Development of Iiyanassa obsoleta embryos after equal distribution of polar lobe material at first cleavage

Ilyanassa obsoleta embryos cleave unequally via the formation of polar lobes, which contain materials essential for the development of larval shell, foot, operculum, statocysts, and eyes. Polar lobe material is shunted to the CD cell during first cleavage and to the D cell during second cleavage. Treatment with cytochalasin B (CB) before first cleavage prevents the formation of the polar lobe and leads to equal cleavage and the equal distribution of lobe material. At second cleavage each cell forms a polar lobe, resulting in a four-cell stage with two large (D) cells and two smaller (C) cells. Embryos equalized with CB frequently display duplications, 68% duplicating two or more larval structures. Embryos with adjacent D cells (CCDD) duplicate statocysts more frequently than embryos with opposite D cells (CDCD), perhaps due to enhanced inductive interactions. When equal cells are separated after first cleavage, resulting larvae develop like CD halves from control embryos. When equal halves are analyzed as pairs and compared with whole, equalized embryos, they duplicate shell, foot, and operculum more frequently. This difference is probably due to masking of duplications of these structures in whole, equalized embryos rather than to general inhibitory interactions between the two D quadrants. These results are discussed with respect to proposals that interactions between D quadrants in equalized embryos may alter developmental capabilities.     

Significance of the polar lobe for the determination of dorsoventral polarity in Dentalium vulgare (da Costa)

The development of dorsoventral polarity in Dentalium dentale has been analyzed after inhibiting first polar lobe formation with cytochalasin B and bisecting the egg into two equal parts at an early trefoil stage. Cleavage pattern and morphogenesis have been studied in both in vivo and permanent cytological preparations. After bisecting the egg, each blastomere may fuse with its adhering polar lobe half and subsequently behave as a CD blastomere. The polar lobe substance may induce both halves to develop an apical tuft and probably also a posttrochal region. Cytochalasin B embryos which pass through an equal first cleavage form a four-cell stage in which the two D blastomeres are situated opposite or adjacent to each other (CDCD or CCDD embryos, respectively). During further development the larvae show a duplication of lobe-dependent structures. It is concluded that dorsoventral polarity originates epigenetically by fusion of the polar lobe with one of the first two blastomeres and is not preformed in the uncleaved egg.     

Organogenesis in normal and lobeless embryos of the marine prosobranch gastropod Ilyanassa obsoleta

The histogenetic and organogenetic capabilities of normal and lobeless embryos of Ilyanassa obsoleta are analyzed. Larvae developed from eggs in which the polar lobe was removed at the trefoil stage of first cleavage were studied in wholemount and section. None showed any evidence of eyes, statocysts, operculum, external shell, heart, or intestine. Sixty per cent of the wholemount larvae observed under polarized light exhibited internal masses of birefringent material resembling shell material. Of eight- to nine-day lobeless larvae studied as stained, sectioned material 100% possessed muscle tissue; 97.5% possessed velar tissue, digestive gland tissue, and style sac tissue; 52.5% possessed two or more style sac areas; 71% exhibited esophagus; 63% possessed stomach tissue; 37% exhibited stomodeal-like invaginations of the ectoderm; 31% exhibited a lightly staining ciliated area at the tip of the prevelar ectoderm; and 21% possessed glandular ectoderm resembling pedal or mantle gland tissue. The evidence presented suggests that the material of the polar lobe has an important role not only in cellular differentiation but also in the organization of tissue types into organs or organ primordia.     

Cell specification and the role of the polar lobe in the gastropod mollusc Crepidula fornicata

