Cellular and Molecular Biology Topics
Blastocyst Formation
In a series of mitotic divisions, the zygote cytoplasm is successively partitioned to form a blastula consisting of increasingly smaller blastomeres. After the 8-cell stage, blastomeres form a morula that has a tightly compacted outer cell mass sealing off the inner mass.
2-cell blastula
4-cell blastula
8-cell blastula
morula
(images from http://www.uco.es/organiza/departamentos/anatomia-y-anat-patologica/embriologia/tutorial/morula/morula.html)
Blastomeres are considered totipotent because they are capable of forming a complete embryo until the 4-8 cell stage. A totipotent cell is capable of forming any of the different cell types and tissues, ultimately needed to generate an entire organism.
The blastocyst is formed when fluid secreted within the morula forms the blastocyst cavity (blastocele). The inner cell mass is referred to as the embryoblast and will develop into the embryo. The outer cell mass is the tropoblast and will become the fetal portion of the placenta. The tropoblast will proliferate into the cytophoblast and the syntiotrophoblast.
morula
blastocyte
(images from http://www.uco.es/organiza/departamentos/anatomia-y-anat-patologica/embriologia/tutorial/blastula/blastula.html)
The hypoblast are cells that separate from the inner cell mass to line the blastocyst cavity, where they give rise to the yolk sac endoderm. The remaining inner cell mass above the hypoblast is now referred as the epiblast.
(images
from http://www.uco.es/organiza/departamentos/anatomia-y-anat-patologica/embriologia/tutorial/blastula/blastula.html)
The epiblast cells are split by a small cleft that functions to divide the epiblast into two layers, one leading to the embryonic epiblast, while the other forms the lining of the amnion. Once the lining of the amnion is completed it fills with amniotic fluid.
Implantation of the blastocyst usually occurs within the posterior superior wall of the uterus by day 7 after fertilization.
Gastrulation
Gastrulation is the process of establishing the three definitive germ cell layers of the embryo: ectoderm, mesoderm and endoderm. These layers form a trilaminar embryonic disk by day 21 of development. Cellular differentiation refers to the process that cells undergo in which changes lead to specialized tissues. Cellular determination is a commitment by an embryonic cell to a particular specialized path of differentiation. Once cellular determination occurs, there is a change in the internal properties of the cell. In the early embryonic environment, cellular determination starts as the cell germ layers are established.
Gastrulation begins with the formation of the primitive streak, which appears as a short line on the dorsal surface of the epiblast. The primitive streak is caused by a convergence of epiblastic cells, and its appearance identifies the antero-posterior and right-left axes.
Epiblast cells migrate to the primitive streak and pass through it on their way to form a new layer of cells between the epiblast and the hypoblast. The movement of cells through the primitive streak contributes to formation of the primitive groove. At the end of the primitive streak is a small accumulation of cells called the primitive node.
With the movement of epiblast cells through the primitive streak, there is a change in the structure as well as the organization of these cells. The majority of the invaginating cells form the mesoderm. The upper layer is the ectoderm (towards the amniotic cavity) and the lower layer is the endoderm (towards the hypoblast).
Epiblast cells passing through the primitive node are channeled into a rod-shaped mass of cells called the notochord.
At about 18 days after fertilization, the primitive streak extends caudally, extending the notochord along the longitudinal axis of the embryo. During that time, formation of the mesoderm continues by cells migrating from the epiblast through the primitive streak. Eventually, the primitive streak will disappear without a trace.
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Ectoderm Differentiaton (Neural Induction)
The rod shape notochord provides inductive signals that transform the overlying surface ectoderm into neural tissue (neural induction). This tissue forms a thickened neural plate overlying the notochord on the dorsal surface of the early embryo. The contours of the neural plate are altered, becoming narrower and longer.
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The process of neurolation (?) involves the lateral folding of the neural plate, elevating the sides along a midline neural groove. The neural tube forms by apposition and fusion of the two lateral apical surfaces of the neural folds. The complete segment of the neural tube separates from the overlying ectoderm sheet and cells of the neural crest separate from the neural tube.
Closure of the neural tube beguins midway along the cranial length of the nervous system at about day 22. The anterior (cranial) and posterior (caudal) neuropores ultimately close so that the entire future central nervous system resembles an irregular cylinder at both ends.
(see movie at http://laxmi.nuc.ucla.edu:8888/Teachers/pphelps/Published_Trays/Basic_Embryology_I/slide_20.html
)
Soon after the formation of the neural tube, the region of the future brain can be distinguished from the spinal cord. That region undergoes a series of subdivisions that provide the gross organization of the brain. Rare failure to close both neuropores can result in severe birth defects. A closure defect of the spinal cord is called rachischisis, and of the brain is called cranioschisis. A number of closure defects can be diagnosed by the detection of elevated levels of a-fetoprotein in the amniotic fluid or by ultrasound scanning.
