Water fleas (Daphnia magna) provide a separate ventilatory mechanism for their brood

The planktonic filter feeder Daphnia magna depends on a steady oxygen supply by convection. In the ventral carapace chamber, this convection is established by the feeding current which is generated by the movement of the thoracic limbs. The present study revealed that this movement can cause an additional flow of medium which passes through the brood chamber of the animal. To visualise this current, ink or fluorescent microspheres were released by a microcapillary near the posterior opening of the brood chamber. The tracks of these probes were monitored by video microscopy. Digital motion analysis was used for the determination of flow velocity and flow rate. Ambient medium entered the brood chamber at the abdomen of the animal and moved then to the anterior end of the brood chamber before entering the ventral carapace chamber. Two horizontal lamellae, which are attached at both sides of the trunk and project laterally to contact the carapace walls, almost completely separate the dorsal brood chamber from the ventral carapace chamber. Water can only pass these barriers through small depressions in these lamellae at the level of the 3rd and 4th appendages. Female daphnids with embryos at late developmental stages showed more rapid water currents in the brood chamber than those with younger embryos. Moreover, animals showed higher flow rates when exposed to hypoxic conditions. As the oxygen uptake rate of older embryos is approximately three times higher than that of younger embryos, the enhanced brood chamber current could improve the oxygen availability for both the mother and its brood under conditions of reduced oxygen availability.List of symbols:a specific oxygen consumption rate (nmol mm-³ h-¹), K Krogh constant for oxygen diffusion (nmol mm-¹ h-¹ kPa-¹, P_c critical oxygen partial pressure (kPa), P o₂ oxygen partial pressure (kPa), r radius (mm), s distance (mm), t time (ms)     

The importance of perivitelline fluid convection to oxygen uptake of Pseudophryne bibronii eggs

The ciliated epithelium of amphibian embryos produces a current within the perivitelline fluid of the egg that is important in the convective transfer of oxygen to the embryo's surface. The effects of convection on oxygen uptake and the immediate oxygen environment of the embryo were investigated in Pseudophryne bibronii. Gelatin was injected into the eggs, setting the perivitelline fluid and preventing convective flow. Oxygen consumption rate (M(.)o?) and the oxygen partial pressure (Po?) of the perivitelline fluid were measured in eggs with and without this treatment. M(.)o? decreased in eggs without convection at Gosner stages 17-19 under normoxia. The lack of convection also shifted embryos from regulators to conformers as environmental Po? decreased. A strong Po? gradient formed within the eggs when convection was absent, demonstrating that the loss of convection is equivalent to decreasing the inner radius of the capsule, an important factor in gas exchange, by 25%. M(.)o? also declined in stage 26-27 embryos without cilia-driven convection, although not to the extent of younger stages, because of muscular movements and a greater skin surface area in direct contact with the inner capsule wall. This study demonstrates the importance of convective flow within the perivitelline fluid to gas exchange. Convection is especially important in the middle of embryonic development, when the perivitelline space has formed, creating a barrier to gas exchange, but the embryos have yet to develop muscular movements or have a large surface area exposed directly to the jelly capsule.     

Regulation of air and blood flow through the booklungs of the desert scorpion, Paruroctonus mesaensis

Injections of dye, latex and India ink were used to reveal the path of hemolymph circulation through the scorpion booklungs. Fine, branched arteries carry blood directly to muscle and other organs. The blood returns through venous channels to the ventral mesosoma where it passes laterally through the booklungs and into the pneumocardial veins just beneath the pleural cuticle. Blood flows dorsally through these veins to the pericardial sinus and heart. The scorpion has four pairs of booklungs located in the anterior segments of the ventral mesosoma. Each booklung has a spiracle which opens into an atrium enclosed by cuticular membrane. Air passes from the atrium into the booklung lamellae. Agitation of the animal or application of CO(2) causes retraction of the anterior and posterior atrial membrane. This expands the atrial chamber and allows gas exchange in the booklung lamellae. The posterior atrial membrane has a specialized region which forms a springy valve. This normally closes the spiracle unless pulled open by contraction of the attached poststigmaticus muscle. The pectens and receptors within the atrium may mediate the responses to CO(2). Slender hypocardial ligaments containing muscle fibers extend from the heart (dorsal mesosoma) to the booklungs in the ventral mesosoma. Heart movements thus cause dorso-ventral movement of the booklungs. The significance of these movements is as yet unclear. They may increase ventilation, help force blood to the heart and/or agitate the blood and booklung lamellae and thereby aid gas exchange. Passage of blood through the booklungs is regulated by dorsal and ventral muscles attached to the atrium at the lateral edge of the booklung. Contraction of the ventral atrial muscle closes the excurrent channel for passage of blood from the booklung into the pneumocardial vein. Electrical stimulation of the segmentai nerves from the subesophageal and first three abdominal ganglia causes spiracle opening and contraction of muscles attached to the atrial membrane. A previous study showed that these same segmental nerves also modulate heart activity. They thus provide a major pathway for regulation of the respiratory and circulatory systems.     