A small polar lobe forms at the first and second cleavage divisions in the gastropod mollusc Crepidula fornicata. These lobes normally fuse with the blastomeres that give rise to the D quadrant at the two- and four-cell stages (cells ultimately generating the 4d mesentoblast and D quadrant organizer). Significantly, removal of the small polar lobe had no noticeable effect on subsequent development of the veliger larva. The behavior of the polar lobe and characteristic early cell shape changes involving protrusion of the 3D macromere at the 24-cell suggest that the D quadrant is specified prior to the sixth cleavage division. On the other hand, blastomere deletion experiments indicate that the D quadrant is not determined until the time of formation of the 4d blastomere (mesentoblast). In fact, embryos can undergo regulation to form normal-appearing larvae if the prospective D blastomere or 3D macromere is removed. Removal of the 4d mesentoblast leads to highly disorganized, radial development. Removal of the first quartet micromeres at the 8-cell stage also leads to the development of radialized larvae. These findings indicate that the embryos of C. fornicata follow the mode of development exhibited by equal-cleaving spiralians, which involves conditional specification of the D quadrant organizer via inductive interactions, presumably from the first quartet micromeres.     

Role of the Vegetal Body in the Regulation of Development in Bithynia tentaculata (Prosobranchia, Gastropoda)

In the first polar lobe of the egg of Bithyma tentaculata thereis a cup-shaped mass of small vesicles rich in RNA, called thevegetal body. It is passed to the CD blastomere with the polarlobe and then its contents are distributed to C and D. Whenthe polar lobe is removed, embryos form neither mesentoblastnor mesoderm bands. They fail to establish bilateral symmetryor to form eyes, foot, operculum and shell. AB half embryosdevelop only the structures found in lobeless embryos, whileCD halves develop lobe dependent adult structures such as eyes,foot and shell. AD- and BC-2/4-embryos develop similarly andboth have lobe dependent structures. Three quarter embryos developas in other molluscs except that ABD (–C) develops poorlyand ABC (–D) develops all lobe-dependent structures. Thus,all evidence available indicates that C receives cytoplasm usuallyrestricted to D in molluscs and therefore has the capacity toexert a similar morphogenetic influence on development.     

The role of unequal cleavage and the polar lobe in the segregation of developmental potential during first cleavage in the embryo of Chætopterus variopedatus

Summary The inequality of the first cleavage division of the Chætopterus embryo is caused by the production of a small polar lobe and the internal shifting of the first cleavage spindle. This division produces a two-celled embryo containing a small AB and a large CD blastomere. These blastomeres have different morphogenetic potentials. Only the larvae resulting from isolated CD blastomeres are able to form bioluminescent photocytes, eyes and lateral hooked bristles. The removal of the polar lobe during first cleavage does not have a great effect on development. These lobeless embryos display a normal pattern of cleavages through the time of mesentoblast formation. The resulting larvae are essentially normal, however they do not form functional photocytes. If the CD cell is isolated after the removal of the first polar lobe, the resulting larva is virtually identical to those formed by the intact CD cell except it lacks the photocyte cells. These results indicate that two separate pathways are involved in the segregation of developmental or morphogenetic potential which takes place during first cleavage. One set of factors, which are necessary for photocyte formation, are associated with the first polar lobe. Other factors that are necessary for the formation of the eyes and lateral hooked bristles are segregated by the unequal cleavage which results from an internal shifting of the cleavage spindle. The removal of a large portion of the vegetal region of the embryo during first cleavage leads to the production of larvae which display a decreased ability to form eyes and lateral hooked bristles. These embryos frequently display an abnormal pattern of cleavages. They do not form the primary somatoblast or the mesentoblast. These results indicate that the vegetal region of the CD cell of Chætopterus is analogous to polar lobes which have been studied in other species, and is therefore important in the specification of the D quadrant. These features of the first cleavage of Chætopterus are a combination of those displayed by forms with direct unequal cleavage and other forms which cleave unequally through the production of large polar lobes. The significance of these findings is discussed relative to the origins of these different types of unequal cleavage.     

