10.3Determination of Embryo Directionality
The direction of the body is determined immediately after fertilization in terms of, for example, which side of the embryo becomes the head/tail, and maternal factors pre-stored in the oocyte play a pivotal role in this process. Since the importance of these factors in development has been studied in depth for fruit flies and frogs, these organisms are used as examples in the discussions below.
Cell Lineage of Nematodes
Cell lineage, like a family tree, is a line that shows the history of a fertilized egg going through cleavage and subsequently being differentiated into various parts of the body. Since humans consist of an enormous number of cells (60 trillion in an adult), it is impossible to trace the history of all cells from fertilized egg to adult body. However, in nematodes, which measure only 1 mm in length, the number of cells in an adult body is only 959. The initial number is 1,090, but 131 cells are programmed to die during the developmental process (this is known as apoptosis; see Chapter 9). It is therefore easy to create a cell lineage for nematodes, as they consist of such small numbers of cells. Indeed, a cell lineage map for nematodes, from the fertilized egg to all cells that make up a mature individual, has already been created (Column Fig. 10-1). Furthermore, the complete DNA sequence of nematodes (consisting of approx. 100 million base pairs) has been determined, and it is estimated that approximately 17,500 genes are distributed over six chromosomes. As multicellular organisms on an easily manageable scale, nematodes have therefore been widely used as a material suitable for analyzing the basic mechanisms of organisms at the molecular level, including developmental mechanisms, functional analysis of the neural network and the causes of diseases.
Development of Fruit Flies
In fruit flies, as shown in Figure 10-1, a number of maternal factor types that determine the directionality of the embryo (i.e., the head and tail sides) are stored in the oocyte during Oogenesis. Among these, the major factors are mRNA for two protein - bicoid and nanos. These mRNAs are stored disproportionately in both sides of the oocyte. After fertilization, they are translated and the concentration gradient of bicoid and nanos is created along the lengthwise axis of the embryo (Fig. 10-4).
In the early stages of development in fruit flies, only the nucleus divides, and the plasma membrane that envelops it is not newly created; the embryo therefore takes on the appearance of a multinucleated cell. At this stage, all the nuclei are in the same cytoplasm. Under these circumstances, when the concentration gradient of bicoid and nanos is created in the embryo, the germ cell nuclei are directly exposed to the gradient. Since bicoid and nanos are factors that regulate gene expression and protein synthesis, the expression of new genes and protein synthesis take place in accordance with their concentration gradients (Fig. 10-4).
Fig. 10-4. The process from determination of directionality in the fruit-fly embryo to segmentation of the embryo into 14 parts
The gene expression pattern observed during the process of bicoid and nano protein expression and the subsequent segmentation of the embryo into 14 parts is shown here. See Column Figure. 10-2 on homeobox genes.
The expression of gap genes occurs in accordance with the concentration gradient patterns of bicoids and nanos, and the proteins translated from gap genes are known as transcription factors. As a result, based on the expression pattern of the gap genes, pair-rule genes are expressed in a way that forms seven stripes in the embryo (this mechanism is discussed later). The proteins translated from the pair-rule genes are also transcription factors. Then, based on the expression pattern, segment-polarity genes are expressed in a way that forms 14 stripes in the embryo. As a result of the cascade expression of these gene groups, 14 regions (or compartments) are formed in the embryo, which is the origin of the 14 basic regions of fruit flies (i.e., the segmental structure of insects). The cascade expression of these gene groups takes place in accordance with the concentration gradient patterns of transcription factors (Fig. 10-5), which are the proteins that regulate the expression of target genes by binding to their regulatory regions (see Chapter 4). Such regulation may enhance or suppress the expression of target genes. Figure 10-5A shows an example of the expression of a target gene being enhanced only in regions where the transcription factor concentration is within a certain range, and otherwise being suppressed. In this case, the expression of target genes is induced when the concentration is within a certain range. Figure 10-5B shows an example of the expression of a target region occurring in an area where the action of enhancing transcription factors dominates that of inhibitory transcription factors.
During the process from the formation of the concentration gradients of bicoids and nanos to the expression of segment polarity genes, 14 regions are formed in the embryo based on the expression pattern of the genes. As the next step, the fate of the 14 regions (in terms of the organs to be created from each) must be determined. A group of genes called homeobox genes play an important role in this process (Column Fig. 10-2).