The Unique Dorsal Brood Pouch of Thermosbaenacea (Crustacea,Malacostraca) and Description of an Advanced Developmental Stage of Tulumella unidens from the Yucatan Peninsula (Mexico), with a Discussion of Mouth Part Homologies to Other Malacostraca

The Thermosbaenacea, a small taxon of crustaceans inhabiting subterranean waters, are unique among malacostracans as they brood their offspring dorsally under the carapace. This habit is of evolutionary interest but the last detailed report on thermosbaenacean development is more than 40 years old. Here we provide new observations on an ovigerous female of Tulumella unidens with advanced developmental stages in its brood chamber collected from an anchialine cave at the Yucatan Peninsula, which is only the third report on developmental stages of Thermosbaenacea and the first for the genus Tulumella. Significant in a wider crustacean context, we report and discuss hitherto unexplored lobate structures inside the brood chamber of the female originating at the first (maxilliped) and second thoracic segments, which are most likely modified epipods, perhaps serving as gills. At the posterior margin of carapace of the female are rows of large spines preventing the developing stages from falling out. The external morphology of the advanced developmental stages is described in much detail, providing information on e.g., carapace formation and early limb morphology. Among the hitherto unknown structures in the advanced developmental stages provided by this study are the presence of an embryonic dorsal organ and rudimentary ‘naupliar processes’ of the second antennae. Since most hypotheses on crustacean (and malacostracan and peracaridan) relationship rest on external limb morphology, we use early limb bud morphology of Tulumella to better establish thermosbaenacean limb homologies to those of other crustaceans, which is a necessary basis for future morphology based phylogenetic considerations.     

Cyst development in the conchostracan shrimp,Eulimnadia texana (Crustacea: Spinicaudata)

The fertilized egg (or cyst) of branchiopods is a highly resistant stage in the life cycle of these aquatic crustaceans. Previous examinations of these cysts have determined that early embryonic development arrests at a late blastula stage, resulting in a small, crescent-shaped body within the egg shell of these shrimp. Herein, we examine the early development of these embryos by sectioning eggs in the ovotestis, brood chamber, and several time periods after exit from the brood chamber in the clam shrimp Eulimnadia texana Packard. The early sections find no evidence of internal fertilization in the ovotestis. Eggs in the ovotestis showed no signs of cell division, whereas eggs sectioned from the brood chamber were found to be undergoing early embryonic development. A number of empty egg shells and the lack of unfertilized eggs in the brood chamber suggested that egg yolks quickly degrade after egg extrusion from the ovotestis. Cysts that were allowed to develop for 24, 48, 72, and 96 h, 1 week and 1.5 years were sectioned, and embryonic development did not change after the 48 h time period. Thus, embryos appear to arrest development somewhere between 24 and 48 h after exiting the brood chamber.     

Oxygen transport to embryos in microdrop cultures

The standard method for culturing small preimplantation mammalian embryos is a system in which they are placed into microdrops of culture medium under oil. Thus the source of oxygen for the embryos lies beyond two liquid phases--medium and oil. If transport of oxygen is not sufficiently rapid to replace that consumed by the embryos, the medium could become depleted of oxygen. Over small distances, the dominant means by which oxygen is transported through liquid is by diffusion. Our calculations show that diffusion alone is sufficient to supply oxygen to mouse embryos; there is virtually no perturbation of the oxygen concentration when there are up to 10 embryos in the drop, and 50 embryos produce a drop in oxygen tension that is not large enough to have a deleterious effect. Furthermore, diffusion is probably not the dominant mechanism by which oxygen is transported to the embryos; on the scale of these microdrops, convection is faster and would serve to mix the drop so that anoxic regions cannot develop. Therefore, we conclude that even a relatively large number of embryos in a culture drop do not significantly deplete oxygen.     