The second polar lobe of theSabellaria cementarium embryo plays an inhibitory role in apical tuft formation

Summary The embryo ofSabellaria cementarium (Polychaeta) forms a polar lobe at each of the first two cleavage divisions which becomes absorbed into one of the blastomeres at the end of the division. Lobe removal experiments show that the polar lobe preceding first cleavage is necessary for the development of the apical tuft and the posttrochal region of the trochophore larva. The polar lobe preceding second cleavage is smaller than the first polar lobe and is necessary only for post-trochal region development. In blastomere isolation experiments, isolates containing the C but not the D blastomere form apical tufts. Isolates containing the D but not the C blastomere do not form apical tufts. When the polar lobe preceding second cleavage is removed and the C and D blastomeres are separated and raised in isolation, each can form an apical tuft. When the second cleavage is equalized with sodium dodecyl sulfate (SDS) such that both the C and the D blastomeres receive second polar lobe material, no apical tuft is formed. These results suggest that apical tuft determinants are distributed to both the C and D blastomeres at second cleavage but that the second polar lobe contains an inhibitor for apical tuft formation which is shunted to the D blastomere after the completion of second cleavage.     

Effect of hexylene glycol-altered microtubule distributions on cytokinesis and polar lobe formation in fertilized eggs of Ilyanassa obsoleta

Some effects of gravity on early morphogenesis are correlated with microtubule locations within cells. During first cleavage in Ilyanassa obsoleta embryos, a transitory polar lobe constriction forms and then relaxes, allowing the polar lobe to merge with one daughter cell. If the polar lobe is equally divided or removed, morphogenesis is severely disrupted. To examine microtuble locations during early Ilyanassa development, eggs were fixed and stained for polymerized alpha-tubulin during first cleavage. The mitotic apparatus assembles at the animal pole. The cleavage furrow forms between the asters, constricting to a stabilized intercellular bridge encircling midbody-bound microtubules, whereas the polar lobe constriction forms below and parallel to the spindle, constricting to a transitory intercellular bridge encircling no detectable microtubules. At metaphase an alpha-tubulin epitope is distributed throughout the spindle, whereas a beta-tubulin epitope is present predominantly in the asters. Incubation in hexylene glycol, a drug that increases microtubule polymerization, during mitosis causes the polar lobe constriction to tighten around polymerized alpha-tubulin and remain stably constricted. If hexylene glycol is removed, alpha-tubulin staining disappears from the polar lobe constriction, which relaxes, whereas microtubules remain in the cleavage furrow, which remains constricted. These observations suggest that asymmetric distribution of microtubules affects early Ilyanassa cleavage patterns, and that continued presence of microtubules extending through an intercellular bridge is important for stabilization of the bridge constriction prior to completion of cytokinesis. These data provide the basis for further analysis of the role of microtubules in possible microgravity disruptions of Ilyanassa development.     

Post-transcriptional regulation of protein synthesis in Ilyanassa embryos and isolated polar lobes

The experimental removal of the polar lobe, an anucleate cytoplasmic protrusion formed in preparation for the first cleavage, from the egg of Ilyanassa obsoleta results in grossly abnormal embryonic development. In experiments reported here normal and delobed embryos, as well as isolated polar lobes, were incubated with [³⁵S]methionine for 4 hr beginning at the completion of the first cleavage or 21 hr later during epiboly. Proteins were extracted and examined by fluorography after resolution by two-dimensional polyacrylamide gel electrophoresis. In normal embryos the synthesis of several proteins begins or ends between the two stages investigated. In isolated polar lobes a subset of these developmental changes in protein synthesis occurs, indicating that the regulation of these events is independent of concomitant nuclear activity and probably involves selective regulation of the translation of mRNA stored in the eggs. The patterns of protein synthesis in normal embryos and delobed embryos are qualitatively extremely similar, though quantitative differences are also observed. No proteins can be detected which are synthesized exclusively in polar lobes.     

Regional specification during embryogenesis in the craniiform brachiopod Crania anomala

