The initial gradients of Bicoid and Nanos mRNAs translate into reiterated segments through a hierarchy of gene activity that occurs. The hierarchy of gene activity follows the pattern that Maternal Effect genes regulate Gap genes, Gap genes regulate Pair-Rule genes, and Pair-Rule genes regulate Segment Polarity genes.
Maternal effect genes, such as Bicoid, act as transcription factors to specify the production of Gap genes throughout the oocyte. Experimental evidence suggest that the production of certain Gap genes is regulated by the concentration of maternal determinants such that some Gap genes are expressed when concentration is high, some are expressed when concentration is low, and some when concentration is between high and low. Additionally, once Gap genes are produced they interact with other Gap genes to further refine their region of expression.
Gap genes, such as hunchback, regulate the expression of Pair-Rule genes. Specifically, Gap genes act as transcription factors by binding to enhancer and repressor regions of Pair-Rule genes to regulate their transcriptional expression. Importantly, each Pair-Rule gene is regulated by several Gap genes such that an individual Gap gene is not solely responsible for the expression of a single Pair-Rule gene. Pair-Rule genes go on to regulate the expression of Segment Polarity genes, which reinforce embryo segmentation and specify the cells of each individual segment.
Upon fertilization the amphibian embryo undergoes a dramatic morphological rotation towards the site of sperm entry that reveals an inner band of grey cytoplasm opposite the site of sperm entry. Ultimately, cortical rotation and grey crescent cytoplasm mark and specify the dorsal region of the amphibian embryo by regulating localizing GPB, Wnt11 and Disheveled proteins to the future dorsal region of the embryo. GPB will inhibit the degradation of B-Catenin, which will lead to specification of dorsal tissue.
Experimentally, researchers noted that when cortical rotation was prevented with UV radiation the resulting embryo formed into a belly piece, which lacked clearly defined axes. However, if cortical rotation was inhibited and then artificially rotated 90-degrees, the resulting embryo developed normally. This experiment highlighted the importance of cortical rotation.
As a follow-up experiment, Spemann and his colleges noted that when an initial embryo was divided into two separate embryos, only embryos that contained grey crescent tissue developed normally, while separated embryos that lacked grey crescent tissue developed into belly pieces. These experiments established that cytoplasmic determinants from the grey crescent region were essential for specification of the embryo axes.
Gastrulation in the frog embryo occurs in four main steps: invagination, involution, convergent extension and epiboly.
Invagination of the frog embryo occurs opposite the site of sperm. The cells regulating invagination, called bottle cells, undergo apical constriction where the apical-facing aspect of the cell tightens "like a purse string" and promotes an inward buckling of the membrane. Experimentally, researchers noted the ability of cells from the dorsal blastopore lip to induce invagination when transplanted into regions of the embryo that do not undergo invagination during normal development.
Involution of the frog embryo occurs when involuting marginal cells migrate along the outer animal hemisphere cells, effectively "towing" a string of cells towards the interior of the embryo. These leading edge cells propel their migration by traveling upon extracellular matrix proteins.
Convergent extension follows involution, occurring when cells at the blastopore lip merge together in a manner similar to cars merging during traffic. The process of numerous, small individual cell movements causes a dramatic overall lengthening of the forming archenteron. Molecularly, convergent extension operates through lamellipodia extensions regulated by PCP and Disheveled signaling.
Lastly, epiboly occurs when animal cap cells and non-involuting cells spread out over the exterior of the embryo. This process is mediated by the flattening of cells such that cells transition from a cuboidal to a squamous shape.
The patterns of embryonic cleavage differ dramatically between amphibian, avian and mammalian embryos.
Cleavage of the amphibian embryo is holoblastic, meaning that the cleavage furrow divides the cells completely during each division. Initial division of the amphibian embryo is radially symmetrical; and while division of the yolk is not as dramatic as is seen in fish or avian embryos, the yolk is divided unequally between cells.
