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This material is from the 4th edition of The Zebrafish Book. The 5th edition is available in print and within the ZFIN Protocol Wiki.

HATCHING PERIOD (48-72 h)

Modified from: Kimmel et al., 1995. Developmental Dynamics 203:253-310. Copyright © 1995 Wiley-Liss, Inc. Reprinted only by permission of Wiley-Liss, a subsidiary of John Wiley & Sons, Inc.

The time of hatching is not useful as a staging index for the zebrafish, in contrast to some other types of embryos, because individuals within a single developing clutch hatch sporadically during the whole third day of development (at standard temperature), and occasionally later. Whether or not an embryo has hatched, its development progresses, hour by hour, and generally individuals that have spontaneously hatched are not more developmentally advanced that ones remaining in their chorions. We arbitrarily call the creatures "embryos" until the end of the third day, and afterwards, "larvae", whether they have hatched or not (Fig. 39).

During the hatching period the embryo continues to grow at about the same rate as earlier (Fig. 16). Morphogenesis of many of the organ rudiments is now rather complete and slows down considerably, with some notable exceptions including the gut and its associated organs. However, these endodermal structures are difficult to visualize in the living embryo because of their deep positions, and we do not consider them completely here. Much easier to see are the rapidly developing rudiments of the pectoral fins, the jaws, and the gills.

Pectoral fin development continues to be a useful feature for staging, especially during the early part of the hatching period. At the onset of the period the paired fin rudiments are elongated buds, each already containing centrally-located mesenchymal condensations that will form the girdle cartilages (arrowheads in Fig. 37C). The distal epithelial fold capping the bud, that developed from the apical ectodermal ridge, now expands into the blade of the fin proper and strengthening actinotrichia appear. At the same time a circulatory channel appears as a continuous loop at the base of the enlarging fin, i.e. its position is proximal to the actinotrichia but distal to the cartilage. This channel will develop both as artery and vein. The subclavian artery takes the blood outward into the fin from the paired portion of the dorsal aorta posterior to the last aortic arch. The subclavian vein returns the blood again, connecting proximally near the origin of the common cardinal.

Prominent changes occur in the pharyngeal region. During the early part of the hatching period the small open mouth can be located with Nomarski optics in a midventral position, between the eyes (Fig. 40), a position that seems surprising, at first, because it is so far posterior in the head. Then, especially during the last 12 hours of embryogenesis, a dramatic repositioning of the mouth occurs. Jaw morphogenesis moves the position of the mouth anteriorwards, and just at the end of the hatching period the mouth protrudes beyond the eye, where it gapes wide open. Rapid jaw and mouth morphogenesis will continue in the early larva.

Examining cartilage development in the jaw primordia is also useful for staging, especially during the last part of the hatching period. As noted above, the two anterior-most pharyngeal arches (the mandibular and hyoid arches) that form the jaw and associated supportive apparatus are always distinctive from the more posterior arches, collectively termed branchial arches. Cartilage development in the jaws lags behind cartilage development in the pectoral fin and behind formation of the first cartilages of the chondrocranium (the latter, basal cartilages located deep beneath the brain, are rather difficult to see in the living preparation and we do not describe them here). On the other hand the jaw cartilages develop ahead of the branchial cartilages. Mesenchymal precartilage condensations appear in both the mandibular and hyoid arches near the beginning of the hatching period and distinctive cartilages, ventral and dorsal elements, develop in each arch by the end of this period (Fig. 41). The ventral cartilage of the mandibular arch (the mandibular or Meckel's cartilage) and the ventral element of the hyoid arch (the ceratohyal cartilage), are large supportive structures of the lower jaw, beneath the pharyngeal or oral cavity. The dorsal elements, the quadrate of the first arch and the hyosymplectic (also called the hyomandibular) of the second, are more delicate and more elaborate in their shapes. A complex set of jaw muscles accompany development of the cartilages (e.g. the adductor of the mandible in Fig. 41).

Earlier, during the pharyngula period, a single bilateral pair of aortic arches formed, and they occupied the jaw primordial region. Now, when the hatching period begins, a second aortic arch has joined the first one on each side of the embryo in the region of the jaw. The first aortic arch passes dorsally just anterior to the promininet ceratohyal cartilage, and the second aortic arch passes just posterior to the same cartilage (Fig. 41). The two vessels join together beneath the eye to form a single artery that dives into the interior of the head as the internal carotid artery.

The next four pharyngeal arches, branchial arches 1-4 (corresponding to pharyngeal arches 3-6) will bear gills. These house prominent aortic arches 3-6, such that the whole series of six pairs of aortic arches is present and carries circulating blood early in the hatching period. A gill slit forms posterior to the hyoid arch and between each branchial arch, making five in all. The rudiments of gill filaments develop late in the hatching period as buds along the posterior walls of the four branchial arches facing the slits (Fig. 42A).

The last arch of the pharyngeal series (branchial arch 5 or pharyngeal arch 7) does not develop gills. There is no gill slit behind it, nor does there appear an aortic arch within it. However, in the fashion of the other branchial arches it does make a supportive cartilage. Pharyngeal teeth develop in association with this particular cartilage, the only teeth in fact that the zebrafish will ever have.

The cartilages of the branchial arches begin to develop morphologically about a half day after the jaw cartilages. Each branchial arch initially develops a single simple linear element, a ceratobranchial cartilage, not dorsal and ventral elements developing nearly together as discussed above for the jaw arches. Moreover, the series of ceratobranchial cartilages does not develop synchronously; the first and cartilages (ceratobranchials 1 and 5) develop ahead of the others (Fig. 42 B & C). The cartilages of middle branchial arches in the series, particularly ceratobranchials 3 and 4, are still undifferentiated by end of the third day.

Detailed description of Hatching stages.