A fate map has been constructed for the embryo of Crania. The animal half of the egg forms the ectodermal epithelium of the larva's apical lobe. The vegetal half of the egg forms endoderm, mesoderm, and the ectoderm of the mantle lobe. The vegetal pole is the site of gastrulation; this site becomes the posterior ventral region of the mantle lobe of the larva. The plane of the first cleavage goes through the animal-vegetal axis of the egg; it bears no relationship to the future plane of bilateral symmetry of the larva. The timing of regional specification was examined by isolating animal, vegetal, or meridional halves from oocytes, eggs, or embryos from prior to germinal vesicle breakdown through gastrulation. Animal halves isolated from oocytes formed either the epithelium of the apical lobe or a larva with all three germ layers. Animal halves isolated from unfertilized eggs and eight-cell embryos formed only apical lobe epithelium. Beginning at the blastula stage, animal halves formed mantle in addition to apical lobe epithelium. In animal halves isolated after gastrulation, the mantle lobe was always truncated. Vegetal halves isolated at all stages prior to gastrulation gastrulated and formed apical and mantle lobes with endoderm and mesoderm; however, the relative size of the apical lobe that formed decreased substantially when vegetal halves were isolated at later developmental stages. When meridional halves were isolated from unfertilized eggs and two- to four-cell embryos, both halves frequently formed normally proportioned larvae. Beginning at the blastula stage, a number of pairs frequently had a member that lacked dorsal setae on its mantle lobe while the other member of the pair formed setae, indicating that the dorsoventral axis had been set up. The process of regional specification in Crania is compared to those of Discinisca and Glottidia in the brachiopod subphylum Linguliformea and Phoronis in the phylum Phoronida.     

Evolutionary change in the process of dorsoventral axis determination in the direct developing sea urchin, Heliocidaris erythrogramma

Embryos of the indirect developing sea urchin, Heliocidaris tuberculata, and of Heliocidaris erythrogramma which develops directly without the formation of a pluteus larva, were bisected at the two- and four-cell stages. Paired half-embryos resulting from the bisection of H. tuberculata embryos along either the first or the second cleavage plane develop identically into miniature prism stage larvae. As in other indirect developing sea urchins, no differential segregation of developmental potential takes place as a result of the first and second cleavage divisions. Although half-embryos resulting from bisection along the second cleavage plane differentiate all cell types and develop equivalently in H. erythrogramma, the isolated first cleavage blastomeres do not. One of these two cells always forms significantly more mesodermal and endodermal cells. These patterns of differentiation are consistent with fate-mapping studies indicating that most mesodermal and endodermal cells are derived from the prospective ventral blastomere. Therefore, a differential segregation of developmental potential takes place at the first cleavage division in H. erythrogramma. When embryos of H. erythrogramma were bisected during the eight-cell stage, isolated tiers of animal blastomeres typically formed only ectodermal structures including the vestibule, whereas vegetal embryo halves formed all differentiated cell types. We propose that animal-vegetal cell determination and differentiation takes place along an axis which has been shifted relative to the pattern of cell cleavages in the embryos of H. erythrogramma. Vegetal morphogenetic potential for the formation of mesodermal and endodermal structures has become more closely associated with the prospective ventral side of the embryo during the evolution of direct development in Heliocidaris.     

Determinative development in the polyclad turbellarian,Hoploplana inquilina

Summary

Blastomere deletion experiments at the two- and four-cell stages were carried out on the embryo of the polyclad turbellarian Hoploplana inquilina to further examine the relationship between spiral cleavage and early embryonic determination in primitive spiralians. Deletion of one cell at the two-cell stage resulted in “half” larvae that were abnormal in body shape, lobe development, and behavior. Deletion of one cell at the four-cell stage produced less abnormal “three-quarter” larvae which were still underdeveloped in one of the quadrants. A 3:1 ratio of one-eyed to two-eyed larvae implies that deletion of any one of three blastomeres results in loss of an eye, with two constituting the eye lineage and the third controlling the development of two eyes. The results demonstrate that the polyclad embryo is determined early in development, though significant cell interactions occur during cleavage, and suggest that determinative development and quartet spiral cleavage are always associated and probably represent a primitive, strongly conserved evolutionary condition.     

