Cleavage Gastrulation and Neurulation

Since the beginning of the 20th century, authors hypothesized that gravity played a role on early embryonic development, in particular on the orientation of first cleavage, the formation of the antero-posterior axis, and the subsequent morphogenesis and organogenesis that are very often characterized by the regular patterning of morphological structures. The basic principles of embryonic development are described in Chapter 1, Section 3.1.

Experiments were done with frog eggs during centrifugation that increases gravitational forces (hypergravity), clinostat rotation that produces a vector-free gravitational environment (simulated microgravity), and true microgravity during orbital flights. All these experiments revealed that gravity was involved in the early developmental stages of embryonic processes.

In the radial-symmetrical mature egg of Xenopus laevis, the polar animal-vegetal axis indicates roughly the embryo's main body axis. Pigment concentrating around the sperm entry point marks the meridian that foreshadows the prospective ventral side. Because in most eggs the blastopore6 forms at the meridian about 180 deg away from the sperm entry point, the embryo's general body pattern is established from that time on. However, the dorsoanterior and ventroposterior polarities can still be altered by the rearrangement of yolk's components due to the influence of gravity and centrifugal forces. This suggests that gravity, in conjunction with the sperm entry point, establishes the dorsoventral polarity.

Another typical feature of early development is the rotation of the egg inside the fertilization membrane by which the animal-vegetal axis aligns itself with gravity. This rotation is not a requirement for normal development,

6 A blastopore is an opening into a developing blastula. The blastula is an early stage of embryonic development in animals. It is produced by cleavage of a fertilized egg and consisting of a spherical layer of cells surrounding a fluid-filled cavity. The blastula follows the morula and precedes the gastrula in the development sequence. A blastula has around 128 cells, with a large central cavity called the blastocoel (see Chapter 1, Section 3.1).

because eggs prevented from rotating can develop normally. Generally, the direction of rotation determines the polarity of the embryonic axis. Eggs inclined with respect to gravity form the dorsal structures on the side of the eggs uppermost in the gravitational field. These observations make it obvious that gravity is used during the early steps of the development of an embryo. However, the answer to the question of whether the presence of gravity is necessary for normal morphogenesis in early development could only be given by conditions of gravity deprivation during spaceflight. Aquatic vertebrate (fish, frogs, salamanders, and newts) and invertebrate species (sea urchins) were first-choice species to answer this question. Experiments with simulated gravity using the fast-rotating clinostat also gave valuable hints to spaceflight experiments (Yokota et al. 1994).

2.2.1 Xenopus laevis

In Xenopus, development under simulated microgravity in a clinostat revealed no change in the cleavage rhythm. At the eight-cell stage, however, the location of the first horizontal cleavage furrow was shifted towards the vegetal pole and completed earlier. Further modifications include:

a. A more centered position of the blastocoels and an increase in the number of cell layers in the blastocoel roof at the blastula stage;

b. A significant smaller blastocoel (Figure 5-03);

c. A dorsal lip that appeared closer to the vegetal pole at the gastrula stage;

d. And head and eye dimensions that were enlarged at the hatching tadpole stage.

Despite of these morphological changes, tadpoles at the feeding stage were largely indistinguishable from controls (Yokota et al. 1994). Similar observations were obtained from studies in Rana dybowskii by exposure to simulated microgravity (Neff et al. 1993).

After successful fertilization of Xenopus eggs in real microgravity during sounding rocket and spaceflights, subsequent embryonic development revealed the same features as seen in simulated microgravity. The cleavage rhythm was normal, but the numbers of cell layers of the blastocoel roof increased from two to three and the blastocoel became smaller (Figure 5-03). Further development in microgravity continued as observed during the Spacelab-J mission. In particular, neurulation7 was not impaired and the

7 A neurula is an embryo at the early stage of development in which neurulation occurs. Neurulation is the development of the nervous system in the embryo. The neural plate will fold to produce the neural tube that will develop into the brain. Remaining tissue will develop into the spinal cord (see Chapter 1, Section 3.1).

neurula at stage 20 appeared normal. After this particular spaceflight with the in-flight fertilization, normal tadpoles were retrieved (Souza et al. 1995).

This observation contrasted somehow the observations following earlier sounding rocket flights. In embryos raised in 1 g after the MASER-3 flight, further development was slightly retarded compared to the ground embryos. In addition, microcephalization and reduced tail formation were observed, while after the MASER-6 flight, embryos developed normally including axis formation (Ubbels 1997, Ubbels et al. 1995).

Simulated microgravity by means of clinostats allows a more detailed analysis of the individual periods of development. In fact, anuran embryos revealed that, in addition to the above mentioned modifications, the dorsal lip approached the vegetal pole at the gastrula, and there was enlarged head and eye dimensions at hatching (Neff et al. 1993).

