Embryonic and larval development of a Japanese spinous loach,Cobitis takatsuensis

The embryonic and larval development ofCobitis takatsuensis, a mountain stream spinous loach, was surveyed by incubating artificially inseminated eggs. The mean diameter of the inflated eggs and mean total length of newly-hatched larvae were 2.7 mm and 5.7 mm, respectively. The eggs were spherical, transparent and unpigmented, with a pale yellow yolk and no oil globule. The daily cumulative temperature to hatching was estimated to be 70–110°C. day. Hatched larvae were unpigmented with outer gill filaments on their cheeks, as in otherCobitis species, but the melanophores were comparatively less obvious at each developmental stage. The larvae started feeding eleven days after hatching yolk absorption being completed sixteen days after hatching. All the fin rays were fully developed and the juvenile stage reached at 16 mm TL, 38 days after hatching. Embryonic and larval developmental traits ofC. takatsuensis, such as egg size, clutch size and larval pigmentation, were similar to the Korean species,Niwaella multifasciata, that lives in the upper reaches of the Nak-tong river, andN. delicata, which inhabits Japanese mountain streams, rather than to its congeners. Among cobitine fishes, the spawning of a small number of larger eggs yielding larger larvae without pigmentation, characteristics shared byC. takatsuensis, N. multifasciata andN. delicata, is attributable to adaptation to cold mountain streams.     

Embryonic and larval development of the sea anemone Haliplanella lineata from Japan

The development of Haliplanella lineata, following fertilization in the laboratory, was studied by light and electron microscopy. Spawned ova were spherical, magenta in color and about 120–150 µm in diameter. Cleavage was holoblastic and radial. Gastrulation occurred by immigration and invagination. Eighteen hours after fertilization, the embryo became a swimming planula larva with an apical organ and ciliary tuft at the aboral end. In the laboratory, planulae lived for about 2 weeks in the swimming state but in no case was there any settlement by larvae in this study. The structural study of planulae concentrated on the development of the aboral ectoderm, because of the functional significance of its cellular organization in larval settlement.     

Early specification of ascidian larval motor neurons

In the tadpole larvae of the ascidian Halocynthia roretzi, six motor neurons, Moto-A, -B, and -C (a pair of each), are localized proximal to the caudal neural tube and show distinct morphology and innervation patterns. To gain insights into early mechanisms underlying differentiation of individual motor neurons, we have isolated an ascidian homologue of Islet, a LIM type homeobox gene. Earliest expression of Islet was detected in a pair of bilateral blastomeres on the dorsal edge of the late gastrula. At the neurula stage, this expression began to disappear and more posterior cells started to express Islet. Compared to expression of a series of motor neuron genes, it was confirmed that early Islet-positive blastomeres are the common precursors of Moto-A and -B, and late Islet-positive cells in the posterior neural tube are the precursors of Moto-C. Overexpression of Islet induced ectopic expression of motor neuron markers, suggesting that Islet is capable of regulating motor neuron differentiation. Since early expression of Islet colocalizes with that of HrBMPb, the ascidian homologue of BMP2/4, we tested a role of BMP in specification of the motor neuron fate. Overexpression of HrBMPb led to expansion of Lim and Islet expression toward the central area of the neural plate, and microinjection of mRNA coding for a dominant-negative BMP receptor weakened the expression of these genes. Our results suggest that determination of the ascidian motor neuron fate takes place at late gastrula stage and local BMP signaling may play a role in this step.     

Embryonic and larval development of the sonic motor nucleus in the oyster toadfish

The sonic motor nucleus (SMN), a likely homologue of the hypoglossal nucleus, provides the final common pathway for sound production in the oyster toadfish (Opsanus tau). SMN neurons increase in size and number for 7-8 years postnatally, and the swimbladder-sonic muscle complex grows throughout life. This study describes the normal embryonic and larval development of the SMN from its initial differentiation on about day 19 through day 40, when the yolk sac is resorbed and the fish is free swimming. In contrast to the rapid development of CNS nuclei in mammals, the SMN gradually increased in maturity with more active growth at the beginning and end of the observation period and a relatively static period in the middle. Consistent with a hypoglossal homology, the SMN differentiated within the spinal cord, added cells rostrally, and eventually extended into the medulla. Immature neurons appeared to originate from precursor cells in the ventral portion of the ventricular zone of the central canal. Such cells were initially round with little cytoplasmic development and later added processes and Nissl substance. The number of neurons increased 10-fold from a median of 35 to 322 cells, and no evidence of cell death was observed. Soma area approximately doubled from 20.6 to 41.2 micron 2, and cell nucleus area followed a similar pattern. [3H]-thymidine autoradiography demonstrated that neurons were added continuously throughout the nucleus during embryonic and larval development.     