Cell contact-dependent positioning of the D cleavage plane restricts eye development in the Ilyanassa embryo

In embryos of the gastropod Ilyanassa obsoleta, the first-quartet micromeres of the A, B and C lineages (1a, 1b, and 1c) are each competent to form an eye in response to signaling from the 3D cell. The first-quartet micromere of the dorsal D lineage (1d) is smaller than the others, divides at a slower rate, and lacks the ability to form an eye. These properties of 1d all depend on inheritance of vegetal polar lobe cytoplasm by its mother cell D at second cleavage. I show that they depend also on the presence of cells adjacent to D during the late four-cell stage: after ablation of the A and/or C cells before this stage, 1d inherits more cytoplasm than normal, divides more rapidly, and frequently forms an eye. In non-D lineages, cleavage plane positioning and micromere division rates are relatively insensitive to cell contacts. Compressing whole embryos during third cleavage also leads to an increase in 1d volume correlated with abnormal eye formation; this suggests that the normal effect of cell contacts is to position the D cell cleavage furrow closer to the animal pole, and the enhanced division asymmetry of the D cell contributes to the suppression of eye development.     

Development of in vitro fertilized embryos of the polyclad flatworm,Hoploplana inquilina,following blastomere separation and deletion

Summary After in vitro fertilization of naked eggs of the polyclad turbellarian, Hoploplana inquilina, both cell separation experiments and deletions of specific blastomeres are possible. With these techniques one can analyze the developmental potential of isolated blastomeres and determine if the embryonic axes have been established at the four-cell stage in this primitive, equally-cleaving spiralian embryo. Two-cell separation experiments with development of both halves resulted in pairs of larvae 1) neither of which had an eye (29%), 2) both of which had one eye (19%), and 3) one of which was eyeless and the other was one-eyed (43%). Deletion of one blastomere at the four-cell stage resulted in 68% one-eyed, 28% two-eyed and 3% eyeless larvae. The one-eyed larvae were asymmetric with respect to eye position with more having right than left eyes. Abnormal or missing ventrolateral lobes occurred with deletion of any of the macromeres at four cells but were significantly more common when A or C rather than B or D was deleted. The experiments support the hypothesis that eye development is a consequence of cytoplasmic localization of both a specific eye precursor and an inducer which segregate independently of cleavage planes, and indicate that the embryonic axes have been determined at the four-cell stage.     

Reproduction and development in a vermetid gastropod, Vermetus triquetrus

Abstract. We report on a study of reproduction and development in the Mediterranean vermetid gastropod Vermetus triquetrus from the SE coast of Spain. It is a gonochoristic species. The egg capsules are attached to the inside of the shell, and females brood up to 22 capsules simultaneously (more often 4–10). The capsules hold 10–61 eggs or embryos; the uncleaved eggs are yolk-rich, with a mean diameter of 377.3 μm. A distinct polar lobe occurs during the first cleavage, and blastomere D has discernible qualities after the 4-cell stage. The formation of the mesentoblast 4d occurs at the transition from the 24-cell stage to the 25-cell stage. Gastrulation begins after the 36-cell stage. Internal yolk is the major source of nutrition for the encapsulated embryos, but some nurse eggs (∼ 12%) and some sibling larvae are also ingested by the developing embryos. Hatching occurs during the swimming/crawling pediveliger stage, and metamorphosis is completed outside the capsules soon after hatching. Hence, larval development in Vermetus triquetrus is lecithotrophic intracapsular, with a short free-swimming/crawling phase.     

Muscle lineage cytoplasm can change the developmental expression in epidermal lineage cells of ascidian embryos

Cytoplasm from muscle lineage blastomeres of an ascidian embryo can cause cells of a nonmuscle lineage to produce larval tail muscle acetylcholinesterase. Muscle cytoplasm was partitioned microsurgically into epidermal lineage blastomeres at the eight-cell stage. Posterior half-embryos (the two B3 cells) of Ascidia nigra were obtained first by separating the anterior and posterior blastomere pairs at the four-cell stage. At third cleavage, the two B3 cells divide into an ectodermal cell pair that gives rise solely to epidermal tissues, and a mesodermal-endodermal blastomere pair from which the tail muscle cells are derived. When the ectodermal and mesendodermal blastomere pairs were isolated from one another by microsurgery and reared as partial embryos, only cells originating from the mesendodermal blastomeres produced a histochemical acetylcholinesterase reaction. Immediately after cleavage of the isolated B3 cells into ectodermal and mesendodermal cell pairs, the cleavage furrows could be made to disappear by pressing firmly on the mesendodermal cells with a microneedle. Repeated up and down pressure with the microneedle at a new position across the mesendodermal cells caused furrows to reestablish in the new position, thereby incorporating mesodermal cytoplasm and increasing the size of the ectodermal cells. The cytoplasmically altered ectodermal blastomere pairs, which became detached from the mesendodermal cells by this microsurgical procedure, continued to divide and were reared to “larval” stages. One-third of these epidermal partial larvae produced patches of cells containing acetylcholinesterase. These results lend further support to the theory that choice of particular differentiation pathways (embryonic determination) in ascidian embryos is mediated by segregation of specific egg cytoplasmic determinants.     

