In the field of developmental biology, regional differentiation, or regional specification is the process by which different areas are identified in the development of the early embryo.Slack, J.M.W. (2013) Essential Developmental Biology. Wiley-Blackwell, Oxford. The process by which the cells become specified differs between organisms.
Cell fate determination
In terms of developmental commitment, a cell can either be specified or it can be determined. Specification is the first stage in cellular differentiation. A cell that is specified can have its commitment reversed while the determined state is irreversible.Slack, J.M.W. (1991) From egg to embryo. Regional specification in early development. Cambridge University Press, Cambridge There are two main types of specification: autonomous and conditional. A cell specified autonomously will develop into a specific fate based upon cytoplasmic determinants with no regard to the environment the cell is in. A cell specified conditionally will develop into a specific fate based upon other surrounding cells or morphogen gradients. Another type of specification is syncytial specification, characteristic of most insect classes.
Specification in sea urchins uses both autonomous and conditional mechanisms to determine the anterior/posterior axis. The anterior/posterior axis lies along the animal/vegetal axis set up during cleavage. The micromeres induce the nearby tissue to become endoderm while the animal cells are specified to become ectoderm. The animal cells are not determined because the micromeres can induce the animal cells to also take on mesodermal and endodermal fates. It was observed that β-catenin was present in the nuclei at the vegetal pole of the blastula. Through a series of experiments, one study confirmed the role of β-catenin in the cell-autonomous specification of vegetal cell fates and the micromeres inducing ability. Treatments of lithium chloride sufficient to vegetalize the embryo resulted in increases in nuclearly localized b-catenin. Reduction of expression of β-catenin in the nucleus correlated with loss of vegetal cell fates. Transplants of micromeres lacking nuclear accumulation of β-catenin were unable to induce a second axis.
For the molecular mechanism of β-catenin and the micromeres, it was observed that Notch was present uniformly on the apical surface of the early blastula but was lost in the secondary mesenchyme cells (SMCs) during late blastula and enriched in the presumptive endodermal cells in late blastula. Notch is both necessary and sufficient for determination of the SMCs. The micromeres express the ligand for Notch, Delta, on their surface to induce the formation of SMCs.
The high nuclear levels of b-catenin results from the high accumulation of the disheveled protein at the vegetal pole of the egg. disheveled inactivates GSK-3 and prevents the phosphorylation of β-catenin. This allows β-catenin to escape degradation and enter the nucleus. The only important role of β-catenin is to activate the transcription of the gene Pmar1. This gene represses a repressor to allow micromere genes to be expressed.
The aboral/oral axis (analogous to the dorsal/ventral axes in other animals) is specified by a nodal homolog. This nodal was localized on the future oral side of the embryo. Experiments confirmed that nodal is both necessary and sufficient to promote development of the oral fate. Nodal also has a role in left/right axis formation.
Tunicates Tunicates have been a popular choice for the study of regional specification because tunicates were the first organism in which autonomous specification was discovered and tunicates are evolutionary related to vertebrates.
Early observations in tunicates led to the identification of the yellow crescent (also called the myoplasm). This cytoplasm was segregated to future muscle cells and if transplanted could induce the formation of muscle cells. The cytoplasmic determinant macho-1 was isolated as the necessary and sufficient factor for muscle cell formation. Similar to Sea urchins, the accumulation of b-catenin in the nuclei was identified as both necessary and sufficient to induce endoderm.
Two more cell fates are determined by conditional specification. The endoderm sends a fibroblast growth factor (FGF) signal to specify the notocord and the mesenchyme fates. Anterior cells respond to FGF to become notocord while posterior cells (identified by the presence of macho-1) respond to FGF to become mesenchyme.
The cytoplasm of the egg not only determines cell fate, but also determines the dorsal/ventral axis. The cytoplasm in the vegetal pole specifies this axis and removing this cytoplasm leads to a loss of axis information. The yellow cytoplasm specifies the anterior/posterior axis. When the yellow cytoplasm moves to the posterior of the egg to become posterior vegetal cytoplasm (PVC), the anterior/posterior axis is specified. Removal of the PVC leads to a loss of the axis while transplantation to the anterior reverses the axis.
C. elegans In the two cell stage, the embryo of the nematode C. elegans exhibits mosaic behavior. There are two cells, the P1 cell and the AB cell. The P1 cell was able make all of its fated cells while the AB cell could only make a portion of the cells it was fated to produce. Thus, The first division gives the autonomous specification of the two cells, but the AB cells require a conditional mechanism to produce all of its fated cells.
The AB lineage gives rise to neurons, skin, and pharynx. The P1 cell divides into EMS and P2. The EMS cell divides into MS and E. The MS lineage gives rise to pharynx, muscle, and neurons. The E lineage gives rise to intestines. The P2 cell divides into P3 and C founder cells. The C founder cells give rise to muscle, skin, and neurons. The P3 cell divides into P4 and D founder cells. The D founder cells give rise to muscle while the P4 lineage gives rise to the germ line.