Embryonic and larval development of the host sea anemones Entacmaea quadricolor and Heteractis crispa

Little information is available on the sexual reproductive biology of anemones that provide essential habitat for anemonefish. Here we provide the first information on the surface ultrastructural and morphological changes during development of the embryos and planula larvae of Entacmaea quadricolor and Heteractis crispa, using light and scanning electron microscopy. Newly spawned eggs of E. quadricolor and H. crispa averaged 794 microm and 589 microm diameter, respectively, and were covered by many spires of microvilli that were evenly distributed over the egg surface, except for a single bare patch. Eggs of both species contained abundant zooxanthellae when spawned, indicating vertical transmission of symbionts. Fertilization was external, and the resulting embryos displayed superficial cleavage. As development continued, individual blastomeres became readily distinguishable and a round-to-ovoid blastula was formed, which flattened with further divisions. The edges of the blastula thickened, creating a concave-convex dish-shaped gastrula. The outer margins of the gastrula appeared to roll inward, leading to the formation of an oral pore and a ciliated planula larva. Larval motility and directional movement were first observed 36 h after spawning. E. quadricolor larval survival remained high during the first 4 d after spawning, then decreased rapidly.     

Embryonic and larval hemoglobins during the early development of the bullfrog, Rana catesbeiana

The main hemoglobin (Hb) found in Shumway (embryonic) stage 25 bullfrogs is that which we have designated Td-4. The other major tadpole Hbs (Td-1, 2, and 3) predominate during Taylor and Kollros (larval) stages I-XVIII. We propose that Td-4 is an embryonic Hb, whereas Td-1, 2, and 3 are larval (fetal-like) Hbs. Embryonic Hb Td-4 continues to be synthesized during the larval stages. During the larval period, the average peripheral blood Hb profile changes very little with morphological stage or general growth. However, there is great heterogeneity in the embryonic:larval Hb ratio among individual tadpoles of a given stage or weight, apparently due to differential Hb and red cell production by the two active erythropoietic sites, mesonephric kidneys (Td-4), and liver (Td-1, 2, 3).     

Embryonic and larval development of Ensis arcuatus (Jeffreys, 1865) (Bivalvia: Pharidae)

The razor clam Ensis arcuatus (Jeffreys, 1865) is distributedfrom Norway to Spain and along the British coast, where it livesburied in sand in low intertidal and subtidal areas. This workis the first study to research the embryology and larval developmentof this species of razor clam, using light and scanning electronmicroscopy. A new method, consisting of changing water levelsusing tide simulations with brief dry periods, was developedto induce spawning in this species. The blastula was the firstmotile stage and in the gastrula stage the vitelline coat waslost. The shell field appeared in the late gastrula. The trochophoredeveloped by about 19 h post-fertilization (hpf) (19°C).At 30 hpf the D-shaped larva showed a developed digestive systemconsisting of a mouth, a foregut, a digestive gland followedby an intestine and an anus. Larvae spontaneously settled after20 days at a length of 378 µm. (Received 5 December 2006; accepted 19 November 2007)     

Embryonic and larval development of the Pacific cod Gadus macrocephalus from Taui Bay,northern Sea of Okhotsk

Experimental data on the embryogenesis and early post-embryonic development of the Pacific cod Gadus macrocephalus Tilesius that breed in Taui Bay are considered. Fertilized eggs at stage II have a diameter of 1.000–1.250 mm, and a mean of 1.103 ± 0.003 mm. Numerous teardrop-shaped inclusions in the plasma of the blastodisc and in blastomeres presumably prevent cod eggs from freezing at low temperatures during incubation in vivo. Embryogenesis of cod in vitro at the temperatures of 2.2 and 5.2°C lasts for 490 and 768 hours, respectively. Newly hatched cod prelarvae have a body length of 4.28–5.10 mm, a mean of 4.34 ± 0.03 mm; they switch to mixed and exogenous nutrition by 6 and 10 days, after reaching a mean length of 4.97 and 5.18 mm, respectively.     

Embryonic, larval and juvenile development of the roughskin sculpin,Trachidermus fasciatus (Scorpaeniformes: Cottidae)

