Left-Right Axis Formation

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Left-Right Axis Formation
BIOL 4061- Molecular and Cellular Principles of Animal Development
Professor: John McDermott
Due Date: March 27th 2015
Shakira Wynter- 210080455

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In developmental biology a problem arises to clarify the cellular behaviour that is
powering morphogenesis and the molecular signals that control it. Important signaling
pathways that control cell community and differentiation have been exposed by genetic
studies, to divulge information about the rudimentary networks of the genes controlling
early mouse development. Interpreting a morphogenetic program is a multifaceted
problem to overcome, as it includes the communication between cells (1). The quest of
elucidation of the cellular behaviours driving morphogenesis and molecular signals is
addressed using microscopy where investigation of the interactions are observed and
contrasted with experimental data (1). This essay will look at early development in mice
with focus on the cellular dynamics, gastrulation and axis formation, anterior-posterior
polarity and signaling center, and transcriptional activity and signaling (WNT).
Gastrulation is an early phase in embryonic development where a blueprint for
embryonic development is made (3). Gastrulation is the early phase where the single
layered blastula is reordered into a three-layered structure that is known as a
trilaminar/gastrula, consisting of the ectoderm, mesoderm and the endoderm. Each layer

aids in the development of different tissues and organs (3).
Embryonic changes of size and shape are observed after the egg has been
embedded in the maternal uterus where basic body plan development can be observed
after implantation (1). After implantation in utero growth is seen in the distribution of
extra embryonic lineages, which is vital for fetal communication and nutrient exchange
and play a part in patterning the embryo (1). This embryonic state observed coincides
with cell polarity thus creating the cell-cell junctions at the epithelial stage. This polarity
is referred to as the Proximal–Distal (PD) polarity which gives rise to the P-D axis (1).

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‘The dorsal-ventral (D-V) axis of the embryo becomes apparent with the proamniotic
cavity surface of the epiblast matches the dorsal side of the embryo and the outside
surface of the visceral endoderm that corresponds to the ventral side of the embryo (3).
Gastrulation utilizes the progression of morphogenesis combined with cell propagation
and differentiation to change an embryo into a gastrula (3).
The mouse will undergo gastrulation by employing the embryonic ectoderm cells
to a fleeting developing cell structures known as the primitive streak. The primitive streak
allows the anterior-posterier (A-P) axis to become morphologically noticeable with the
steak on the posterior side of the embryo with the left-right axis being obvious (3). The
primitive streak also allows the “epiblast cells to undergo an epithelial to mesenchymal
transition,” allowing entrance amid the epiblast and endoderm to become combined with
either the mesoderm or the endoderm germ layers (3). The new tissue formed from the
mesoderm allows expansion of the primitive streak forming the mesodermal wings. It is
important to note that mouse embryo develops from the inside to the outside, and after
gastrulation occurs, there is a transposal of the germ layers cause reorganization of
endoderm to the inside and the mesoderm to the middle (3).
When looking at the formation of the mouse body plan, the axes of the preimplantation embryo must be asymmetric in order to generate the primitive streak. This
confined asymmetry results in the intimate association of ICM with a subdivision of
trophectodem cells, which in effect causes the confinement. The confinement of the
ICM/polar trophectoderm shows the arrangement of the embryonic-abembryonic (EmAb) axis (3). After the confinement there is differentiation of the endoderm on the
blastocoelic surface, which ultimately enhances the asymmetry. Further differentiation

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occurs of the primitive endoderm into the visceral endoderm as it moves to the
phectoderm. The two endoderms contribute to the extra-embryonic tissues and are
notably different from the endoderm and the embryo proper (3). When the mouse embryo
shows symmetry it can attach to the uterine epithelium of the maternal uterus, where it
develops an extended axis of the egg cylinder. However the pri-implantation embryo
unlike the implantation embryo does not show symmetrical arrangement and is located on
the opposite site of the embryo (3). The titled positioning of the ecto-placental cone and
the unequal bends are suggestive of the A-P axis but not its polarity (3).
Organization of the A-P axis indicates the initial appearance of the mature body
plan of the mouse embryo (4). The question of whether early polarity in the mouse
related to the later anterior-posterior axis is asked. The answer to this is answered by
observing organisms such as Xenopus laevis and Caenorhabditis elegans, which have
separation of the cytoplasmic elements that is located in the eggs or the zygote. They also
have established a critical role in the polarity (4). This segregation causes disruption of
the experimental aspects of the early embryo and can stop the normal development of the
embryo (4). However, the early progression of the mouse embryo is extremely controlled
and diverts to multiple experimental disruptions including the elimination of the polar
cytoplasm and the combination or the elimination of the blastomeres (4). With all of the
various disruptions that occurs it is presumed that the axis of the embryo does not begin
from the polarity in the pre-implantation embryo (4). The place of sperm synthesis may
deliver the situational information that affects the alignment and the timing of the
blastoma cleavage, which will allow determining the polarity in the embryo (4).

