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.     

Electron microscopic study of larval eye development in Turbellaria Polycladida

The appearance and development of the embryonic and larval eyes of the polyclad turbellarian Stylochus mediterraneus were studied. In the embryo, the left epidermal eye appears first. Subsequently, the right epidermal eye appears, and within hours it sinks into the parenchyma and turns into a cerebral eye. Newly hatched Götte's larvae possess both the left epidermal and the right cerebral eye. Three days after hatching, an incomplete eye appears adjacent to the left epidermal eye. The left cerebral eye then originates from this incomplete eye as it sinks into the parenchyma. This third eye is believed to originate through a process of induction.     

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.     

Mating behavior,spawning, parental care,and embryonic development of some marine pseudocerotid flatworms (Platyhelminthes: Rhabditophora: Polycladida) in Singapore

The reproductive biology of tropical marine cotylean polyclads is presently poorly known. Reproductive aspects of 16 polyclad species from the family Pseudocerotidae in the genera Acanthozoon, Nymphozoon, Phrikoceros, Pseudobiceros, Pseudoceros, Thysanozoon, and Tytthosoceros from Singapore were documented for the first time. Penis fencing was observed to be just a mating ritual and not necessary for insemination, not always aggressive, and could also result in eventual reciprocal insemination. Results showed that all species underwent similar embryonic developmental stages and hatched as Müller's larvae. Only Pseudoceros concinnus and Pseudoceros laingensis, with mean developmental times of >20 days and mean brood sizes of <1,000 eggs, displayed long‐term parental care. Polyclads producing larger brood sizes had shorter developmental times and only covered their egg masses for about one day. Phrikoceros baibaiye and Pseudobiceros spp. produced egg capsules with pointed opercula, whereas all other species possessed smooth, rounded opercula. All genera hatched with brownish‐orange larvae, except for Pseudoceros spp., which hatched with reddish‐purple larvae regardless of the initial embryo color (either grayish‐yellow or reddish‐purple). These could potentially complement current taxonomic characters in distinguishing polyclad genera and species.     

Myogenesis in two polyclad platyhelminths with indirect development,Pseudoceros canadensis and Stylostomum sanjuania

SUMMARY Myogenesis of two representatives of Platyhelminthes, Stylostomum sanjuania and Pseudoceros canadensis, was followed from egg deposition until well‐differentiated free‐swimming larval stages, using F‐actin staining and confocal laserscanning microscopy. Zonulae adhaerentes are the only structures to stain before 50% of development between egg deposition and hatching in S. sanjuania, and before 67% of development in P. canadenis. Subsequently, irregular fibers appear in the embryo, followed by a helicoid muscle close to the apical pole. Three longitudinal muscle pairs form, of which the dorsal pair remains more pronounced than the others. Gradually, new muscles form by branching or from double‐stranded muscle zones adjacent to existing muscles. This results in an elaborate muscular bodywall that consists of a single helicoid muscle as well as multiple circular and longitudinal muscles. Diverse retractor muscles insert at the sphincter muscles around the stomodeum. The overall arrangement and formation mode of the larval musculature appears very similar in both species, although only P. canadensis has a primary circular muscle posterior to the helicoid muscle. Muscle formation in the apical region of the embryo precedes that at the abapical pole and the primary longitudinal muscles form slightly later than the primary circular muscles. Myogenesis and larval myoanatomy appears highly conserved among polyclad flatworms, but differs significantly from that of other trochozoan clades. Our data suggest that the larval muscular ground pattern of polyclad larvae comprises a bodywall consisting of a helicoid muscle, circular and longitudinal muscles, several retractor muscles, and sphincter muscles around the stomodeum.     