Fig. 10-5. Patterns of a new gene being expressed in accordance with the transcription factor concentration gradient
A) An example of a gene being expressed only in a region where the transcription factor concentration is within a certain range. In this case, upper and lower limits (thresholds) of the transcription factor concentration causing gene expression exist.
B) An example of a gene being expressed in a region where the concentration balance of transcription factors that inhibit gene expression and those that enhance it is within a certain range. The enhancement of gene expression is based on the combined effects of the different promoting factors.
During the long history of research into fruit flies, many mutants with structural abnormalities have been reported. Recent gene-level analysis has identified a series of genes that cause structural abnormality in the body of fruit flies. These are called homeotic gene complexes (HOM-C), and consist of a group of eight genes (Column Fig. 10-2A). It has also been found that gene groups equivalent to these exist in four clusters (HoxA-D) in mammals.
In addition to this correspondence in the sequence of genes in chromosomes between HOM-C in fruit flies and Hox in vertebrates, their expression patterns in the body are also similar. As an example, the expression pattern of HOM-C in fruit flies (which is expressed in an anteroposterior direction along the body) and that of Hox in vertebrates (which is expressed along the head-tail axis) are very similar (Column Fig. 10-2B).
The series of genes contained in HOM-C and Hox are called homeobox genes, as all proteins translated from such genes have a region consisting of 60 amino acids called a homeodomain. The homeodomain is a region with a special tertiary structure for binding to DNA (Column Fig. 10-2C). The proteins translated from homeobox genes are transcription factors that regulate the expression of other genes by attaching to them. It is believed that homeobox genes determine which region becomes which organ after the rough regional division that takes place in the embryo.
Column Fig. 10-2. Homeobox genes
A) HOM-C in fruit flies and HoxA-D in mammals are shown here. ↔ indicates the correspondence between the two.
B) The similarity of gene expression between HOM-C in fruit flies and Hox in vertebrates. The homeobox genes in fruit flies are expressed in the body in accordance with the gene sequence on the chromosomes. This is also the case with homeobox genes in vertebrates. As an example in mice, a part of the homeobox genes expressed in the brain and the spinal cord is shown. The arrow shows the directionality of homeobox gene expression along the head-tail axis.
C) The tertiary structure of the region known as the homeodomain, which all proteins translated from homeobox genes have, is shown here. It has three α-helices with which it binds to DNA.
Development of Frogs
Next, we discuss development of frog embryo as an example of vertebrate development. The upper hemisphere of the frog egg, which is rich in pigment and therefore dark, is called the animal pole, while the opposite hemisphere, which is white, is called the vegetal pole. Before fertilization, the egg has only animal-vegetal-pole directionality. However, upon fertilization, the future dorsal and ventral sides are determined in the embryo. This is because the surface part of the cytoplasm in the egg moves in a particular direction after fertilization. This in turn causes the movement of the maternal factors that determine the dorsal side of the embryo from the vegetal pole to near the equator on one side of the embryo (Fig. 10-6). As a result, the side opposite the sperm entry point becomes the future dorsal side. Thus, in the development of frogs too, maternal factors stored locally in the egg play an important role in determining the directionality of the embryo.
A protein called Dishevelled is among the important maternal factors that are stored in the vegetal pole of the embryo and turn the region to which they move into the dorsal side. This protein moves to a region where it causes the expression of new genes, thereby inducing that side of the embryo to become the dorsal side (Column Fig. 10-3). As a result, a region called the organizer, which determines the dorsal side of the embryo, is created on the side that the Dishevelled moves to. The basic structure of the frog’s body is formed around this region.
Roles of Maternal Factors in Determining the Dorsal Side of the Frog Embryo
Dishevelled suppresses the degradation of a transcription factor called β-catenin. This means that β-catenin increases in the cytoplasm on the side of the embryo to which Dishevelled moves. The increased amount of β-catenin moves into the nucleus and causes the expression of its target, the Siamois gene. Siamois (a transcription factor) causes the expression of its target (the goosecoid gene) in cooperation with other transcription factors known as Smads. The goosecoid gene has a strong effect in inducing the formation of the dorsal side in the embryo.
Column Fig. 10-3. Process of Dishevelled determining the dorsal side of the embryo
The process of maternal factors determining the dorsal side of the embryo takes place mainly through the regulation of gene expression by transcription factors. During the process, maternal factors and extracellular signals collaborate.