Mesoderm Differentiation (Bones, Muscle, Limbs, Kidneys and Gonads)
After passing through the primitive streak, the mesodermal cells spread laterally as a continuous layer between the ectoderm and endoderm. There are three regions of the mesoderm: paraxial, intermediate and lateral. Nearest to the neural tube is the paraxial mesoderm. The intermediate mesoderm and lateral mesoderm are adjacent to the paraxial region.
The paraxial mesoderm becomes organized into segments known as somitomeres that form in the cranial region. Somitomeres 1-7 contribute mesoderm to the pharyngeal arches. The remaining somitomeres further condense in a cranio-caudal sequence to form pairs of somites.
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Somites are one of a series of paired blocks of mesodermal tissue that form
on either side of the notochord. A pair of somites first appears around day
20, and are established at the expense of eight somitomeres. Thereafter, somites
are generated at a rate of about 3 per day.
The somites give rise to vertebrae, with each somite generating one vertebrae, associated musculature and connective tissue. To derive these tissues, somites differentiate into sclerotome (cartilage and bone), myotome (muscle) and dermatome (dermis).
The intermediate mesoderm forms a longitudinal dorsal ridge known as the urogenital ridge, which is involved in the formation of the kidneys and gonads. The lateral mesoderm divides into two layers that will contribute to the formation of the heart, body walls and limbs.
Endoderm Diferentiation (Gut and Lungs)
Early in the third week, the endodermal sheet constitutes the roof of the yolk sack. Lateral folding of the embryonic body and the ventral bending of the cranial and caudal ends of the embryo into a C-shaped structure contributes to the formation of the tubular foregut and hindgut.
(see movie at http://laxmi.nuc.ucla.edu:8888/Teachers/pphelps/Published_Trays/Basic_Embryology_I/slide_21.html)
This process also begins the delineation between the yolk sack and the gut proper. The constricted region becomes the yolk stalk, with the embryonic gut above and the yolk sack below. The portion of the gut that is still open into the yolk sack is referred to as the midgut.
The anterior end of the foregut remains temporarily sealed by the ectodermal-endodermal bilayer called the oropharyngeal membrane. The membrane separates the future mouth from the pharynx, the endodermally lined anterior part of the foregut. The bilayer is unstable and eventually breaks.
(see
movie at
http://laxmi.nuc.ucla.edu:8888/Teachers/pphelps/Published_Trays/Basic_Embryology_I/slide_22.html)
As the gut becomes increasingly tubular, a series of local inductive interactions between the epithelium of the digestive track and the surrounding mesenchyme initiate the formation of most of the major digestive and endocrine glands, the respiratory system and the liver.
Advance Topics:
Egg-Polarity Genes
The anterior-posterior and dorso-ventral axes of the embryo are established through the localization of the mRNA encoding morphogenes. Morphogens are substances that specify cell identity as a function of its concentration. A continuous gradient of a morphogen can elicit a set of unique responses at a finite number of threshold concentrations. In drosophila, the body axes are established by the morphogenic egg polarity genes dorsal and bicoid.
Follicular cells that surround the egg produce a localized signal at the ventral surface of the egg. This results in a graded concentration of the transcription factor known as dorsal across the embryo. The concentration of dorsal is highest in the ventral area and lowest at the dorsal area.
Where the concentration of dorsal is highest, it switches on expression of transcription factors (for example twist) that lead to mesoderm induction. Where the dorsal concentrations are low, other signals (like dpp, TGFb homologue) are allowed to be active and specify dorsal structures.
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Bicoid is localized at the anterior end of the embryo, and its concentration gradients allows for the development of anterior-to-posterior structures in a manner similar to dorsal.
Advance Topics:
Segmentation Genes
The drosophila embryo is divided by overlapping segments and parasegments which will eventually give rise to the different anatomical structures, i.e. head, thorax and abdomen and their subdivisions. A parasegment is about 4 cells wide and actually correspond to developmental boundaries, while the segments correspond more with the anatomical features (head, thorax, abdomen). Segmentation genes act after egg-polarity genes. There are three classes: gap genes, pair-rule genes and segment polarity genes.
gap genes act first and affect one or more groups of parasegments. They mark the coarsest division of the embryo, each gene affecting several consecutive segments and parasegments. For example, the hunchback gene is transcribed in the earlier segments that will eventually become the head and upper parts of the thorax, while the Kruppel gene is expressed in the segments corresponding to the lower thorax and upper abdomen. These genes still affect regions beyond their transcription areas.
Pair-rule genes act after gap genes, and affect alternating parasegments. Their expression is staggered and overlapping in such a way that combinations of expression define unique regions one-cell wide. For example, even-skipped (eve) is expressed in odd segments while fushi-tarazu (ftz) is expressed in even segments.
The combination of which pair-rule genes are expressed tells the cell where iot is in the embryo. The concentrations will be sligthly different from one cell to the next bercause of the gradient, giving each cell a location label.
This pattern of expression of pair-rule genes governs the expression of segment polarity genes such as engrailed, hedgehog and wingless. If a pair-rule gene is mutated or deleted, the result is a lack of alternating parasegments. For example, eve mutants lack odd numbered segments.
