Rate of development
(Fig. 1) - The developmental rate for Lepisosteus, as is common for fish, proceeds at a pace dictated partly by the developmental program and partly by temperature. Our temperature control regime was of necessity weather-related, for specimens were raised either on a water table or in running lakewater. Developmental rates under such conditions can be measured fairly accurately over a short time period of up to several hours; but their accuracy suffers over longer periods because of environmental temperature fluctuations. We list short-term rates for early stages of development, at several temperatures, in the following paragraphs. Longer-term rates are shown in figure 1, which is based on selected developmental stages for a single batch of eggs taken in 1983. The temperatures listed each represent the average of two temperature readings taken in the running lakewater system every day, at 9 AM and 9 PM. Water temperature, at any time during a 24-hour period, fluctuated as much as a degree away from the average, i.e. warmer during the day and cooler at night. Development for this batch was not timed past six days because travel arrangements precluded it.
Stage 1, one cell
(Fig. 2A, 2B) - This stage begins at fertilization and lasts until the first cleavage furrow is readily visible, about three hours post-fertilization at 17°. The eggs are spherical, about 3 mm in diameter. As with other fish species, there is some variation in egg size from female-to-female. We measured a range of 2.8-3.1 mm for batches from various females. The eggs become quite sticky when they are placed in water, and adhere firmly to whatever substrate is available. This is true even for those that remain unfertilized.
The egg envelopes enlarge slowly over the next hour, revealing a narrow perivitelline space about 0.1 mm wide. A cushioning, nonadhesive jelly fills the perivitelline space. The principle envelope that surrounds the egg is the chorion, derived from the vitelline envelope of the unfertilized ovum. It is clear, allowing the egg to be viewed easily. This layer has similar optical and mechanical properties to the chorion of the medaka, Oryzias latipes, though it lacks the medaka's characteristic filamentous ornamentation. A second layer of jelly surrounds the chorion. This jelly layer is of variable thickness, up to 0.3 mm, and it provides the sticky character of the eggs.
Dean [6] described "slaty gray" eggs. This is true, except that each has a white blastodisc at the animal pole. The intersection of the white and gray regions establishes a distinctly visible border for the blastodisc. The blastodisc, itself, is about 2.3 mm in diameter and occupies about 100° of the egg's circumference. A dimple occurs at its apex, and marks the animal pole. The dimple is the point of sperm entry. It lies immediately adjacent to the micropyle, which is easily seen in the chorion and jelly layers.
Loosening of the vitelline envelope from the egg surface at fertilization allows gravity- induced egg rotation to occur, but the rotation is quite slow, perhaps impeded by the perivitelline jelly's thick consistency. It takes two hours or more to complete.
Stage 2, two cells
(Figs. 2C, 10A) - This stage begins with appearance of the first cleavage furrow, about four hours after fertilization at 17° (2 hours at 21°, 5.5 hours at 14°). The furrow first appears at the animal pole, and gradually extends to the edge of the blastodisc, which we now call the blastoderm. It will ultimately cut further, but by then subsequent furrows appear. Cleavage in this species is meroblastic; furrows divide the blastoderm similar to the teleosts. Unlike the teleostean pattern, however, the cleavage furrows continue past the blastoderm margin as grooves in the yolk cell surface. The first two grooves sometimes meet at the vegetal pole, but we found no evidence in living or fixed eggs that they extend significantly beneath the yolk cell cortex. This phenomenon led to some confusion in 19th century literature concerning whether garpike cleavage is holoblastic or meroblastic. It is meroblastic.
Stage 3, four cells
(Fig. 2D) - The second cleavage furrow appears about 6 hours after fertilization at 17° (9 hours at 14°). Dean [6] reported its appearance at three-hours, but his specimens were subject to an uncertain temperature regimen. The second furrow proceeds like the first, dividing the blastoderm deeply and extending past its margin outward across the gray yolk cell. Like the first groove, this one will ultimately reach the vegetal pole in some specimens.
Stage 4, eight cells
(Figs. 2E, 10B) - The paired third cleavage furrows appear about 7.5 hours post-fertilization at 17° (10.5 hours at 14°). They are generally parallel to the first furrow, and produce two rows of four blastomeres each. However, a few specimens (up to 10% in some batches of eggs) show an oblique third-cleavage orientation that produces a morula with a rosette of blastomeres at its animal pole. Dean first reported this in 1895 [6].
The first cleavage groove extends to the egg's equator by now, while the second meets the blastoderm margin. The vegetal half of the morula, the future yolk cell, still exhibits a smooth surface.
