changes in the cell. It is not the only property of multicellular organisms. Many unicellular organisms undergo phenotypic changes along with changes in physiological processes.

Any change in the environment of unicellular or­ganisms whether—physical or at the nutrient level—can undergo remarkable physical cellular changes like the formation of different types of spores, sporulation in bacteria, fungi etc.

These are the example of cellular differentiation in unicellular organisms. The differentiation observed in higher or­ganisms, particularly animals, is different and complicated. It has attracted developmental bi­ologists to study the development of an embryo from a single cell, i.e., zygote.

This process takes place in several steps:

i. Fertilisation:

Fusion of sperm and egg.

ii. Cleavage:

Development of zygote to form blastula (group of undifferentiated cells).

iii. Gastrulation:

Differentiation and move­ment of cells to form specialised cell layers.

iv. Differentiation:

Development of specialised cells into tissues, organs and growth of the embryo.

v. Growth, Maintenance and Regeneration of some cells.

Molecular Changes During Oogenesis and Fertilisation:

The process of female gamete formation is known as oogenesis. The primordial germ cells are called oogonia. When the oogonia start meiosis they are called oocytes; where an exten­sive growth phase occurs.

During this growth phase of oocyte, large number of metabolic and morphological changes occur.

These are:

i. Increase of RNA synthesis—along with polytene-like changes, are found in the chromosome.

ii. Amplification of Ribosomal genes takes place leading to the increased synthesis of ribosomal RNA. Associated cytological changes are the occurrence of many nucle­oli in the nucleus.

iii. Size of the nucleus is increased indicating the metabolic changes of the nucleus.

iv. Accumulation of protein, lipid and carbo­hydrate takes place which will be utilised during the formation of embryo. In higher animals all those nutrients are produced by the liver and follicle cells of the developing oocyte. However, mammalian oocytes do not accumulate yolk proteins as they come through mother’s bloodstream and thus storage of nutrients is not necessary here.

v. In non-mammalian oocyte the asymmetry in polarity is found during its development. One end of the cell is called the Vegetal pole containing most of the nutrients and yolk platelets. The other end is called the Animal pole containing ribosomes and mitochondria besides nucleus.

The embryo is developed at this end. After these developmental changes, the meiosis takes place in the oocyte to produce mature egg which is a highly differentiated specialised cell.

The Spermatogenesis—Spermatogonia:

increase their number by mitosis and then undergoes meiosis to form sperms. Cellular and other molecular changes are not so significant like oogenesis. Considerable changes are found after fertilisation during the development of embryo.

Role of Cytoplasm in Cellular Differentiation:

The importance of cytoplasm on cell differen­tiation has been demonstrated in large number of experiments.

The egg of snail produces a lobe-like struc­ture at the vegetal end during cell differentia­tion which is known as Polar lobe. If this lobe is excised, defective embryo is produced. Again, a coloured area is produced in the amphibian egg during cell differentiation after fertilisation. This is known as Grey Crescent. If the grey crescent is injured it induces abnormality in the nervous system.

In case of amphibian egg, if the first cleavage is perpendicular to the grey crescent and the resulting blastomere is separated, each blastomere will produce a normal animal. But if the cleavage takes place parallel to the grey crescent and if the two blastomeres are separated—the one having grey crescent will produce normal animal. Thus the differentiation of cell depends on the partitioning of substances in the cyto­plasm.

Another observation has been made in case of the embryonic development of egg of the round worm, Ascaris. During the development of embryo, the first cleavage occur perpendicular to the animal vegetal axis.

When the animal pole of the new blastomere starts dividing, the heterochromatic portion of the chromosome becomes degenerated. The euchromatic portion of the chromosome is fragmented into numerous small chromosomes by a process known as chromosome diminution.

Thus chromosome diminution occurs during embryonic develop­ment. Now Theodor Boveri made an exper­iment in which eggs are centrifuged before cleavage in order to disturb the polarity of the cell.

By centrifugation, the mixture of both animal and vegetal cytoplasm occurs and the first cleavage occurs along the animal vegetal axis not perpendicular to it. No chromosome diminution occurs—this indicates the role of cytoplasm in chromosomal behaviour.

Again, in case of embryonic development of Drosophila, the primordial germ cells generally arise from the posterior end of the egg. Illmensee and Mahowald made an experiment by removing some cytoplasm from the posterior end of the egg of Drosophila and injected then into the anterior end of another egg. It has been noted that germ ceils are then produced from the anterior end of the injected egg.

Thus it can be said that the cytoplasm has an important role in inducing differentiation in cells. Its role has also been noted in adult cells. When the inactive nuclei of mature erythrocytes are injected into the cytoplasm of active cells, the inactive nuclei become transcriptionally active.

Similar effect has also been noted in other types of cells. All these results clearly reveal that cytoplasmic factors are responsible for cell differentiation. But in case of mammals, embryonic cells are totipotent after the first cleavage division till the development of the embryo.

