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Throughout the animal kingdom, there is a remarkable variety in the morphogenetic processes that take place during embryonic development. However, for most species, the patterns of early embryogenesis tend to follow a common sequential thread.

After fertilization, embryonic development begins with the cleavage phase. During this phase, a series of rapid cell divisions takes place giving rise to a substantial increase in cell number. By the end of the cleavage phase these cells, termed blastomeres, are generally arranged in a sphere known as the blastula (Fig. I.1a) (Gilbert, 2003).

The initial stages of animal development occur in the absence of de novo transcription. During this period, the progression of embryogenesis relies entirely on maternally inherited mRNAs and proteins. As early development progresses, maternal mRNAs and proteins are gradually degraded, and zygotic transcription is activated, thus progressively diminishing the maternal influence over embryogenesis. This gradual shift from maternal to zygotic control is known as the Maternal to Zygotic Transition (MZT). The MZT spans the period from the onset of maternal mRNA degradation to the first major developmental requirement for zygotic transcripts. For instance, in the zebrafish the MZT begins at fertilization, spanning the entire cleavage and blastula phases and coming to an end during the gastrula phase (Fig. I.1a,b) (Langley et al., 2014, Tadros and Lipshitz, 2009).

The gastrula phase begins after the rate of cell divisions has diminished, and is characterized by extensive cell rearrangements. These highly coordinated cell movements are termed gastrulation. Gastrulation is accompanied by a series of specification and patterning events which enable the establishment of a multi-layered body plan containing three germ layers: the outer ectoderm, the inner endoderm, and the interstitial mesoderm (Fig. I.1c) (Gilbert, 2003, Solnica-Krezel, 2005).

Although the patterns of cell rearrangement during the gastrula phase vary throughout the animal kingdom, there are four evolutionarily conserved gastrulation movements: internalization, epiboly, convergence and extension. Internalization

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movements carry prospective mesoderm and endoderm cells inward, beneath the prospective ectoderm. Epiboly movements lead to an expansion and thinning of the germ layers. Convergence and extension movements narrow the germ layers medio-laterally and elongate the embryo from head to tail (Fig. I.1c) (Solnica-Krezel, 2005).

In vertebrate embryos the final stages of gastrulation are either accompanied with, or followed by, the onset of neurulation – the formation of the neural tube – and segmentation – the formation of the somites. The neural tube is formed from ectodermal precursors situated above a rod-shaped mesodermal structure termed notochord, which demarcates the anterior-posterior embryonic body axis. The somites are spherical mesodermal structures which form on both sides of the notochord, and contain the precursors of the vertebrae, skeletal muscles, and dermis (Wolpert, 2002) (Fig. I.2a,b).

Embryonic development subsequently progresses to the organogenesis phase, during which, extensive cell rearrangement, differentiation and specialization

Gastrula zebrafish embryonic development. (a) Representation of key embryonic stages of the cleavage, blastula and gastrula phases of zebrafish embryonic development (adapted from (Kimmel et al., 1995)). (b) Maternal to Zygotic transition in zebrafish.

The blue curve represents the degradation profiles of

destabilized maternal transcripts. The red curve illustrates the minor and major waves of zygotic genome activation (adapted from (Tadros and Lipshitz, 2009)). (c) Representation of the gastrulation movements and process of germ layer specification in the zebrafish embryo. YSL, yolk syncytial layer (adapted from (Solnica-Krezel, 2006, Kimelman, 2006)).

5 processes take place to form the different tissues and organs of the embryo (Fig.

I.2a). During this phase, the ectoderm will give rise to the epidermis, nervous system and pigmented cells. The endoderm will contribute to the gastrointestinal, urinary and respiratory systems, as well as several endocrine glands. The mesoderm will give rise to the heart, kidneys, gonads, axial skeleton, cartilage, connective tissue, trunk muscles and blood cells. In addition, the formation of various organs will involve interactions between the different germ layers (Gilbert, 2003, Kiecker et al., 2016).

The next subchapter will focus on two critical developmental processes: the elongation and segmentation of the anterior-posterior axis; and the establishment of internal organ asymmetry along the left-right axis. In addition, it will address the specific contributions of the fibroblast growth factor (FGF) signalling pathway to different aspects of embryonic development.