Stage 5, 16-32 cells
(Figs. 2F, 10C) - The fourth round of cleavage begins about 11 hours post-fertilization at 17° (12 hours at 14°), and lasts for about two hours. By now, the first cleavage groove reaches halfway between the equator and the vegetal pole.
Stage 6, 64-512 cells
(Fig. 3A, 3B, 3C) - This stage begins the blastula phase of garpike development. Cleavage divisions carry the blastula from 64 to 512 cells. In a 64-cell embryo, about 30 complete blastomeres appear on the surface, surrounded by marginal cells that are continuous with the yolk cell. Many central blastomeres are also separated from the yolk cell by tangential cleavage divisions. Dissection shows that the deep blastoderm cells are still connected to the yolk cell, and appear to bud from it.
Stage 7, small-cell blastula
(Fig. 3D) - The surface of the blastoderm has acquired a lumpy appearance, which indicates the steady decrease in blastomere size. An enveloping cell layer can be distinguished. Many deep cells lie beneath the enveloping layer. Cleavage furrows on the yolk cell are regressing by this time; they extend only to the egg's equator, no further.
Stage 8, smooth-surfaced blastula
(Fig. 3E) - This is the final stage before epiboly. Embryos reach it by about 20 hours at 17° (16 hours at 20°). Superficial blastomeres are tiny and the blastoderm surface consists of a thin enveloping layer, similar to that in teleosts. The yolk furrows gradually regress until they are no longer visible.
A virtual, uninflated, segmentation cavity forms, floored with large yolky cells. In many specimens, the blastoderm forms a peaked mound at the animal pole. The blastoderm has an irregular rim and a flat bottom where it joins the yolk cell. Dissection reveals a continuing presence of deep central cells that appear to be budding from the yolk cell. Many of them show broad connections with the yolk cell, and many join it via a narrow stalk.
Stage 9, epiboly begins
(Fig. 3F) - The blastoderm begins epiboly by 36 hours at 18°, but has not reached the egg's equator. Some specimens show the first indication of dorsal/ventral asymmetry, as a result of a lag in epiboly of the dorsal blastoderm margin. This produces a slight bulge in the blastoderm margin at that location, as though epiboly were being retarded by its attachment to the yolk cell. The bulge is the first external expression of the embryonic shield. The germ ring appears internally at this stage as well.
Dissected specimens reveal an extensive subgerminal cavity, whose floor is paved by large yolky cells that are adherent to the yolk syncytial layer (ysl). The blastoderm can be cleanly separated (by dissection) from the ysl surface beneath the germ ring and embryonic shield, but the deep yolky cells at the animal pole adhere to the ysl. Some of them are difficult to remove, for they are still attached by stalks to the yolk cell.
Stage 10, epiboly reaches the equator
(Figs. 3G, 10D) - The hint of dorsal/ventral asymmetry seen at stage 9 is now definite. The blastoderm overhangs its margin at the dorsal midline, having a crinkled edge where it attaches to the yolk cell. The overhang appears similar to the dorsal blastopore lip in amphibian embryos, and we give it that name. The exposed yolk cell surface beneath the dorsal lip is the first external appearance of the yolk syncytial layer.
Stage 11
(Fig. 4A, 4B) - The blastoderm reaches 1/3 the distance from the equator to the vegetal pole. The germ ring is wide, approaching 1 mm; and the embryonic shield is broader still.
The external ysl now occupies a greater portion of the blastoderm margin, and is broader at the dorsal midline than elsewhere. Viewed from the dorsal side, the blastoderm margin at the dorsal lip has the appearance of a horseshoe; its sidewalls face each other.
Deep cells anterior to the future forebrain region begin to migrate away from the animal pole, producing an evacuation zone that is devoid of cells. The evacuation zone is not visible in living specimens at this stage, and some deep cells still remain tightly attached to the ysl at the animal pole. The zone ultimately enlarges to a diameter of 1 mm at stage 14.
Stage 12
(Fig. 4C) - The blastoderm margin reaches 2/3 the distance from equator to vegetal pole. The dorsal lip overhangs a pit floored by the external ysl, a virtual gastrocoel. The embryonic shield extends above the equator. It can be dissected into two principal layers, the epiblast and the hypoblast. The roof of the evacuation zone at the animal pole is much thinner than before. The exposed surface of the yolk cell now comprises a large yolk plug.