Brooding in the Chilean Oyster Ostrea chilensis: Unexpected Complexity in the Movements of Brooded Offspring within the Mantle Cavity

Brooding in invertebrates serves to protect embryos from stressful external conditions by retaining progeny inside the female body, effectively reducing the risk of pelagic stages being exposed to predation or other environmental stressors, but with accompanying changes in pallial fluid characteristics, including reduced oxygen availability. Brooded embryos are usually immobile and often encapsulated, but in some Ostrea species the embryos move freely inside the female pallial cavity in close association with the mother’s gills for as long as eight weeks. We used endoscopic techniques to characterize the circulation pattern of embryos brooded by females of the oyster, Ostrea chilensis. Progeny at embryonic and veliger stages typically circulated in established patterns that included the use of dorsal and ventral food grooves (DFG, VFG) to move anteriorly on the gills. Both embryos and veligers accumulated around the mother’s palps, and remained there until an active maternal countercurrent moved them to the gill inhalant area. Both food grooves were able to move embryos, veligers, and food-particle aggregates anteriorly, but the DFG was more important in progeny transport; early embryos were moved more rapidly than veligers in the DFG. A microcirculation pattern of embryos was apparent when they were moved by gill lamellae: when they were close to the VFG, most embryos lost gill contact and ´´fell´´ down to the DFG. Those that actually reached the DFG moved anteriorly, but others came into contact with the base of the lamellae and again moved towards the VFG. The circulation pattern of the progeny appears well-suited for both cleaning them and directing them posteriorly to an area where there is more oxygen and food than in the palp region. This process for actively circulating progeny involves the feeding structures (gill and palps) and appears to be energetically costly for the female. It also interferes with feeding, which could explain the poor energy balance previously documented for brooding females of this species.     

Ultrastructural evidence of the ion-transporting role of the adult and larval neck organ of the marine gymnomeran Cladocera (Crustacea,Branchiopoda)

The neck organ of adult and immature forms of four marine gymnomeran Cladocera species (Podonidae) has been characterized morphologically by light-scanning- and electron microscopy. Although displaying some special features, a comparison with organs assuming a comparable regulating function in other organisms indicates that the neck organ of the adult Podonidae exhibits ultrastructural evidence of involvement in ion-transporting mechanisms. The extensive ventral plasma membrane infoldings closely connected with the chondriom and the peculiar organization of the cuticular site associated with the subjacent apical cell limits of the organ are among the chief structural specializations. The fact that similar structures are already present in the embryonic neck organ confirms previous indirect evidence that podonid embryos can hypo-osmoregulate as soon as the ion concentration of the brood chamber rises to that of the outside medium.     

Distinct effects of ectopic expression of Wnt-1, activin B, and bFGF on gap junctional permeability in 32-cell Xenopus embryos.

A polarity in gap junctional permeability normally exists in 32-cell stage Xenopus embryos, in that dorsal cells are relatively more coupled than ventral cells, as measured by transfer of Lucifer yellow dye. The current study extends our analysis of whether gap junctional permeability at this stage can be modulated by secreted factors, and whether the polarity in gap junctional permeability correlates with the effects of ectopic expression of these secreted factors on the subsequent phenotype of the developing embryo. Following ectopic expression of activin B or Wnt-1, but not bFGF, the transfer of Lucifer yellow between ventral animal pole cells is detected in a greater percentage of 32-cell stage embryos. This increased incidence of dye transfer between ventral cells correlates with axial duplications later in development. However, there are differences in the extent of Lucifer yellow transfer between animal and vegetal hemisphere blastomeres which is dependent on whether activin B or Wnt-1 RNA had previously been injected. These results suggest that enhanced gap junctional permeability between ventral cells of 32-cell Xenopus embryos correlates with subsequent defects in the dorsoventral axis, although there are at present no direct data demonstrating a role for gap junctions in establishment or maintenance of this axis. Moreover, while both activin B and bFGF are mesoderm-inducing growth factors, only activin B has effects on gap junctional permeability in 32-cell embryos following ectopic expression, demonstrating an interesting difference in physiological responses to expression of these factors.     

Simple flow chamber for embryos]

The chamber is made of two subject glasses arranged in such a way that nutritional medium can flow through it. In order to prevent washing out of the embryos by the stream of nutritional medium, a porous packing is inserted through which the nutritional medium is flowing. The upper subject glass has a hole through which the embryos are put into the chamber. The hole is covered with a covering glass. The chamber can be preserved for a long time "avaiting" for the cultivated material at any regime of the stream or at a static regime.     