Axis specification The anterior/posterior axis is specified by the sperm at the posterior side. At the two cell stage, the anterior cell is the AB cell while the posterior cell is the P1 cell. The dorsal/ventral axis of the animal is set by a random position of cells during the four cell stage of the embryo. The dorsal cell is the ABp cell while the ventral cell is the EMS cell.
Localization of cytoplasmic determinants The autonomous specification of C. elegans arises from different cytoplasmic determinants. PAR proteins are responsible for partitioning these determinants in the early embryo. These proteins are located at the periphery of the zygote and play a role in intracellular signaling. The current model for the function of these proteins is that they cause local changes in the cytoplasm that lead to different protein accumulation in the posterior vs. the anterior. Mex-5 accumulates in the anterior while PIE-1 and P granules (see below) accumulate in the posterior.
Specification of germ line P granules were identified as the cytoplasmic determinants. While uniformly present at fertilization, these granules become localized in the posterior P1 cell prior to the first division. These granules are further localized between each division into P cells (ex. P2, P3) until after the fourth division when they are put into the P4 cells which become the germ line.
Specification of EMS and P1 cells Other proteins that are likely to function as localized cytoplasmic determinants in the P1 lineage include SKN-1, PIE-1 and PAL-1.
SKN-1 is a cytoplasmic determinant that is localized in the P1 cell lineage and determines EMS cell fate. PIE-1 is localized in the P2 cell lineage and is a general repressor of transcription. SKN-1 is repressed in P2 cells and is unable to specify an EMS fate in these cells. The repressive activity of PIE-1 is required to keep the germ line lineage from differentiating.
Specification of C and D founder cells PAL-1 is required to specify the fates of the C and D founder cells (derived from the P2 lineage). PAL-1, however, is present in both EMS and P2. Normally, PAL-1 activity is repressed in EMS by SKN-1 but not repressed in P2. Both C and D founder cells depend on PAL-1 but there is another factor that is required to distinguish C from D.
Specification of E lineage The specification of the E lineage depends on signals from P2 to the EMS cell. Components of Wnt signaling were involved and were named mom genes. mom-2 is a member of the Wnt family of proteins (i.e. the signal) and mom-5 is a member of the frizzled family of proteins (i.e. the receptor).
Specification of ABa and ABp The specification of ABa and ABp depend on another cell-cell signaling event. A difference between these two cell types is that ABa gives rise to anterior pharynx while ABp does not contribute to pharynx. A signal from MS at the 12-cell stage induces pharynx in ABa progeny cells but not in ABp progeny. Signals from the P2 cells prevent the ABp from forming pharynx. This signal from the P2 was discovered to be APX-1 within the Delta family of proteins. These proteins are known to be ligands for the Notch protein. GLP-1, a Notch protein, is also required for specification of the fate of ABp.
Drosophila
Anterior/posterior axis The anterior/posterior patterning of Drosophila come from three maternal groups of genes. The anterior group patterns the head and thoracic segments. The posterior group patterns the abdominal segments and the terminal group patterns the anterior and posterior terminal regions called the terminalia (the acron in the anterior and the telson in the posterior).
The anterior group genes include bicoid. Bicoid functions as a graded morphogen transcription factor that localizes to the nucleus. The head of the embryo forms at the point of highest concentration of bicoid and the anterior pattern depends upon the concentration of bicoid. Bicoid works as a transcriptional activator of the gap genes hunchback (hb), buttonhead (btd), empty spiracles (ems), and orthodentical (otd) while also acting to repress translation of caudal. A different affinity for bicoid in the promoters of the genes it activates allows for the concentration dependent activation. Otd has a low affinity for bicoid, hb has a higher affinity and so will be activated at a lower bicoid concentration. Two other anterior group genes, swallow and exuperantia play a role in localizing bicoid to the anterior. Bicoid is directed to the anterior by its 3' untranslated region (3'UTR). The microtubule cytoskeleton also plays a role in localizing bicoid.
The posterior group genes include nanos. Similar to bicoid, nanos is localized to the posterior pole as a graded morphogen. The only role of nanos is to repress the maternally transcribed hunchback mRNA in the posterior. Another protein, pumilio, is required for nanos to repress hunchback. Other posterior proteins, oskar (which tethers nanos mRNA), Tudor, vasa, and Valois, localize the germ line determinants and nanos to the posterior.
In contrast to the anterior and the posterior, the positional information for the terminalia come from the follicle cells of the ovary. The terminalia are specified through the action of the Torso receptor tyrosine kinase. The follicle cells secrete Torso-like into the perivitelline space only at the poles. Torso-like cleaves the pro-peptide Trunk which appears to be the Torso ligand. Trunk activates Torso and causes a signal transduction cascade which represses the transcriptional repressor Groucho which in turn causes the activation of the terminal gap genes tailless and huckebein.
Segmentation and homeotic genes