This has been evidenced by segregating mouse or rabbit embryo at 8 or 16 cells stage. Each blastomere gives rise to blastocyst if the proper cultural conditions are provided and these blastocysts will form normal animal after implantation into the uterus of another female rat. This property of totipotency of egg cell is due to the homogeneity of its cytoplasm.

The retention of totipotency by an individual cell has been demonstrated in case of plant systems. Any cell from any part of the plant can be grown in culture where they form at first the mass of undifferentiated tissue, known as callus.

This callus can be regenerated into a whole plant if the proper concentration of the hormone is supplemented in the media. But single animal cells after the first cleavage division will not form full animal even if the proper nutrients and other conditions are given in culture.

Genetic totipotency of animal cell has been demonstrated by John Gurdon in the nuclear transplantation experiment in toad; showing that each and every cell possesses all the genes required for the development of the whole or­ganism.

Still the development or differentiation into whole organism has not been in animal sys­tem. Thus researchers thought that the pattern of gene expression in each cell type is different, although all the genes are present in each cell. So we see that the control of gene expression in higher organisms, particularly animals, is very complicated.

In other words, many types of post-transcriptional modifications of the pri­mary transcript of the gene are regulated in the formation of the final functional gene product necessary for a particular cell differentiation or development.

Two model organisms are used for the study of the specific genes involved for a particu­lar cellular development. One is the round­worm, Caenorhabditis elegans, and another is Drosophila, C. elegans.

These are used as a model in the developmental genetics for the following reasons:

i. Short life cycle (3 days).

ii. Ease of maintenance like E. coli.

iii. Can reproduce by self or by cross- fertilization.

iv. Hermaphrodite (XX)—contains 5 pairs of autosomes and 1 pair of X chromosomes.

v. The male (XO) contains 5 pairs of auto­somes and a single X chromosome.

vi. Haploid genome is about 8 x 10⁷ bp.

vii. More than 600 genes have been identified.

viii. Easy to obtain homozygous populations, as self-fertilization is possible.

ix. In-breeding is automatic in hermaphrodite population.

x. DNA transformation through microinjec­tion at the selected stage of development is possible in this animal.

The reproductive system of the hermaphro­dite animal produces a bilobed structure in early stages of development. The oocytes and embryos in each lobe start developing from the distal end to the uterus. All stages can be detected easily and thus microinjection of DNA can be done at the selected stage of development.

Another important work on Drosophila made the discovery of new class of genes called Homeotic genes. These genes were identified through mutations where one part of the body is replaced by a structure that is found somewhere else. This unusual thing occurs during development of the embryo. The mutations of homeotic gene (Antp) result in the formation of middle legs in place of antenna of Drosophila.

The molecular analysis of homeotic gene has resulted in the discovery of a 180bp sequence present in other homeotic genes. This sequence is known as Homeobox. Since the homeobox is present in higher animals also, gene cloning and sequencing has been done from mice to man. The function of these genes has been found to be the same as in Drosophila.

The homeobox genes are expressed in highly differentiated cells in case of Amphibians and Mammals. Home­obox genes are found to play very important role in developmental processes and they are highly conserved during evolution.

Homeotic Genes of Cellular Differentiation:

During embryonic development in Drosophila, the identification of different segments of the body has been found to be under the control of many genes. Two complexes of these genes have been identified in Drosophila.

One is Antennapedia Complex (ANT-C) located in the chromosomal position 84AB. Another group of genes is known as Bi-thorax complex (BX-C) located at chromosomal position 89E. The first group (ANT-C) is responsible for the develop­ment of the head and thoracic segments while the BX-C are responsible for the development of the trunk segments.

The analysis of this homeotic complex has been done in Beetle also. One of the important features of these genes is the large size—about 50 to 100 kb and very large introns. The next important feature is the presence of con­served region, i.e., Homeobox.

The homeobox generally codes for a DNA binding protein domain, whose product binds to DNA. Another important characteristic is the presence of cis- acting regulatory regions.

Cellular differentiation, pattern formation and morphogenesis were studied in detail in case of plant Arabidiopsis as model system. It involves some factors which help in causing different cell types, organs etc. to originate at specific locations. This is also done by cell shape changes and planes of cell division. Cell differentiation is clearly noted during embryo- genesis.

Different and molecular observations were studied in maize and rice in case of monocotyledonous plants. In dicots, several genes involved in storage proteins have been cloned and their expressions noted in Soybean. However, detailed studies have been done in Crucifers, particularly in Arabidiopsis thaliana, in identifying embryo developmental genes but mutagenesis.

Arabidopsis has been accepted as a model system in having the following criteria:

i. Small size of the plant.

ii. Small size of the nuclear genome.

iii. Short generation-time.

iv. Self-fertilizing bisexual flowers.

v. Large number of progeny in each flower.

vi. Starchy endosperm is absent.