Although gap and pair-rule proteins are initially used to determine segment polarity, they are also used laters in other capacities. For example, eve and ftz are used in the central nervous system.
Advance Topics:
Regulation of Gene Expression
The early morphogens
(dorsal and bicoid) activate segmentation genes. Segmentation genes of one tier
can regulate the expression of genes in lower tiers.
For example, Kruppel,
a gap gene, regulates expression of ftz, a pair-rule gene. Kruppel mutants have
no ftz stripes. Segmentation
genes can also regulate genes in the same tier. For example, ftz and eve, both
pair-rule genes, can regulate each other expression, in such a way that an initial
irregular (blurred pattern) expression is resolved into sharply defined stripes.
Some genes can regulate each other at parasegment boundaries. For example, lets say wingless is produced in the last cell of a parasegment. Then it will activate hedgehog to be produced in the adjacent cell in the next parasegment.
Regulation of expression of segmentation genes occurs via modular transcription
elements. There is actually a gene regulatory module in each promoter that regulates
expression of each individual stripe. For example, each stripe of eve expression
is regulated by a separate module that responds to a specific concentration
of signals that occur at a particular location: high hunchback and bicoid, and
low giant and Kruppel.
Transcription regulatory modules sense a gradient of signals. Products of egg-polarity genes provide global positional values. This results in gap genes being expressed in particular regions. The gap genes then regulate additional genes, such as the pair-rule genes. Combined gradients of gap and pair-rule signals then regulate segment polarity genes.
Advance Topics:
Positional Labels
Once the parasegments are established and the cells are told were they are in the embryo, the pattern of gap and pair-rule genes breaks down. Labels are left behind that are remembered throughout development. There are two major labels: segment polarity genes and homeotic selector genes.
Segment polarity genes act after pair-rule genes and affect part or all of a single parasegment. They include engrailed, wingless and hedgehog. Some segment polarity genes, like engrailed, maintain expression throughout development. Segment polarity genes self-maintain their transcription later in life by interacting with enhancer elements in their own regulatory regions.
Some segment polarity genes encode for transcription factors like engrailed, but several are secreted ligands and transmembrane receptors required for communication between cells.
Homeotic selector (Hox) genes code for transcription factors that determine the anterior-posterior character of the parasegment and are activated by pair-rule and gap genes. Hox genes and are defined by a common homeobox domain that codes for the DNA binding domain of the transcription factor. Many other genes also contain the homeobox domain, for example engrailed (a segment-polarity gene) and paired/pax (homologous paired-rule genes in Drosophila/mammals).
Drosophyla Hox genes are composed of two complexes clustered on one chromosome: the bithorax complex and the antennapedia complex. The bithorax complex controls differences between abdominal and thoraxic segments. The antennapedia complex controls differences between the thoraxic and head segments. The chromosome can be aligned with the anterior-posterior expression of the genes.
Specific combinations of homeotic selector genes control a cell’s address. Each has a specific domain of action and deletions result in loss of distinction between parasegments.
Regulatory regions store memory trace and modulate response under different conditions. Autoregulation (i.e. the gene products activate their own expression) and maintenance by the products of the polycomb and trothorax gnes are important for memory. Polycomb and Trithorax regulate chromatin structure to de-repress and maintain Hox gene expression.
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Advance Topics:
Homeotic Genes in Mammals
Four groups of mammalian Hox genes are homologous to the fly homeotic genes: Hox A, Hox B, Hox C and Hox D. Each complex has sections that code for homologues to the fly proteins. For example, labial (the first Drosophila Hox gene) is analogous to Hox A1, Hox B1 and Hox D1. The order along the chromosome is also conserved, and like in Drosophila, the chromosome can be aligned with the anterior-posterior expression of the genes.
In the nomenclature for mammals, the most anterior expressed Hox genes have the lowest numbers, and the posterior expressed genes have the highest. Mammalian Hox genes with the lower numbers (more anterior) are expressed earlier because they are more sensitive to activation by retinoic acid, a secreted signaling molecule that regulates Hox genes.
In mammals, segmentation is evident in developmental structures like rhombomeres and somites, which are controlled by Hox genes. Somites form the axial skeleton and muscles.
Rhombomeres are segments in the hind brain that correlate with patterns of neuronal development. They are lineage-restricted compartments because the cells of adjacent units do not mix with each other. In addition to Hox genes, several other proteins are important for specifying the natue of individual rhombomeres.
The boundary between midbrain and hindbrain also marks a segmentation boundary. This region is the site of production of two secreted signaling molecules: FGF-8 and Wnt-1 (a homologue of wingless in drosophyla, a segment polarity gene). These signals regulate the expression gradient of 2 engrailed genes (also segment polarity genes) that are critical for the development of the midbrain and cerebellum.
Hox gene mutations cause homeotic transformations. If a Hox gene is deleted, the corresponding segment will assume a more anterior structure. Altered Hox expression results in developmental abnormalities like the Klippel-Feil syndrome: short neck with a reduced number of cervical vertebrae.
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