EVOLUCIÓN DE LA FUNCIÓN DE RECURSOS HUMANOS

“It is not birth, marriage, or death, but gastrulation which is truly the most important time in your life.” (Lewis Wolpert)

Gastrulation is a central process during development and highly conserved across species. The three germ layers (ectoderm, mesoderm, endoderm) are specified and positioned correctly with respect to each other during gastrulation. All future embryonic and adult tissues and organs derive from one of the germ layer precursor cell populations. Major patterning events establish the dorsal-ventral (D/V) and anterior-posterior (A/P) axes, resulting in the formation of the major initial embryonic axis. Organ precursor cell populations are specified in the correct head-to-tail organization along the embryonic axis. Importantly, the well-coordinated cell rearrangement and fate specification events taking place during gastrulation are essential for all future developmental processes (somitogenesis, organogenesis etc.). Disturbances of gastrulation processes usually translate into severe developmental defects, which accumulate as development proceeds. Three evolutionarily conserved morphogenetic movements are essential for zebrafish gastrulation and account for the strictly regulated cell migration processes: Epiboly, Internalization, and Convergence and Extension (C/E).

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Figure 1.6 The first day of zebrafish embryogenesis. Camera lucida drawings of selected developmental

stages from the zygote (1-cell stage) to the early, bilateral organized zebrafish larva (24 hpf). Nomen- clature according to morphological features. Images modified from reference [99]. For details refer to section 1.4.1 and to reference [99]. The arrows indicate important anatomical structures. 1: dome formation at the beginning of epiboly; 2, 3: germ ring; 4, 5: shield (dorsal organizing center); 6: extending dorsal axis; 7: blastoderm margin closing over the yolk; 8: anterior pole of the dorsal embryonic axis; 9: eye primordium; 10: otic placode; 11: somites; 12: yolk extension; 13: midbrain- hindbrain boundary

Epiboly [101]: Towards the end of the blastula period epiboly begins, the first major

morphogenetic movement reorganizing the early embryo (Fig. 1.7). Epiboly is the thinning and spreading of mono- and multilayered cell sheets over the yolk cell, along the animal-to-vegetal axis.

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During epiboly, the blastoderm as well as the YSL collectively spread over the yolk cell, resulting in global cell movements towards the vegetal pole (Fig. 1.7). The spreading of the blastoderm over the yolk cell, along the animal-to-vegetal axis, is referred to as “% epiboly”. Epiboly is considered a two-phase process. The first phase, initiation, is marked by the morphological transition from sphere (4 hpf) to dome stage (4.3 hpf) (Fig. 1.7 A). Radial intercalation is the major cell movement during the initiation phase. Radial intercalation is the intermixing of deep cells through cell migration of more centrally located deep cells towards the blastoderm periphery (Fig. 1.7 B). The second phase is epiboly progression, which begins at 30% epiboly and progresses in synchrony with the other gastrulation movements, which are initiated at 50% epiboly (Fig. 1.7 C- E). Epiboly ends with the closure of the blastopore at the vegetal pole at the end of gastrulation, when the coherent movements of blastoderm and YSL have fully engulfed the yolk cell (tailbud stage; 10 hpf).

Figure 1.7 Zebrafish epiboly. (A) At sphere stage, blastomeres sit as a coherent cell mass on top of the

yolk cell and form a flat interface with the underlying yolk cell. (B) Radial cell intercalation drives inner blastomeres towards the outer periphery, increasing tension globally, and resulting in epiboly initiation. (C) The blastoderm spreads over the yolk cell, thinning and expanding its surface area. (D) Once the blastoderm has reached 50% epiboly, having covered half of the yolk cell, gastrulation is initiated (indicated by shield formation at the future dorsal axis). (E) During gastrulation, epiboly continues in coordination with the other gastrulation movements until embryonic cells have fully covered the yolk. Black arrows indicate the direction of cell migration movements. d: dorsal; dc: deep cells; dcm: deep cell margin; evl: enveloping layer; ysn: yolk syncytial nuclei; e-ysn; external yolk syncytial nuclei; ep: epiblast; hyp: hypoblast; i-ysn: internal yolk syncitial nuclei; vp: vegetal pole; yc: yolk cell. For details refer to section 1.4.2. Modified from [101].

Internalization [102]: In zebrafish, the onset of gastrulation is marked by the

formation of the “germ ring” at 50% epiboly. The germ ring is formed by a local thickening of the blastoderm margin around the entire circumference (equivalent of the blastopore in Xenopus and primitive streak in mouse and chick).

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Mesendodermal progenitor cells within the margin internalize by moving as a continuous stream over the margin down towards the yolk cell, resembling a “folding- in” of the blastoderm margin. Germ ring formation coincides with the transient slowing down of blastoderm epiboly progression. Germ ring cells that have internalized are termed “hypoblast cells” and non-internalized cells are termed “epiblast cells” (Fig. 1.7 E). Hypoblast cells (specified as mesendoderm) eventually migrate coherently towards the animal pole of the gastrula, counterbalancing the vegetally moving epiblast cell layer. In the future dorsal region of the gastrula’s germ ring, blastoderm cells converge and form a compact population of cells, the embryonic “shield” (Fig. 1.8 A). The shield is the zebrafish major dorsal organizing center and is the functional equivalent to the Spemann–Mangold organizer in Xenopus and Hensen’s node in amniotes. The shield marks the region of the future dorsal axis and the first axial mesendoderm structures are formed by collective cell migrations of a tightly packed group of cells specified as prechordal plate. Internalization in the dorsal shield region occurs by the ingression of individual cells [103], while cells in ventro-lateral regions internalize via involution of cell sheets [104] (Fig. 1.8 A). Around 70% epiboly, mesoderm and endoderm cells in lateral regions of the embryo adjust their migratory trajectories towards the future dorsal axis and globally migrate dorsally in a directed manner (Fig. 1.8 B). These combined cell migration processes are termed Convergence and Extension movements.

Convergence and Extension (C/E) [105]: C/E is the morphogenetic process which leads

to the narrowing (convergence) of a tissue in one dimension, while lengthening (extension) it with respect to the perpendicular axis (Fig. 1.8). During C/E, all three germ layers are narrowed in the medio-lateral direction towards the dorsal midline and extended along the anterior-posterior axis to shape the dorsally located embryonic body axis (Fig. 1.8 A-C). The type of cell migration which accounts for C/E movements in the dorsal gastrula domain is called medio-lateral intercalation. Ectodermal and mesodermal cells become highly elongated, acquire a bipolar shape and align their long axes perpendicular to the dorsal midline. In parallel, cells from both sides of the dorsal midline intercalate with each other, compacting the dorsal midline domain and extending it along the anterior-posterior axis.

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In par-axial dorsal domains of the gastrula, medio-lateral and polarized radial intercalations account cooperatively for C/E. Mesoderm and endoderm cells positioned in lateral domains contribute to C/E via directed cell migration towards the emerging dorsal axis. In the most ventral domain of the gastrula, C/E movements are absent and mesoderm cells migrate directly towards the animal pole. C/E movements are considered to be a combined process in which convergence and extension occurs simultaneously and interdependent (as shown in Xenopus), but evidence from work in zebrafish suggests a possible separation of these two processes [106]. For example, in the zebrafish Smad5 mutant somitabun (disrupted BMP signaling) convergence is impaired while extension remains largely unaffected [107]. In summary, C/E movements establish the dorsal embryonic axis and depend on intact developmental signaling.