Stages During the Pharyngula Period

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.

Prim-5 stage (24 h): EL = 1.9 mm, HTA = 120 degrees, and OVL = 5. The advancing end of the lateral line primordium overlies myotome (somite) 5. There are about 30 somites, 13 of them in the tail. The length of the yolk extension just equals the greatest diameter of the yolk ball.

The median fin fold is easily recognized (Fig. 28C). By 26 h (prim-8), mesenchyme gathers specially at the paired locations where the pectoral fin buds will form, lateral to the third somites. The buds first appear as mounds by 28-29 h.

Pigment formation begins in cells of the pigmented retinal epithelium and then in melanophores dorsolaterally in the skin. Melanophores appear in an approximate AP gradient, first just posterior to the otic vesicle. At the prim-5 stage the pigmentation is so light that a compound microscope may be required to see it. Use bright field, not Nomarski, transmitted illumination so that background contrast will be minimal. The pigment cells develop rapidly, such that within a few hours they are a prominent feature of the embryo (Fig. 30A); readily apparent at low magnification by the prim-12 stage (28 h; Fig. 29B). From a dorsal view at the same stage (Fig. 29C) one can see that melanophores now migrate to reach the dorsal and more anterior part of the yolk sac.

The heart is first visible as a cone-shaped tube deep to the brain, seemingly more dorsal than its later location, and prominently occupying a pericardial sac on the anterior-most region of the yolk. The heart begins to beat just prior to this stage, at first with no apparent direction to the beat, and the rhythm may be interrupted. By 26 h (prim-7), the heart tube is elongated and the contraction occurs as an AP-going wave. Most blood cells are in the blood island (Fig. 17D and Fig. 20A), and by 24-25 h some of them move more anteriorly and dorsally, into the region between the yolk and notochord, where the major vessels supplying the trunk and tail are forming. Blood cells also occupy the yolk ball, over very ill-defined and broad regions that herald the development of the common cardinal veins. The blood cells will begin circulation very soon after the stage when they first appear on the yolk ball.

The prim-5 embryo continues to exhibit spontaneous side-to-side contractions involving the trunk and tail. The contractions often occur in bursts, at a rate of about 8 episodes per minute (Grunwald et al., 1988). Sensory-motor reflexive circuits are becoming functional. At the prim-5 stage one cannot clearly distinguish responsiveness to light touches to the head or body of the dechorionated embryo, possibly because of a fairly high background level of spontaneous contractions. However, by 27 h (prim-10), this background lowers (it remains transiently high just after dechorionationation), and the first consistent tactile responses appear. The reflexive movements have approximately the same nature as the spontaneously occurring ones.

Prim-15 stage (30 h): EL = 2.5 mm, HTA = 95 degrees, OVL = 3. The yolk extension is just longer than the yolk ball. In dorsal view the yolk ball appears about twice as wide as the head.

The ventral portion of the median fin fold extends anteriorly, to underlie the yolk extension. The pectoral fin buds have the form of shallow domes, each with a height less than a third of the diameter at its base.

The embryo now begins to slow its overall rate of lengthening as tail morphogenesis comes to an end. A distinctive tail bud is no longer present, rather, some degenerating cells, evident by their bright appearance with Nomarski optics, occupy its position. The cell death may represent a natural process of elimination of the excess cells remaining in the bud after tail somitogenesis is complete (Kimmel et al., 1989).