Figure 5-03. Gastrulae from Xenopus laevis fixed in microgravity (¡.ig) and on the ground (Ig) showing the thickening of the hlastocoel roof'in microgravity. Note: 1, blastocoel; 2, hlastocoel roof'; 3, blastopore. Adapted from Ubbels et al. (1995)

2.2.2 Pleurodeles

Some other aquatic animals gave hints to the extent of modifications in the embryo caused by spaceflight. Some were similar to those found in Xenopus, while other were absent or not detectable due to the analysis methods used. In Pleurodeles, 24 out of 25 eggs fertilized in 0 g exhibited normal location of the first furrow. However, subsequent cleavages were irregular and 3, 5, or 7 cells were observed in the animal hemisphere (Figures 5-04 and 1-18). About 35% of microgravity eggs exhibited large unpigmented areas in the animal pole, and movements of the pigment towards the animal pole were amplified up to the morula stage. As in Xenopus, the blastocoel roof in gastrulae was thicker in the 0-g eggs than in the 1-g controls, but the blastocoel was still composed of two cell layers. In contrast to Xenopus, however, neurulation was strongly affected by microgravity (Gualandris-Parisot et al. 2002).

2.2.3 Fish and Newts

Poor or even absent sensitivities to microgravity were observed in the Medalca fish (Oryzias latipes) during the IML-2 mission (STS-65 in 1995) and in the newt Cynops. After the successful mating of Medaka fish in microgravity (Ijiri 1998), the subsequent developmental steps were similar in flight and ground-control fish. Newly laid eggs formed a cluster on the belly of the female fish. After detachment from the female's body, young fish hatched in microgravity (Ijiri 2003) (see Figure 2-12).

This lack of microgravity effects contrasts with the changes in the plane of bilateral symmetry and the orientation of the microtubules in the vegetal pole region of zygotes induced by tilting or centrifiigation (5 g) (Fluck et al. 1998).

In-flight video-recordings of early Cynops stages also revealed normal morphological shapes of the late morula, early blastula, gastrula, neurula and tail bud stage up to the stage shortly before the first gill (respiratory organ) ramification appeared (Yamashita et al. 2001).

2.2.4 Conclusion

All these modifications seem to occur only transiently, because after spaceflights or simulated microgravity hatched Xenopus tadpoles at the feeding stage are largely indistinguishable from controls (Souza et al. 1995, Yokota et al. 1994). Long-term microgravity exposure revealed that Pleurodeles larvae were able also regulate the morphological changes of the gastrula and neurula stages while being in microgravity. Even the time of hatching in microgravity was identical to that in the ground controls. Histological and immunohistochemical studies with larvae fixed within five hours after landing showed no microgravity specific effects in their central nervous system, eyes, somites, pronephros8, and gut (Dournon 2003).

These facts demonstrate the efficiency of self-regulatory genetic mechanisms during development in altered gravitational environment (see Horn 2005 for review). The reasons for the observed developmental

8 Pronephros is the first temporary stage of kidney development.

modifications during early development are not yet understood. In both gastrulae and cultures of presumptive ectoderm cells9 of Cynops pyrrhogaster, TUNEL staining10 and electron microscopy revealed apoptotic cells, but the number of these cells was always smaller in clinostat-treated samples than in the controls (Komazaki 2004).

Figure 5-04. Morphological effects during early emhrvogenesis. Left: Light micrographs of Pleurodeles eggs fertilized in space (fig) and on the ground (Ig). In microgravity, the pigmentation concentrated around the animal pole and an unpigmented area covered a large part of the animal hemisphere (arrow). Bar = 600 ¡urn. Right: Note the incomplete closure of the neural tube (*) in an embryo of salamander Pleurodeles wait! fixed in microgravity (fig) compared to the ground control (lg). a and p, anterior and posterior pole of the embryo, respectively (see also Figure 1-18). Courtesy of C. Dournon, Henri Poincare University-Nancy 1, Vandoeuvre-les-Nancy, France.

Figure 5-04. Morphological effects during early emhrvogenesis. Left: Light micrographs of Pleurodeles eggs fertilized in space (fig) and on the ground (Ig). In microgravity, the pigmentation concentrated around the animal pole and an unpigmented area covered a large part of the animal hemisphere (arrow). Bar = 600 ¡urn. Right: Note the incomplete closure of the neural tube (*) in an embryo of salamander Pleurodeles wait! fixed in microgravity (fig) compared to the ground control (lg). a and p, anterior and posterior pole of the embryo, respectively (see also Figure 1-18). Courtesy of C. Dournon, Henri Poincare University-Nancy 1, Vandoeuvre-les-Nancy, France.

9 The ectoderm is the outer most of the three primaty germ layers of the embryo, from which the skin, nerve tissue and sensory organs develop

10 TUNEL staining is a procedure for detecting apoptotic cells. Because DNA fragmentation is a hallmark of apoptosis, the TdT-mediated UTP-biotin nick end-labeling (TUNEL) uses the enzyme deoxynucleotidyl transferase (TdT) to directly label the fragmented DNA ends. The apoptotic cells can then either be quantified using flow cytometry or visualized in tissue sections by using colorimetric reagents.

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Responses

  • julia hueber
    What is morula, blastula, gastrula, neurulation, incubation?
    1 year ago
  • gary
    What are the observation of cleavage,blastulation,gastrulation,neurulation?
    1 year ago
  • Ester
    Does gravitational force affect neurulation?
    3 months ago

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