Stage 13
(Fig. 4D) - The yolk plug is half or less the diameter of the equator. The germ ring is narrow now and the embryonic shield is longer, nearly reaching to the evacuation zone above the equator. There is a hint of a neural groove on its surface. The crinkled sidewalls of the dorsal lip flatten out as epiboly in the dorsal midline catches up to that in other areas of the blastoderm margin.
Stage 14
(Fig. 4E) - The yolk plug is 1/8 or less the equator diameter but is still visible. The embryonic shield is long, intruding anteriad on the evacuation zone, which has a jumble of floor cells. They can easily be dislodged with a hair loop. The shield extends over about 100° of the yolk cell surface. Although its neural groove has deepened somewhat, it is still quite shallow.
The subgerminal cavity, distinct from the evacuation zone, begins to inflate under the embryonic shield, but little outside it. It has a smooth floor, which is formed by the surface of the ysl.
Stage 15, yolk plug closure
(Fig. 4F) - The yolk plug is no longer visible inside the tiny blastopore. The neural folds are distinct, but feeble. They close in a posterior-anterior sequence at this stage, remaining open last at their anterior end. Internally, the posterior part of the notochord segregates from the neural keel. A crescent of cells extends forward, anterior to the neural field. Later, these cells will contribute to the adhesive organ. The subgerminal cavity inflates lateral to the neural keel. This cavity is not a blastocoel; it is a product of morphogenetic cell movements, not of cleavage.
Stage 16
(Fig. 5A, 5B) - The embryo's axis occupies from 150° to 180° of the egg circumference. The neural folds disappear and the neural keel pushes deeper, intruding on the subgerminal cavity. The prospective diencephalon enlarges slightly where the optic lobes will form.
The inflated subgerminal cavity extends the full length of the embryonic axis. The cavity extends laterally as a wide pellucid area on each side of the axis, and anterior to the brain, it extends into the evacuation zone. Somites 1-10 appear during this stage, and pronephric ducts occur from somite 4 posteriad. A slight swelling, the trunk-tail bud, lies at the posterior end.
Stage 17
(Fig. 5C) - New somites form, from the 10th to the 20th. The length of the axis exceeds 180° of the egg circumference. Expansion of the optic vesicles is obvious; they enlarge to hemispherical proportions. The telencephalon lies anterior to them. Development of the adhesive organ produces a bean-shaped swelling anterior to the brain. Thickened protrusions in the subgerminal cavity roof, lateral to the axis, mark the beginning of the pharyngeal arches.
Stage 18
(Fig. 5D) - New somites form, from the 20th to the 25th. The brain and trunk-tail bud rise prominently above the main curvature of the embryo, but neither is undercut. The single curved mass of the adhesive organ wraps tightly around the telencephalon. The optic swellings have become rounded vesicles.
The hyomandibular and hyobranchial pharyngeal pouches are visible externally, but they are best seen from below after dissection of the embryo from the yolk. The walls of the pharynx, as seen in dissection, project down laterally and begin to fold mesad to form the pharynx floor.
Stage 19
(Fig. 5E) - New somites form, from the 25th to the 30th. The trunk-tail bud projects from the embryo; it is slightly undercut, but not bent down around the yolk sac's contour. The head end is markedly raised and appears knobby because of the swollen brain vesicles, the spherical optic vesicles, and the adhesive organs. The operculum appears, extending a free edge. Ventral fusion of the pharynx sidewalls begins, signaling the appearance of the anterior intestinal portal. Lateral plate mesoderm extends about halfway out over the yolk sac. A vascular network shows on the dorsal portion of the yolk sac, but the heart is straight and not beating.
Stage 20
(Fig. 5F) - New somites form from 30th to 40th. The trunk-tail projection bends down in a curve that follows the yolk mass, and is undercut for a distance equal to that from adhesive organ to hindbrain. The posterior intestinal portal appears. The pronephros begins to bend. Lenses can be seen in the eyes. The opercular edge begins its extension. This is the stage of first motility, a slight one-sided squirm at best.
Stage 21
(Fig. 6A) - The trunk-tail outgrowth is now longer than the whole brain, but still curved along the contour of the yolk. Post-cloacal somites are now forming and the dorsal fin fold appears. The trunk is almost straight. The entire trunk is still attached to the yolk mass, but the cloacal region is beginning to lift away from it. There is a strong pronephric bend.
The head begins to project freely over the mouth area. The adhesive organ is larger than the eyeball, but it is not sticky. The epiphysis appears and olfactory placodes are visible. One can find pigment on the yolk sac and posterodorsal to the eye. The heart begins to beat. It is hardly curved at all. Blood vessels cover all but the midventral part of the yolk. Motility increases, and embryos move in a strong, single coil.