Embryonic, larval and juvenile development of the catadromous roughskin sculpin,Trachidermus fasciatus, were described using eggs spawned in an aquarium. The eggs, measuring 1.98–2.21 mm in diameter, were light reddish-yellow and had many oil globules, 0.05–0.18 mm in diameter. Hatching occurred 30 days after spawning at 2.3–11.3°C. The newly-hatched larvae, measuring 6.9–7.3 mm BL, had a single oil globule, 9–10+25–26=34–36 myomeres and 6 or 7 large stellate melanophores dorsally along the gut. The yolk was almost resorbed, number of pectoral-fin rays attained 16–17, and two parietal, one nuchal and four preopercular spines were formed, 5 days after hatching, at 8.2–8.4 mm BL. The oil globule disappeared, and one supracleithral spine was formed, 11 days after hatching, at 8.9–9.5 mm BL. Notochord flexion began 15 days after hatching, at 9.7–10.3 mm BL. A posttemporal spine was formed 20 days after hatching, at 10.7–10.9 mm BL. The first dorsal fin spines (VII–VIII), second dorsal fin and anal fin rays (18–19, 16–18, respectively) appeared 23 days after hatching, at 12.0–13.7 mm BL. The pelvic fin spine and rays (I, 4) were formed and black bands on the head and sides of the body began to develop 27 days after hatching, at 13.8–15.8 mm BL. Newly-hatched larvae swam just below the surface in the aquaria. Preflexion larvae (8.9–9.5 mm BL), in which the oil globule had disappeared, swam in the middle layer, while juveniles (13.8–15.8 mm BL) began swimming on the bottom of the aquaria. Swimming behavior observed in the aquaria suggested that the fish started to change to a demersal existence at the juvenile stage.     

Embryonic skin development and repair

Fetal cutaneous wounds have the unique ability to completely regenerate wounded skin and heal without scarring. However, adult cutaneous wounds heal via a fibroproliferative response which results in the formation of a scar. Understanding the mechanism(s) of scarless wound healing leads to enormous clinical potential in facilitating an environment conducive to scarless healing in adult cutaneous wounds. This article reviews the embryonic development of the skin and outlines the structural and functional differences in adult and fetal wound healing phenotypes. A review of current developments made towards applying this clinical knowledge to promote scarless healing in adult wounds is addressed.     

Voltage-dependent calcium influx mediates maturation of myofibril arrangement in ascidian larval muscle

Calcium signaling is important for multiple events during embryonic development. However, roles of calcium influx during embryogenesis have not been fully understood since routes of calcium influx are often redundant. To define roles of voltage-gated calcium channel (Cav) during embryogenesis, we have isolated an ascidian Cav beta subunit gene (TuCavbeta) and performed gene knockdown using the morpholino antisense oligonucleotide (MO). The suppression of Cav activity by TuCavbetaMO remarkably perturbed gastrulation and tail elongation. Further, larvae with normal morphology also failed to exhibit motility. Phalloidin-staining showed that arrangement of myofibrils was uncoordinated in muscle cells of TuCavbetaMO-injected larvae with normal tail. To further understand the roles of Cav activity in myofibrillogenesis, we tested pharmacological inhibitions with ryanodine, curare, and N-benzyl-p-toluensulphonamide (BTS). The treatment with ryanodine, an intracellular calcium release blocker, did not significantly affect the motility and establishment of the myofibril orientation. However, treatment with curare, an acetylcholine receptor blocker, and BTS, an actomyosin ATPase specific inhibitor, led to abnormal motility and irregular orientation of myofibrils that was similar to those of TuCavbetaMO-injected larvae. Our results suggest that contractile activation regulated by voltage-dependent calcium influx but not by intracellular calcium release is required for proper arrangement of myofibrils.     

The effects of larval abundance, settlement and juvenile mortality on the depth distribution of a colonial ascidian

The colonial ascidian Didemnum candidum (Savigny) is more abundant at shallow depths on floating docks than at greater depths along pilings in Pearl Harbor, Oahu, Hawaii. To compare the effects of selective settlement and postsettlement mortality on adult distribution, I determined if settlement was nonrandom relative to depth, if differences in adult abundance were responsible for the depth distribution of settlement, and if juvenile mortality varied with depth. A plankton pump was used to measure variation in larval abundance with depth. Acrylic settling plates were suspended at different depths and sampled nondestructively to measure settlement intensity and juvenile mortality. Settlement and mortality of the didemnid ascidians Diplosoma listerianum Milne-Edwards and Diplosoma sp. were also measured in the field and compared to that of D. candidum. Settlement of D. candidum was indeed nonrandom over depth. Both planktonic larvae and settled juvenils were more numerous at 0.5 than 3 or m. Settlement intensity on vertical plates was greatest within 1–2 cm of the water surface in the laboratory and within 2–3 cm in the field. Directly adjacent to the floating dock, where adults were equally abundant, settlement on horizontal plates was greater at shallow depths, suggesting directed movement of larvae upward. However, the ratio of settled juveniles to planktonic larvae (number of settlers: number of larvae) did not significantly differ with depth, suggesting that larvae were not more likely to settle at a particular depth. Settlement of the Diplosoma species was also heaviest near the surface. Juvenile mortality was greater at 0.5 than at 3 or 6 m for both D. candidum and the Diplosoma species. Experimental settlement showed that mortality, per se, of D. candidum was independent of depth. Rather, mortality was density-dependent, and the higher mortality near the surface was due to the greater number of larvae settling there. At this location, nonrandom settlement appears to determine the adult distribution of D. candidum, despite greater juvenile mortality at shallow depths. The pattern of settlement over depth is largely determined by adult proximity, rather than active larval behavior.