Time-lapse analysis and normal stages of development of cleavage and blastocyst formation in the marsupials the brown antechinus and the stripe-faced dunnart

The first six normal stages of cleavage and blastocyst formation in vitro of the brown antechinus and the stripe-faced dunnart are described based on 429 embryos from 37 dunnarts and 143 embryos from 32 antechinus and an information from previous studies. Embryos were cultured in Dulbecco's modified Eagle's medium with high glucose and 10% foetal calf serum at 35 degrees C and 5% CO2 in air. Seven antechinus and 18 dunnart embryos were cultured in Rose chambers and filmed by time-lapse cinematography, with an exposure of 0.2 sec and at a rate of 1 frame/30 sec. A rim of zona was formed around the yolk mass during early cleavage. Blastomeres attached to this rim, ensuring that the zona in the hemisphere containing the yolk mass was the first to be lined by blastomeres during blastocyst formation, which is completed at about the 32-cell stage. Two foci were established during cleavage. The descendants of the first cell to divide at the four-cell stage included, at both the fourth and fifth division, the first cell to divide and the cell with the shortest cell cycle time and had the shortest average cell cycle time at each division. The descendants of the cell opposite the first cell to divide at the four-cell stage included, at both the fourth and fifth divisions, the last cell to divide and the cell with the longest cell cycle time and had the longest average cell cycle time at each division.     

Prospective fate of early blastomeres in Hydractinia echinata (Cnidaria,Hydrozoa)

Summary Blastomeres of two-cell, four-cell, and eight-cell embryos of Hydractinia echinata were injected with horseradish-peroxidase (HRP) or fluorescein isothiocyanate (FITC)-dextran. The fate of the descendants of the injected blastomeres was followed until the planula larva had developed. The results obtained after HRP or FITC-dextran injection were essentially the same. Blastomeres are equivalent up to the four-cell stage, i.e. half-blastomeres produce half of the ectoderm of the planula larva and quarter-blastomeres give rise to one quarter of the larval ectoderm. During normal embryogenesis, the larval anterior-posterior axis corresponds to the animal-vegetal axis of the zygote. Thus, the labelled areas of larvae consisting of the progeny of injected half or quarter blastomeres normally stretch along the larval anterior-posterior axis. Normally, material giving rise to anterior or posterior larval parts, respectively, is separated at the third cleavage. Irrespective of the type of experiment, the progeny of injected blastomeres always contributed to endoderm formation, i.e. in larvae resulting from injected embryos the endoderm was more or less uniformly labelled. Application of vital stains locally to the exterior of zygotes and following these markers through first and second cleavage, produced evidence that in the vast majority of cases, the second cleavage is meridional. Offprint requests to: A. Schlawny     

Development ofDentalium following removal of D-quadrant blastomeres at successive cleavage stages

Summary Each primary micromere and macromere of the D-quadrant ofDentalium was deleted, through the mesentoblast stage, to investigate the way in which the polar lobe cytoplasm exerts its influence on development.-D and -1D embryos form an apical tuft but no posttrochal structures.-2D embryos form an apical tuft and a reduced posttrochal region without a shell. -3D and -4D are externally similar to control embryos. -1d embryos and -1c embryos have an apical tuft with a reduced number of cilia. Embryos in which both 1c and 1d are deleted lack the apical tuft.-2d embryos lack shell and most other posstrochal structures. -3d and-4d embryos appear externally equivalent to controls.The polar lobe cytoplasm exerts its influence sequentially, and as inIlyanassa the maximal effect is at the third quartet stage.