Use of Hybrids between Xenopus laevis and Xenopus borealis in Chimera Formation: Dorsalization of Ventral Cells

The combination of Xenopus borealis and X. laevis provides an excellent cell marking system. The potential availability of this system for chimera formation has also been suggested. However, eggs and early embryos of these species differ in size and the fusion of blastomeres of different sizez results in some disturbance in arrangement of blastomeres of a chimera. This disturbance was avoided by use of embryos from X. laevis eggs fertilized with X. borealis sperm, instead of X. borealis embryos. The cells of these hybrids could also be distinguished from the cells of X. laevis.
The fate of animal ventral cells placed in the dorsal region was followed by making a chimera by fusing a right lateral half of an 8-cell X. laevis embryo with that of an 8-cell hybrid embryo. The animal ventral cells in the "dorsal" region were found to become "dorsalized", giving rise to a lateral half of dorsal axial structures. This observation explains a previous finding that the replacement of dorsal cells by ventral ones had no effect on embryogenesis in a composite embryo.     

Reproductive strategy of the semelparous clam Gaimardia bahamondei (Bivalvia,Gaimardiidae)

Abstract. Semelparity is one of the most drastic reproductive strategies found among marine invertebrates. It is frequently found in species whose members have small adult sizes and brood embryos internally. In this study, we describe the reproductive strategy of the bivalve Gaimardia bahamondei to explore the possible causes of the association between semelparity and internal brooding. Males of this species exhibit continuous gonadal activity throughout the breeding season. Apparently continuous spawning of sperm is associated with an abundance of captured sperm in the adfrontal region of the gill filaments of both males and females. Females are capable of brooding three cohorts of embryos simultaneously while also producing three new cohorts of oocytes. This suggests that females are able to generate at least six cohorts of embryos during the breeding season. Embryos are brooded in the suprabranchial chamber and are individually surrounded by a membrane with a projecting peduncle; embryos are anchored by this peduncle to the adfrontal region of the gill filaments. Members of the youngest cohort differ in size, color, and shell ornamentation from members of the two older cohorts. There is no difference between members of the two older cohorts in size, but there is with respect to coloring and shell ornamentation. The importance of the embryonic cohorts in terms of their percentage of the total number of embryos varied among brooding females, suggesting among‐female variation in the timing of release of the oldest cohort of embryos. Members of this cohort break loose from the gills, lose their surrounding membrane, and fall into the ventral region of the suprabranchial chamber, from which they are evacuated to the exterior. Continuous sperm production in males and the production of at least six cohorts of embryos in females suggest that the costs of reproduction are high, which may partly explain the semelparity identified in this species.     

Functional morphology of Palaeozoic ostracods: phylogenetic implications

Recent discussions of ostracod systematics have focused on soft anatomy, both as seen in extant groups and as recorded by rare examples of special fossil preservation. The position of the fossil Palaeocopina and Leperditicopida, for which no substantial soft part evidence has yet been found, remains in the view of post-Palaeozoic workers uncertain, with some doubt as to whether they should be retained within the Ostracoda. The evolution of carapace bauplans (e.g. the development of brood pouches and lobal structures in palaeocopids as well as the development of adductor muscle scar patterns, calcified inner lamellae and carapace incisures in podocopines) is discussed in relation to presumed soft anatomy. It seems possible to distinguish between plesiomorphic (ancestral, simple) and apomorphic (derived, advanced) characters and consider their significance in ostracod systematics. Although the presumed ‘protostracod’ is not known, the combination of soft anatomy, carapace architecture and behaviour (feeding techniques, brood care) provide evidence of a general body plan which appeared (at the latest) during the Ordovician and continuously evolved towards the anatomy of modern ostracods. In parallel lineages, plesiomorphic forms have died out (leperditicopids and most palaeocopines as well as metacopines), while apomorphic lineages (‘drepanellid archetype’ of palaeocopines; resistant platycopines, podocopines and myodocopines) have survived all extinction events. The evidence supports the retention of the Palaeocopina (and probably the Leperditicopida) in the Ostracoda.     

Secretory and inductive properties of Drosophila wingless protein in Xenopus oocytes and embryos.