Cells throughout the pigmented layer of the retina develop visible pigment granules. One can now very readily distinguish many of these pigmented epithelial cells, as well as the mesenchymal-looking melanophores in the skin by their dark pigmentation. A distinctive dorsal clustering of melanophores presages the formation of the median dorsal stripe, lengthwise and overlying the tops of the myotomes, and at this stage extending posteriorly to about somite 12. Other melanophores have appeared anteriorly in the head; a few of them are present overlying the dorsal midbrain and occasionally the forebrain. Yet others have migrated ventrally and begin to fill in a ventral stripe of pigmentation that underlies the myotomes bilaterally, but still is dorsal to the yolk. This ventral stripe is now very ill-defined and incomplete, particularly in the tail. Other melanophores migrate superficially to the myotomes but are not yet organized into a lateral stripe that will eventually overlie the horizontal myosepta. Melanophores also continue to accumulate on the dorsal yolk sac. Migration proceeds faster anteriorly than in more posterior regions, and results in a ventrally projecting anterior horn subregion of the ventral stripe containing broad, smoothly outlined melanophores extending to the bases of the eyes on each side of the midline (Fig. 29E, arrow in F).

The heart is still a straight tube, and its beat is becoming more prominent. Each heart-beat is in two parts, the first indication of development of chambers. There is a single aortic arch and complete arterial system between the pharynx and the tail, through which blood slowly circulates. A bit more than half-way along the tail a labyrinth-like network of channels connect between the caudal artery and vein. An apparently specialized set of very refractile cells line these particular channels (Fig. 38). We term the elements the tail reticular cells, because they very rapidly take up latex beads injected into the circulatory system (unpublished observations of CBK and BU), in the manner of reticulo-endothelial elements of the adult blood vascular system.

The frequency of spontaneous body contractions now diminishes to 1-2 episodes per minute. A light touch placed anywhere on the body or head usually elicits a robust reflexive side-to-side bout of wriggles. However, the embryo cannot yet effectively move its body through the water.

Prim-25 stage (36 h): EL = 2.7 mm, HTA = 75 degrees, OVL = 1. The length of yolk extension now markedly surpasses the diameter of the yolk ball. In dorsal view, the width of the yolk ball exceeds that of the head by a factor of about 1.3.

Actinotrichia develop posteriorly in the median fin fold where they can be revealed by Nomarski optics (Fig. 28D). Each pectoral fin bud is a dome-shaped mound raised about three-fourths as high as its width. As the bud grows outwards it loses the symmetrical appearance it had earlier, beginning to form an apical tip posterior to the center of the bud (Fig. 37B). The apical ectodermal ridge is now present at the tip, and easily visible with Nomarski optics if the orientation is correct, because the ridge runs obliquely across the bud, in a ventroposterior to dorsoanterior direction (Hatta et al., 1991a).

Pigmentation is prominent in the eye, but is still light enough so that one can readily visualize the unpigmented cell nuclei in this retinal epithelium. Melanophores on the body elaborate a bolder and longer (but still not sharply defined) dorsal stripe; now usually extending from the diencephalic region of the head to the tip of the tail, excluding the fin fold. There are still gaps along this dorsal stripe. Melanophores also appear laterally on the trunk and tail, some of them lodged in the region of the horizontal myoseptum where the lateral stripe will form. Others fill in the ventral stripe, making its anterior horn more readily apparent (Fig. 29 H, I), and extending the ventral stripe posteriorly to about about half way to the tip of the tail. Melanophores are also present on the dorsal and lateral surfaces of the yolk sac. Beginning at this time it be useful to examine the embryo with epi-illumination, using either the dissecting or compound microscope, not just with transmitted light. This is because the first reflective pigment cells, iridophores, soon appear on the eye. However, one cannot identify them at 36 h.

The pericardial cavity is not yet prominently inflated. Blood circulation is strong. The heart, previously a straight tube, now bends slightly. There is still but a single pair of aortic arches. The circulatory loop connecting the caudal artery and vein has moved to about 3/4 of the way to the end of the tail. Returning blood through the trunk and across the yolk, the pathway that includes the axial vein, the posterior cardinal vein and common cardinal vein are well-defined channels.

Spontaneous episodes of lashing activity of the embryo are now infrequent, occurring at less than one episode per min. The reflexive escape response to a touch lasts longer than before, and now effectively serves to displace the embryo, usually up to several body-lengths. The wriggles still occur slowly enough that one can easily resolve their rhythm; later the reflex is snappier and individual swimming beats occur so quickly that they simply cannot be resolved by eye.