Cleavage of the avian embryo occurs in the oviduct prior to encasing of the embryo in an eggshell. Division of the embryo is telolecithal, meaning that the cleavage furrow does not completely divide the cells during division. Initial cleavage is discoidal, with a large yolk cell forming the base of the embryo, and several, rapidly dividing animal pole cells comprise a disc that sits atop the yolk. At this time, some blastoderm cells absorb water to form the subgerminal cavity, which later helps to form the area pellucida where most of the actual embryo is formed.
The mammalian embryo undergoes holoblastic rotational cleavage, where the second cleavage has one cell divide equitorally and the other cell divide meridionally. Several characteristics of mammalian cleavage are unique. Specifically, the timing of division is significantly slower than observed in other animals, and cells do not divide synchronously. Additionally, the mammalian embryo undergoes compaction, where cell adhesion proteins (cadherins) are expressed promoting the tight grouping of cells.
Scientist initially identified the existence of tissue interactions during neural crest cell formation through a series of in vitro isolation experiments. These experiments demonstrated that several neural crest cell markers were not in isolated neural tube tissue - indicating that, by itself, neural tube tissue was unable to induce neural crest cell formation. However, when tissue from the neural tube was co-cultured with tissue from non-neural epiderm, markers for neural crest cell induction were expressed. These results indicated that the induction of neural crest cells was dependent upon neural tissue, epidermal tissue and the interactions that take place between the two tissues.
Wnt signaling was also identified to play a role in neural crest cell induction. Researchers noted that, when isolated, neural tube tissue could not develop neural crest cells - a result similar to the above experiment. However, when neural tube tissue was bathed in wnt-rich media, neural crest cells formed and dispersed away from the neural tube. This result indicated a role for wnt signaling during neural crest cell induction. Further experiments validated this result. Specifically, insertion of beads that inhibited wnt activity prevented the formation of neural crest cells, and knocking down the expression of frizzled (a wnt receptor) via morpholino injection led to a loss of pigmentation (neural crest cell derived) in zebrafish.
BMP signaling plays important roles in both the induction of neural crest cells and in the initial dispersal of neural crest cells from the neural tube.
Based on the previously studied role of bmp signaling in amphibian axis specification, researchers suspected that bmp signaling might play a role in the specification of neural tube. Researchers determined that a gradient of bmp signaling specifies neural crest cells - specifically high levels of bmp and non-existent bmp signaling do not specify neural crest cell development, while low, "Goldilocks-levels" of bmp signaling specify neural crest cell development. Experimentally, researchers discovered that embryos that artificially overexpressed bmp failed to develop neural crest cells, while embryos that expressed limited amounts of bmp developed neural crest cells throughout the embryo.
During the initial dispersal of neural crest cells, the regulation of bmp expression plays a critical role in the timing of dispersal. Researchers have determined that, while bmp is dispersed throughout the embryo, a variety of other factors are localized to neural crest cells. These factors act to regulate the activity of bmp, which ultimately promotes dispersal of neural crest cells. Specifically, somatic cells inhibit that activity of noggin, which in turn inhibits the activity of bmp. Therefore activation of somatic cells activates bmp activity in neural crest cells by preventing noggins inhibition of bmp. After activation, bmp activates an unidentified protease enzyme that degrades N-cadherin, which under normal circumstances acts as a cell adhesion molecule to attach neural crest cells to the neural tube. In sum: somatic cells activate bmp by inhibiting noggin activity. And bmp facilitates the dispersal of neural crest cells by degrading their N-Cadherin mediated attachment to the neural tube.
Ethanol exposure has been shown to have two main effects on the development of neural crest cells. First, ethanol exposure increases cell death, specifically for neural crest cells. One experiment noted that exposure to ethanol in chick embryos led to a 3-fold increase in neural crest cell death. Therefore, ethanol exposure leads to fewer migrating neural crest cells. Second, ethanol exposure has also been shown to disrupt the migration of neural crest cells by disrupting several key signaling molecules that normally direct the migration of neural crest cells. Interestingly, researchers have conducted experiments on SHH (a signaling messenger) that demonstrated that restoration of SHH expression after ethanol exposure restored normal phenotype.
From a public health perspective, ethanol exposure in utero can result in mental retardation, craniofacial anomalies, heart defects, deficient myelination, and limb or join defects.