Like its vertebrate homologues, Xenopus wnt-8 and murine wnt-1, we find that Drosophila wingless (wg) protein causes axis duplication when overexpressed in embryos of Xenopus laevis after mRNA injection. In many cases, the secondary axes contain eyes and cement glands, which reflect the induction of the most dorsoanterior mesodermal type, prechordal mesoderm. We show that the extent of axis duplication is dependent on the embryonic site of expression, with ventral expression leading to a more posterior point of axis bifurcation. The observed duplications are due to de novo generation of new axes as shown by rescue of UV-irradiated embryos. The true dorsal mesoderm-inducing properties of wg protein are indicated by its ability to generate extensive duplications after mRNA injection into D-tier cells of 32-cell embryos. As revealed by lineage mapping, the majority of these D cell progeny populate the endoderm; injections into animal blastomeres at this stage are far less effective in inducing secondary axes. However, when expressed in isolated animal cap explants, wg protein induces only ventral mesoderm, unless basic fibroblast growth factor is added, whereupon induction of muscle and occasionally notochord is seen. We conclude that in intact embryos, wg acts in concert with other factors to cause axis duplication. Immunolocalisation studies in embryos indicate that wg protein remains localised to the blastomeres synthesizing it and has a patchy, often perinuclear distribution within these cells, although some gets to the surface. In oocytes, the pool of wg protein is entirely intracellular and relatively unstable. When the polyanion suramin is added, most of the intracellular material is recovered in the external medium.     

A Computational Study of the Hydrodynamics in the Nasal Region of a Hammerhead Shark (Sphyrna tudes): Implications for Olfaction

The hammerhead shark possesses a unique head morphology that is thought to facilitate enhanced olfactory performance. The olfactory chambers, located at the distal ends of the cephalofoil, contain numerous lamellae that increase the surface area for olfaction. Functionally, for the shark to detect chemical stimuli, water-borne odors must reach the olfactory sensory epithelium that lines these lamellae. Thus, odorant transport from the aquatic environment to the sensory epithelium is the first critical step in olfaction. Here we investigate the hydrodynamics of olfaction in Sphyrna tudes based on an anatomically-accurate reconstruction of the head and olfactory chamber from high-resolution micro-CT and MRI scans of a cadaver specimen. Computational fluid dynamics simulations of water flow in the reconstructed model reveal the external and internal hydrodynamics of olfaction during swimming. Computed external flow patterns elucidate the occurrence of flow phenomena that result in high and low pressures at the incurrent and excurrent nostrils, respectively, which induces flow through the olfactory chamber. The major (prenarial) nasal groove along the cephalofoil is shown to facilitate sampling of a large spatial extent (i.e., an extended hydrodynamic “reach”) by directing oncoming flow towards the incurrent nostril. Further, both the major and minor nasal grooves redirect some flow away from the incurrent nostril, thereby limiting the amount of fluid that enters the olfactory chamber. Internal hydrodynamic flow patterns are also revealed, where we show that flow rates within the sensory channels between olfactory lamellae are passively regulated by the apical gap, which functions as a partial bypass for flow in the olfactory chamber. Consequently, the hammerhead shark appears to utilize external (major and minor nasal grooves) and internal (apical gap) flow regulation mechanisms to limit water flow between the olfactory lamellae, thus protecting these delicate structures from otherwise high flow rates incurred by sampling a larger area.     

Evolutionary ecology of pipefish brooding structures: embryo survival and growth do not improve with a pouch

For animals that reproduce in water, many adaptations in life‐history traits such as egg size, parental care, and behaviors that relate to embryo oxygenation are still poorly understood. In pipefishes, seahorses and seadragons, males care for the embryos either in some sort of brood pouch, or attached ventrally to the skin on their belly or tail. Typically, egg size is larger in the brood pouch group and it has been suggested that oxygen supplied via the pouch buffers the developing embryos against hypoxia and as such is an adaptation that has facilitated the evolution of larger eggs. Here, using four pipefish species, we tested whether the presence or absence of brood pouch relates to how male behavior, embryo size, and survival are affected by hypoxia, with normoxia as control. Two of our studied species Entelurus aequoreus and Nerophis ophidion (both having small eggs) have simple ventral attachment of eggs onto the male trunk, and the other two, Syngnathus typhle (large eggs) and S. rostellatus (small eggs), have fully enclosed brood pouches on the tail. Under hypoxia, all species showed lower embryo survival, while species with brood pouches suffered greater embryo mortality compared to pouchless species, irrespective of oxygen treatment. Behaviorally, species without pouches spent more time closer to the surface, possibly to improve oxygenation. Overall, we found no significant benefits of brood pouches in terms of embryo survival and size under hypoxia. Instead, our results suggest negative effects of large egg size, despite the protection of brood pouches.     

Xwnt-8 modifies the character of mesoderm induced by bFGF in isolated Xenopus ectoderm.