High-pec stage (42 h): EL = 2.9 mm, HTA = 55 degrees, OVL = 3/4. The name for this stage, as well as for the next two, draws attention to the appearance of the rudiments of the pectoral fins. Now, at the high-pec stage, the fin bud's height is about equal to the width of its base. The apical ridge is readily visible using just the dissecting microscope. The ridge has increased prominence because it now incorporates, in addition to ectodermal epithelium, an underlying loose mesenchyme that will participate in development of the blade of the fin.

The yolk extension retains a distinctly cylindrical shape along most of its length, but its posterior end begins to taper, to take on a more conical appearance. Its length is about 1.5 times the diameter of the yolk ball. A dorsal view reveals that the yolk ball is about 1.3 times wider than the head.

The migrating primordium of the posterior lateral line gets smaller as it migrates, making it harder to identify (its identification never was particularly easy), and by 42 h it has reached the posterior end of the axis, so that its position can no longer be used as a staging index. The mandibular and hyoid arches (the first and second pharyngeal arches) are well defined. Inspection of the olfactory pits with Nomarski optics reveals the first formation of cilia by the epithelial cells. There is little or no ciliary beat. The same method also reveals a thickening in a dorsolateral region along the inner side of the otic vesicle's epithelial wall, that preludes development of the semicircular canals.

The primordia of the liver and probably the swim bladder make their appearances along the gut tract, posterior to the pharynx in the region of the anterior yolk ball. However, they are rather hidden by the dorsal part of the yolk ball; sectioned material reveals endodermal morphogenesis much more readily rather than do views of the living embryo. Hindgut endoderm is now recognizeable (first at the prim-28 stage at 39 h) as a solid plug (not a hollow tube) of midline cells in the anal region, thicker than the adjacent pronephric ducts.

The dorsal stripe of melanophores now is very well filled out to the end of the tail. The ventral stripe, now filled out anteriorly from the eye, also may contain pigment cells to the end of the tail. However, gaps along it in the tail region are conspicuous, one of these persisting at its posterior end. At this stage the dorsal and ventral stripes come together at the end of the tail to make an incompletely pigmented posteriorly pointed V. Melanophores along the lateral aspect of the myotomes now begin to organize distinctively along the horizontal myosepta as the lateral stripe (Fig. 29H). Only a very few cells are in it, 3 or so on each side of the body. The first few melanophores also reach the ventral surfaces of both the yolk ball and yolk extension, and will later organize into a yolk stripe that joins the ventral stripe at the level of the anus. Very bright illumination will for the first time reveal that the head has a very pale yellow cast, marking the first appearance of xanthophores. Also for the first time, bright incident illumination occasionally reveals a very few faintly reflective pigment spots, iridophores, scattered in a disorganized fashion on the eye. Iridophores are not yet apparent elsewhere on the body. Both xanthophores and iridophores will be much more easily and consistently recognized at the long-pec stage (48 h).

The bend in the heart is now prominent and marks the division between the atrium and ventricle, and Nomarski optics reveals an endothelial cushion between them. The heart beats (about 180 per minute) with the atrial beat preceding the ventricular one. There is still only one pair of aortic arches. The common cardinal vein on the yolk sac is very broad and prominent. Segmental arteries and veins are present along the trunk and tail. These vessels are deep to the myotomes, and are most easily recognized overlying the spinal cord near each transverse myoseptum. Those in the anterior tail begin to carry circulating blood.

Dechorionated embryos lie on their sides when at rest, yolk sac obliquely upwards. A touch elicits a vigorous and rapid response, with a wriggling rhythm just slow enough that its beat is still barely discernable. The response propels the embryo from a few body lengths in many cases, or up to the whole width of a 5 cm dish in others. At the end of the bout of swimming the embryo comes to rest on its side, not dorsal-up.