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The AP axis in the mouse is not aligned with its long axis at the pre-streak stage
and can reflect a change in the developmental stages, the angular distribution of the
expression domains of both posterior and anterior markers. Studies have shown that
although Ca2+ is typically involved in the regulation of the “ciliary movement, the beat
frequency” in the node and changes to the Ca2t signaling with ionomycin is not affected.
It can thus be concluded that Ca2t signaling does not appear to regulate the movement of
the nodular cilia but signaling may regulate the asymmetric patterning (5).
The modeling of the right-left body axis occurs through the early somitogenisis
process in the mouse embryo. During embryogenesis a node emerges posterior end to the
developing notochord (5). Nodular cells have motility of its primary cilia such that the
one directional rotation that is generated with external liquid flow which is called the
nodal flow. This nodal flow is critical for obliterating the left-right symmetry such that
the route of flow determines the left-right embryo pattern (5). Normal left-right
organization is referred to as situs solitus (7). The lack of nodal flow in mutants with
deficits in ciliary motility shows that there is random laterality. However, this random
laterality can be saved by a supplemental or artificial external nodal flow, which will
result in tissue polarization of the node along the left-right axis (7). ‘The mutation that is
seen in the left-right dynein results in the iv mouse results in non-functional nodal monocilia and left-right axis malformations’ (7). In the iv mouse similar mutant genes will
show ‘situs inversus and while nodal monocilia are motile the left nodal flow is slower in
comparison to the wild-type embryos’ (7). This information solidifies that the nodal
monocilia produces a leftwards flow that disrupts the left-right symmetry. If there is
disruption of the flow of symmetry by establishing a chemical gradient or by the

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production of the mechanical forces are unidentified (7). The sonic hedgehog (shh) will
perform as early right-sided determinants and the FGF8 will perform as a early left-sided
determinants (7).
Genes such as the Nodal and Cerl-2 are represented as being asymmetrical along
the edge of the node and ought to find a way to convert the nodal flow into a signal
generating tissue polarization. The process or the way in which this occurs is still
indistinguishable (5). The way in which Cerl-2 is expressed in the node is in opposition
of the Nodal flow such that the asymmetric Cerl-2, which is highly expression on the
right side of the node is thought to be more important than the left side (5). There is also
disruption in the asymmetric pattern when ionomycin is present resulting in embryos with
lower expression levels (5). This Cerl-2 that is present is reduced when ionomycin is
increased. The Ca2t signal is basically down-regulating the Cerl-2 expressions in the
node causing up-regulation of the nodular signaling. Cerl-2 is therefore a major factor in
the targeting of the left-right signaling pathway that is mediated by the polycystin-2 (5).
The formation of an embryonic body plan is a consequence of communications
between the originator tissues and morphogenesis; gastrulation. Intracellular signaling
activities, that drives the interactions between the progenitor and morphogenesis are
utilized in a timely and site specific manner where the signal strength is meticulously
controlled (2). Secreted antagonists that adapt ligand and receptor functions, allow for a
“globally controlled and locally graded signals onto the tissue of early post-implantation
mouse embryo”(2). The embryo develops its body arrangement in response to WNT
signaling, nodal and bone morphogenetic protein (BMP) signaling cascades, seen as
changes in the developmental outcome of cells situated at different locations of the

