Specification in development

Development is the process by which higher organisms transition from a single celled zygote to the complex, multicellular adult. During this transition the cells of the developing embryo will undergo periods of patterning, specification, migration, rapid division, apoptosis and differentiation. These processes are regulated in a precise and concerted manner by a remarkably small set of developmental genes1,2. The expression of these developmental genes must be tightly restricted to specific spatial and temporal locations within the developing embryo. The result of these processes is the specification of populations of cells, cell lineages, which will go on to form all of the >200 cell types found in the complex metazoans, known colloquially as ‘higher animals’..

The specification of a cell lineage is the result of several processes acting in concert3. Firstly, the cell(s) must interpret the developmental cues from their surroundings to derive their ‘identity’. Secondly, the expression of specific regulatory genes necessary for this identity must be activated and then stabilised. Thirdly, alternative regulatory genes for alternative identities must be excluded. Finally, various lineage specific genes necessary for proper development of the lineage within the context of the overall embryo must be activated. When a decision is made to express a given regulatory gene, a pleiotropic gene regulatory network (GRN) is initiated where a cascade of interregulating genes are expressed that result in the appropriate course of development for the given lineage3.

2 The processes of development are initiated in response to a range of inter- and intracellular signalling cues. Many cues are derived from the overlapping gradients of signalling molecules throughout the embryo generated by specific groups of cells (such as dorsal-ventral orientation). Other cues, however, are transmitted via direct cell-cell contacts (such as Notch/delta signalling in pigmentation). A cell must integrate information from various competing and cooperating signals and determine the appropriate response. One mechanism of this integration process occurs during signal transduction: different signalling pathways might share common elements in their cascades and by affecting the activity of these elements, the information from different signalling sources is merged. The p38 mitogen activated protein kinase (MAPK) pathway is an example of a signalling pathway with multiple inputs4. The activation of specific regulatory factors within the nucleus, however, is the terminus of many signalling cascades. In the nucleus, integration of complementary and competing signals is achieved at the promoters, enhancers, silencers and other regulatory modules associated with specific target genes. The result of these regulatory interactions is the expression of genes that result in the assumption of a cellular identity. Although selected, in some cases the identity of a cell is still plastic, as demonstrated by tissue grafting experiments in the chick embryo5. Cells previously expressing genes specific for one location can be induced to express genes specific to another location once grafted to the new location and the alternative signalling cues are internalised and interpreted.

A cell lineage is maintained by a permanent alteration of the cell’s response to signalling. Elements of a signalling cascade may be sequestered, degraded or expression deactivated in order to make a cell deaf to a specific signal. Activation of a specific regulatory factor may initiate a positive feedback loop, reinforcing its own expression that, by virtue of its effect on the expression of downstream genes, locks in the identity of the cell to a specific lineage. Furthermore, epigenetic modification of the DNA and histones is

3 known to lead to the silencing of whole regions of DNA, preventing the expression of regulatory genes therein6–8. Silencing is usually achieved by a combination of epigenetic and histone modification driven by the recruitment of DNA methylases and histone deacetylases that serve to favour the packaging of DNA into silent, non-expressing heterochromatin9.

The spatial and temporal expression of genes necessary to specify a cell lineage is usually tightly controlled. Figure 1.1 exemplifies this using the specification of muscle progenitor cells in the developing embryo. The specification of a region of tissue in the embryo that will become the adult skeletal musculature is achieved by the overlapping presence of several signals; bone morphogenetic protein-4 (BMP4), noggin, Shh and the Wnt proteins. This process is discussed further in chapter 3.

Figure 1.1: Overlapping signals that lead to the specification of muscle cells in the developing embryo. Image shows a transverse section through a developing embryo; A central neural tube (top centre) and notochord (bottom centre) are flanked by a pair of somites on each side followed by a pair of lateral limb buds. Left half of the image shows the morphogenic fields of various transcriptions factors: Red; BMP4, cyan; noggin, yellow; Shh and blue; Wnt proteins. Right side of image shows the relative concentration of these factors in the different portions of the somite. Letters denote the tissues that are the sources of the different signalling molecules. The section highlighted in green on the left and by 1111 on the right is the area where muscle cell progenitors are specified. Image taken from Piran et al.10.

4 Bacteria are able to achieve a sufficiently sophisticated suite of regulatory control mechanisms by direct interactions between transcription factors and the core transcriptional machinery11. A population of bacteria may respond to changes in environment by evolving their responses appropriately, trimming excess genetic code and altering regulatory interactions. In contrast, the responses to environmental change of each of the >200 cell types of the higher animal must be encoded in each of the individual cell types. Remarkably, this function is achieved with relatively fewer transcriptional genes2. As a result, the regulatory interactions that ensure the appropriate responses occur are significantly more complex in higher animals than prokaryotes. In higher animals correct spatiotemporal gene regulation is achieved through the complex interactions of multiple DNA-binding proteins and their cognate binding sites in regulatory modules in the non-coding DNA. This is achieved with a relatively small amount of genes operating in pleiotropic networks and the mechanisms that result correct regulation of each gene are likely to be complex. To understand how genes are regulated, the mechanisms of their expression must first be understood.