Stage 22
(Fig. 6B) - The free portion of the trunk is fairly straight, but the tail is bent down beyond the cloaca, which is 1/2 to 2/3 of the way out along the free ventral surface of the trunk. The dorsal finfold is obvious and the intestine is visible between the yolk mass and the cloaca. Post-cloacal somites have formed halfway out along the tail.
When viewed face on, the adhesive organ has the shape of a horseshoe. The heart begins to coil and blood flows on to the dorsal face of the yolk sac. Dissection reveals a bulging liver, although this is not visible externally. Trunk somites are arranged as chevrons halfway down the attached portion of the trunk. Motility increases to one or two coils, but there is no backlash.
Stage 23
(Fig. 6C) - The tail is finally straight. Both the dorsal and ventral fin folds of the tail are taller than the caudal somites and a ventral fin notch appears at the cloaca. Inside the chorion, the tail tip reaches the eye.
The somites become dusted with pigment. Somites are chevron-shaped only on the attached part of the trunk. The margin of the operculum is thickened but does not yet flare. Blood circulation is established over most of the yolk mass and the flow is strong in the subintestinal vein.
Stage 24
(Fig. 6D) - Tail segmentation is virtually complete; it never reaches the tail tip. The yolk mass has become ovoid. The adhesive organ becomes sticky. The free edge of the operculum begins to expand, but does not yet cover the branchial arches. Dechorionated embryos can swim clumsily when prodded.
Stage 25
(Fig. 7A) - Somites are chevron-shaped as far as the middle of the trunk. Pectoral fin buds are visible after fixation. Specimens can swim in circles when prodded.
Stage 26
(Fig. 7B) - Pectoral fin buds are visible in the living embryo. Somites are chevron-shaped past the cloaca. There is a prominent bulge in the operculum edge below the branchial arches.
Stage 27
(Fig. 7C) - Pigment aggregates in the ventral fin fold, indicating the location of the future anal and caudal fins. Pectoral plates now possess barely detectable ridges. The adhesive organ is equal to or larger than the eye.
Stage 28
(Fig. 7D) - Anlagen of the anal and caudal fins are clearly visible in the ventral fin fold. Erosion of the dorsal fin fold begins, back beyond the pectoral fin level. The pectoral fin is now a low disc without a membranous flange. The opercular flap, which is as wide as the eye, obscures the branchial arches in side view. Slight swellings on the gill arches presage gill filaments.
Stage 29
(Fig. 8A, 8B) - The anlage of the dorsal fin is now visible in the dorsal fin fold. Although the pectoral fin has acquired the shape of a half-moon, it still has no flange. Gill filaments are sprouting on branchial arches one and two. Most hatching occurs at this stage.
Stage 30
(Figs. 8C, 10E) - The dorsal fin fold has eroded as far as the mid-trunk. The height of the yolk mass now has been reduced nearly to the height of the tail including its fin folds. The pectoral fin is larger than a half-moon, with a flange narrower than its muscle mass. The operculum is nearly twice as wide as the eye. Gill filaments occur on branchial arch three.
Stage 31
(Fig. 8D) - The dorsal fin fold has eroded beyond the mid-trunk; and the height of the yolk mass is now less than or equal to the height of the tail including its fin folds. Somites are chevron shaped clear to the caudal fin. The pectoral fin flange is now equal to or wider than the fin's muscular disc. The edge of the operculum flap reaches the base of the pectoral fin. The diameter of the adhesive organ is now less than that of the eye. The nasal pit is still single and round, as it has been since stage 27.
Stage 32
(Fig. 9A, 9B) - Pelvic fin mats are visible as smooth bulges. The yolk mass is still perhaps twice the volume of the head. Nasal openings are still single, but elongating; and the snout begins to lengthen. The lower jaw reaches the adhesive organ. Rhythmic movements of the jaw and operculum begin. Bump-like tooth primordia are barely visible on the jaws. The pectoral fins tremble at the end of a swim.
Stage 33
(Fig. 9C, 9D) - The fin fold of the tail is shrinking between the anal and caudal fins, and around the permanent dorsal fin. Rudiments of fin rays become visible in these unpaired fins. No dorsal fin fold remains anterior to the level of the cloaca. The yolk mass is now smaller than the head. The nasal apparatus finally has separated into incurrent and excurrent openings.
Stage 34
(Fig. 9E, 9F) - The yolk is finally exhausted. Pelvic fins are half-moon shaped with narrow membranous flanges. The jaws are studded with sharp teeth.