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anterior-posterior body axis (2). The two signaling centers that are the source of opposed
factors and the centre of transcriptional activities are contrasted with the progenitor
tissues of the head structures in the mouse embryo and ultimately responsible for the
formation of the anterior structures in the mouse embryo. The formation of the anterior
structures is fundamentally dependent on the morphogenetic activity that occurs at the
signaling centers (2). These signaling factors also negatively control the WNT, nodal and
BMO signaling cascades. The organized events that occur forms a framework for the
embryonic head by the early-somite stage of development leading to continues tissue
interactions and development with aid to support the growth, regionalization,
differentiation and morphogenesis needed for a recognizable head structure (2).
The signaling cascade of the Wnt there is attachment of ligand and similar
receptor, which stimulates an assortment of intracellular responses. These intracellular
responses include the study of the activation of the tcf/lef, b-catenin transcriptional
complexes that are present in the official Wnt pathway (8). The interweaving of the
pathways is shared amongst various response modes.
Deregulated Wnt/b-catenin signaling demonstrates phenotypic effects such that
the signaling pathway is crucial for gastrulation and axis development in the mouse
embryo. ‘Loss-of-function mutations in activating components of the pathway blocks
primitive streak formation indicating that Wnt signal’ are vital (8). ‘Gain-of-function
mutations in an activator or loss-of-function mutations of negative regulators of canonical
Wnt signaling result in two distinctive phenotypes, namely anterior truncation and
posterior axis duplication, in the mouse and other vertebrates’ (8). The physical
appearance produced by high eptopic signaling sows that that there is preservation of a

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low level of Wnt signaling which stops the posterorisation of the anterior neurectoderm
and prevents the supernumerary boxy axis from forming (8).
Wnt signaling continue to control pathway activity and one of the difficulties is
assessing action of the different components of the Wnt network to attain the correct
signaling activity in biological setting. A way of observing Wnt/b-catenin target gene
regulation in vertebrates is by looking at the absence of ligands, promoter targets are
composed or stifled by transcriptional mediators from the Tcf/Left family in performance
with association of the TLE family of Groucho associated co-repressors (8). Studies show
differences in the traditional gene regulation method of the subjugation and activation of
the Wnt/b-catenin targets early mouse development (8). A transformational analysis has
recognized that of the four Tcf/Lef family, only Tcf711 is needed during the
embryogenesis process. The deficit of Tcf711 caused the embryo containing the primitive
streak morphology, anterior truncation and posterior axis duplication work as a repressor
of the target promoters. Mouse embryos having same genes for a mutant allele of Tcf7l1
that also lack the b-catenin communication domain continues during gastrulation and
anterior axis formation with no obvious defects. Physical interaction of the Tcf711 is not
required for interaction of with the b-catenin and its use in repressor activity at the
various stages of development (8). Over a period of time additional head and brain
structures are missing in the mutant embryos that contain dissimilar allelic combinations
of Dkkl, Lrp6 and Ctnnb1. The allelic combinations are associated with the anterior
expansion of the Wnt reporter expression domain and the degree to which the Wnt
activity occurs (8).

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Hereditary investigations have given indication that the signaling pathways
observed in the Wnt is at the forefront of the control of gastrulation in the mouse embryo.
Restoration of normal signaling in Wnt3 results in the lack of the primitive streak and a
failure in gastrulation. This means that there will be inactivation of the Wnt co-receptor
and/or a lack of their chaperones (9). Restoration of normal axis of a negative regulator of
the official Wnt signaling pathways in the mouse embryo will cause replication of the
primitive streak. Epiblast expression is limited to the area that is adjoining the posterioranterior visceral endoderm. The twofold expression patterns of the Wnt3 in the A-P axis
configuration are unable to be assigned to tissues in the standard knock out experiments.
Molecular downstream of the events that are occurring in the Wnt3 signaling growth of
the mouse embryo are not understood (9). Function of the Wnt3 is determined by knock
out Wnt3 in the epiblast using Sox2cre transgene. Though embryos containing the Wnt3
have the ability to undergo gastrulation they are unable to complete the process and are
reabsorbed at E9.5 (9).
The assumption that the anterior visceral endoderm (AVE) cells breaks the radial
symmetry of the mouse embryo marks the anterior and the circumstances that allow the
formation of the primitive streak on the contrasting site. Traverse sections of the
gastrulating mouse embryo fits the plan with the primitive streaks located at one end of
the longest axis. The relationship between the anterior-posterior (AP) polarity and the
morphology of the post implantation embryo is unclear (6). The modification in the
alignment of the AP axis is only evident with the consequences of a significant
transformation of the entire epiblast with cell migrations taking no part (6).

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In conclusion early development in mice focusing on the cellular dynamics,
gastrulation and axis formation, anterior-posterior polarity and signaling center, and
transcriptional activity and signaling (Wnt) provide the steps of development of the
mouse embryo. It also provides information about the body structure and plan and the
various steps required for axis formation.

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Arkell R.M., Fossat N., and Tam P. (2013).Wnt signalling in mouse gastrulation and
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