Molecular studies show that a number of genes are involved during early and late stage embryogenesis in both zygote and somatic em­bryo. Different cell division patterns have been noted in somatic embryos. Many pattern genes have been identified in Arabidopsis through mutagenesis, which show seedling abnormali­ties.

These mutants were named gnom (gn), affecting the apical and basal region, gurke (gk) affecting the apical region, faeckel (fx) affecting the central part of the seedling. There is the other mutants which change the shape of the seedling.

Several methods have been used for cloning pattern genes which has been used as an in­serted probe to isolate the flanking DNA in Arabidopsis. T-DNA tagging approach is used to clone the first embryo pattern gene of Ara­bidopsis. The study of plant developmental genetics and cell differentiation have been changed con­siderably with the isolation of several mutant phenotypes and biochemical mutants in Ara­bidiopsis.

More than 500 embryo-defective mutants and many mutants showing aberration in vegetative development have been analysed in Arabidiop­sis. Arabidiopsis Biological Resource Centre has been established in the Ohio State Univer­sity for the supply of mutant seeds and DNA.

Some of these mutants have been found to alter in basic cellular functions necessary for normal growth and development, i.e., the function of ‘house-keeping genes’.

The molecular basis of these mutants have also been studied to find out the relationship between gene function and nor­mal development. The first known biochemical mutant noted in Arabidiopsis is bio I auxotroph (mutant 122 G-E).

This mutant bio I stops growth of the embryo at the heart stage of development but shows normal growth when biotin is added in the medium. The normal bio-synthetic pathway of biotin in bacteria is shown in Fig. 19.1.

It has been noted that bio I mutants were defective in the synthesis of 7, 8- diaminopelargonic acid from 7-keto 8 diaminopelargonic acid. This step is regulated in bacteria by bio A gene. When the bio A gene from bacteria is introduced into the bio I mutant plants, the transgenic plants do not require biotin for their growth.

Thus this experiment has shown that a plant mutant can be recovered to normal by the introduction of cloned bacterial gene.

Another mutant ‘gnom’ or emb 30 shows another type of defect in basic cellular function. Meinke (1985) first observed the morphologi­cal structure as fused cotyledons and rootless plants which can be grown in culture. He also identified the first allele (112A-2A) of this mutant.

Many other alleles have been identified in other laboratories. This mutant and other embryo-defective mutants showed alterations in embryonic pattern formation and altered pattern of cell division during embryo development.

DNA sequence analysis of this mutant (EMB 30) shows homology with the SEC 7 gene of yeast which helps in the protein transport from the Golgi, indicating its role in transporting some proteins to the cell surface. It may also have an alteration in the signal transduction pathway.

Another interesting mutant is ‘fusca’ which shows insufficient accumulation of anthocyanin in developing cotyledons. Seeds of this mutants germinate to produce defective seedling which fail to complete the life cycle. Sev­eral genes of ‘fusca’ mutants have now been cloned and sequenced. These are: FUSI/COPI FUS2/DETI, FUS6/COP11, FUS7/COP9 etc.’ These genes encode some novel proteins.

The product of COPI gene has N-terminal zinc binding domain and a C-terminal domain which shows homology with the B-sub-unit of G” proteins. Again, the sequence of FUS6 gene shows similarity with that of human gene GPSI. Thus, it can be said that G proteins also play an important role in signal transduction pathways of plants.

The pattern formation and morphogenesis go hand in hand during development in multicel­lular organisms. Cyto differentiation is nothing but a division of labour between component cells. During cyto differentiation, some alter­ations in the biochemical and structural prop­erties occur leading to functional specialisation.

In the developmental stage the formation of cell diversity is the process of cell pattern or shape formation where positional information determines the final development of a cell. In the plant systems, the presence of any devel­opmental memory has not been clearly pointed out.

But the involvement of some localised activ­ity of specific regulatory proteins in Cyto differentiation and development has been estab­lished. It has been observed that the de­velopment of floral organs in Arabidiopsis is regulated by some genes encoding transcription factors.

The generation of individual cell types within an organ is dependent on the cell autonomous expression of regulatory molecules. Larkin (1993) has shown the role of transcription factors in trichome differentiation.

The mutational studies have shown that mutations in a specific gene can inhibit the development of individual cellular domains, keeping the other domains normal. For example, monopteros mu­tation shows no development of hypocotyl and root but the shoot meristems and cotyledons are not affected.

But there are some mutants like emb 30/gnom, hydra/fuss rootless and ‘monopteros’ which show defective shape and also abnormalities in cell differentiation, par­ticularly in vascular tissue organisation.

That means the respective gene products are es­sential both for morphogenesis and cellular differentiation. Thus, the relative positioning of cells in plant development is very important to continue the cell-cell signalling events during morphogenesis.