I.1.1 Posterior body elongation and Somitogenesis

The first morphogenetic events that define the shape of the embryonic body are thought to take place during the gastrula phase, as a result of convergence and extension movements. However, this process carries on after gastrulation, with posterior body elongation and segmentation presenting as two major aspects of vertebrate development (McMillen and Holley, 2015, Bénazéraf and Pourquié, 2013).

I.1.1.1 Posterior body elongation

The development and elongation of the posterior body is achieved through the progressive deposition of cells from a posterior growth zone in the embryo. This posterior leading edge of the growing embryo, named tailbud, contains the progenitors of the musculature, axial skeleton, vasculature, spinal cord and blood (McMillen and Holley, 2015, Beck, 2015).

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Interestingly, studies done in chick and zebrafish embryos have found that posterior body elongation is primarily driven by cell migration rather than cell proliferation, with tailbud musculoskeletal progenitors exhibiting only a modest level of proliferation during posterior body elongation (Bouldin et al., 2014, Kanki and Ho, 1997, Bénazéraf et al., 2010, McMillen and Holley, 2015). In these organisms, instantaneous cell velocities are greater in the posterior tailbud, with posterior growth occurring as these highly motile progenitors lessen their motility and assimilate into more anterior tissues, namely the paraxial mesoderm (Lawton et al., 2013, Dray et al., 2013, Mara et al., 2007, Bénazéraf et al., 2010, Delfini et al., 2005).

For instance, during zebrafish posterior body elongation, the progenitor cells of the dorsal medial tailbud dive ventrally as a coherent posterior flow. At the posterior ventral tailbud there is a loss in cell flow coherence which leads to an increase in cell mixing (Lawton et al., 2013, Dray et al., 2013). These mesodermal progenitors subsequently lose velocity as they enter the posterior paraxial mesoderm, concomitantly with the assembly of an extracellular matrix composed primarily of Fibronectin and Laminin (Dray et al., 2013, Latimer and Jessen, 2010, McMillen and Holley, 2015).

The entry of these tailbud cells into the paraxial mesoderm territory appears to include a process of convergence and extension, akin to what is observed during gastrulation, with this process being regarded as an important contributing factor to posterior body elongation (Steventon et al., 2016, Kanki and Ho, 1997).

Furthermore, paraxial mesoderm assembly is accompanied by the formation of the notochord from axial mesoderm precursors. The vacuolation and rearrangement of the notochord cells has also been proposed as a contributing factor to the progression of posterior body elongation (McMillen and Holley, 2015, Kanki and Ho, 1997, Dray et al., 2013).

I.1.1.2 Somitogenesis

As posterior body elongation progresses, the paraxial mesoderm, also known as presomitic mesoderm (PSM), is subdivided into metameric structures, termed somites. In vertebrates, somites form sequentially along the anterior-posterior

7 embryonic axis, budding off in bilateral pairs from the unsegmented PSM. Each somite presents as a spherical cell mass surrounded by an epithelial sheet, and contains the precursors of the vertebrae, skeletal muscles, and dermis (Fig. I.2a,b) (Yabe and Takada, 2016).

The process of somite formation – somitogenesis – is tightly regulated, both spatially and temporally, with the frequency of somite formation and the total number of somites formed being species-specific traits. For instance, in zebrafish a new pair of somites is formed every 25 minutes until a total of approximately 33 somite pairs is reached, whereas in mice a new somite pair is formed every 2 hours resulting in the formation of approximately 65 somite pairs (Yabe and Takada, 2016, Hubaud and Pourquié, 2014).

To account for this spatiotemporal regulation of somitogenesis, a theoretical model termed “Clock and Wavefront model” was proposed. In this model, rhythmic and sequential somite formation is achieved by two regulatory mechanisms: a segmentation clock and a wavefront of differentiation. The cyclic activation of the segmentation clock provides temporal information, which is integrated with the spatial information provided by the continuous regression of the wavefront that results from posterior body elongation. A consequence of this model is that the size of each newly formed somite is fixed by the distance travelled by the wavefront during one period of the segmentation clock (Fig. I.2b) (Cooke and Zeeman, 1976, Yabe and Takada, 2016, Hubaud and Pourquié, 2014).

Since the Clock and Wavefront model was proposed, several genes have been associated with the establishment of the segmentation clock and wavefront mechanisms.