In Xenopus, growth factors of the TGF-beta, FGF and Wnt oncogene families have been proposed to play a role in generating embryonic pattern. In this paper we examine potential interactions between the bFGF and Xwnt-8 signaling pathways in the induction and dorsal-ventral patterning of mesoderm. Injection of Xwnt-8 mRNA into 2-cell Xenopus embryos does not induce mesoderm formation in animal cap ectoderm isolated from these embryos at the blastula stage, but alters the response of this tissue to mesoderm induction by bFGF. While animal cap explants isolated from non-injected embryos differentiate to form ventral types of mesoderm and muscle in response to bFGF, explants from Xwnt-8 injected embryos form dorsal mesodermal and neural tissues in response to the same concentration of bFGF, even if the ectoderm is isolated from the prospective ventral sides of embryos or from UV-ventralized animals. Our results support a model whereby dorso-ventral mesodermal patterning can be attained by a single mesoderm inducing agent, possibly bFGF, which is uniformly distributed across the prospective dorsal-ventral axis, and which acts in concert with a dorsally localized signal, possibly a Wnt protein, which either alters the response of ectoderm to induction or modifies the character of mesoderm after its induction.     

Embryology of Chaoborus-induced spines in Daphnia pulex

Daphnia pulex (Crustacea: Cladocera) embryos were found to be sensitive to a chemical cue (kairomone) in an extract of the predator Chaoborus americanus (Insecta:Diptera). Sensitivity of embryos to the kairomone remains throughout embryonic development. Apparently declining sensitivity as development proceeds may be due to the amount of time the embryos are exposed to the kairomone. Male embryos were also found to be sensitive to the kairomone. The smallest eggs within a brood produced small offspring, which showed the antipredator morphology to a significantly lower degree than largest eggs. The production of the neckteeth is described, at the developmental stage in the maturation of the Daphnia coinciding approximately with the escape of the embryos from the brood chamber.     

Ovoviviparity and the structure of the brood pouch in Melanoides tuberculata (Gastropoda: Prosobranchia: Thiaridae)

The freshwater gastropod Melanoides tuberculata broods its young in a pouch located in the anterodorsal region of the head-foot. The wall of the brood pouch is composed of smooth muscle surrounded by connective tissue. The lumen of the brood pouch is incompletely partitioned by trabeculae, formed by extensions or folds in the chamber wall that are composed of smooth muscle, connective tissue, nonciliated squamous epithelial cells, and some storage cells containing lipid and glycogen. The lumen of the chamber also contains a few cells with storage products. The general absence of secretory cells suggests that embryos derive little nutrition from the mother, and therefore embryonic development is probably ovoviviparous. Embryos in various stages of development were found within brood pouches, with later stage embryos varying in size. There was a negative relationship between embryo size and number of embryos in the brood pouch.     

Two-step induction of primitive erythrocytes in Xenopus laevis embryos: signals from the vegetal endoderm and the overlying ectoderm

Primitive blood cells differentiate from the ventral mesoderm blood islands in Xenopus embryos. In order to determine the tissue interactions that propagate blood formation in early embryogenesis, we used embryos that had the ventral cytoplasm removed. These embryos gastrulated normally, formed a mesodermal layer and lacked axial structures, but displayed a marked enhancement of alpha-globin expression. Early ventral markers, such as msx-1, vent-1 and vent-2 were highly expressed at the gastrula stage, while a dorsal marker, goosecoid, was diminished. Several lines of experimental evidence demonstrate the critical role of animal pole-derived ectoderm in blood cell formation: 1) Mesoderm derived from dorsal blastomeres injected with beta-galactosidase mRNA (as a lineage tracer) expressed alpha-globin when interfaced with an animal pole-derived ectodermal layer; 2) Embryos in which the animal pole tissue had been removed by dissection at the blastula stage failed to express alpha-globin; 3) Exogastrulated embryos that lacked an interaction between the mesodermal and ectodermal layers failed to form blood cells, while muscle cells were observed in these embryos. Using dominant-negative forms of the BMP-4 and ALK-4 receptors, we showed that activin and BMP-4 signaling is necessary for blood cell differentiation in ventral marginal zone explants, while FGF signaling is not essential. In ventralized embryos, inactivation of the BMP-4 signal within a localized area of the ectoderm led to suppression of globin expression in the adjacent mesoderm layer, but inactivation of the activin signal did not have this effect. These observations suggest that mesodermal cells, derived from a default pathway that is induced by the activin signal, need an additional BMP-4-dependent factor from the overlying ectoderm for further differentiation into a blood cell lineage.