The embryonic development of the polyclad flatworm Imogine mcgrathi

In this paper we describe the embryonic development of the polyclad flatworm Imogine mcgrathi. Imogine is an indirect developer that hatches as a planctonic Goette’s larva after an embryonic period of approximately 7 days. Light and electron microscopic analyses of sections of staged embryos were combined with antibody stainings of wholemounted embryos to reconstruct the origin and movement of the primordia of the various organ systems, with particular emphasis on the nervous system. We introduce a system of morphologically defined stages aimed at facilitating future studies and cross-species comparisons among flatworm embryos. Imogine embryos undergo typical spiral cleavage. Micromere quartets 1–3 form an irregular double layer of mesenchymal cells that during gastrulation expands over micromere quartet 4. Micromere 4d divides into several large mesendodermal precursors whose position defines the ventral pole of the embryo. These cells, along with the animal micromeres that obtained a sub-surface position during cleavage, form a deep layer of cells that gives rise to all internal structures, including the nervous system, musculature, nephridia, and gut. Micromeres 4a–c are large yolky cells that are incorporated into the lumen of the gut, but do not themselves contribute to the gut epithelium. Shortly after gastrulation, cell differentiation sets in. Cells located at the surface adopt epithelial characteristics and form cilia that result in continuous movement of the post-gastrula stage embryo. Deep cells at the lateral margins of the embryo become organized into a protonephridial tube. A cluster of approximately 50 deep cells at the anterior pole forms the brain, in which we have identified sets of founder neurons of the brain commissure and the dorsal and ventral connectives. The early differentiating neurons, along with other cells forming stabilized microtubules (ciliated cells of the epidermis, gut and protonephridia; apical gland cells) could be analyzed in detail because of their labeling with an antibody against acetylated α-tubulin. Our findings indicate that, despite significant differences in the cleavage pattern and arrangement of blastomeres in the early embryo, morphogenesis and organ formation of a polyclad embryo follows a pattern that is very similar to the pattern observed by us and others in phylogenetically more evolved rhabdocoel flatworms. Received: 10 February 2000 / Accepted: 10 April 2000     

Removal of the polar lobe leads to the formation of functionally deficient photocytes in the annelidChaetopterus variopedatus

Summary The light emitting photocytes ofChaetopterus variopedatus larvae are bilaterally situated within the ectoderm of the post-trochal region. Their histological appearance is similar to that of the adult photocytes. The larval photocytes contain a large quantity of membranous secretory vesicles (photosomes), which probably contain the photoluminescent protein. The two-cellChaetopterus embryo contains a small AB and a large CD blastomere. Previous studies have shown that only the “larvae” resulting from isolated CD blastomeres are able to luminesce. Consistent with these findings, morphologically distinct photocytes are only found in the CD larvae. The removal of the small polar lobe that forms during first cleavage leads to the production of a “larva” that is unable to produce light. All delobed larvae contain morphologically distinct photocytes, which are identical to those in normal larvae except they appear to contain only a small quantity of photosomes. Experimental equalization of first cleavage leads to the production of a double embryo. While photocytes are found in both of the duplicated post-trochal regions, usually only one of these is capable of emitting luminescence. Apparently, the highly localized vagetal material (determinants) responsible for functional light emission is distributed to both halves in only a few cases when first cleavage is experimentally “equalized”. These results indicate that the determinative action of the polar lobe is not required for the formation of the photocytes themselves, but rather for their ability to function as emitters of light. The determinants in the polar lobe ofChaetopterus may control some aspect of the photoluminescence reaction itself, such as the production of the photoprotein.     

Large‐scale ciliary reversal mediates capture of individual algal prey by Müller's larva

We documented capture of microalgal prey by several species of wild‐caught Müller's larvae of polyclad flatworms. To our knowledge, this is the first direct observation of feeding mechanism in this classical larval type. High‐speed video recordings showed that virtually all captures were mediated by large‐scale transient ciliary reversal over one or more portions of the main ciliary band corresponding to individual lobes or tentacles. Local ciliary beat reversals altered near‐field flow to suck parcels of food‐containing water mouthward. Many capture episodes entailed sufficient coordinated flow disruption that these compact‐bodied larvae tumbled dramatically. Similar behaviors were recorded in at least four distinct species, one of which corresponds to the ascidian‐eating polyclad Pseudoceros canadensis.     

Uncovering the Post-embryonic Role of Embryo Essential Genes in Arabidopsis using the Controlled Induction of Visibly Marked Genetic Mosaics: EMB506, an Illustration

The embryo essential gene EMB506 plays a crucial role in the transition of the Arabidopsis embryo from radial symmetry to bilateral symmetry just prior to the early heart stage of development. In addition to influencing embryo development EMB506 also affects chloroplast biogenesis. To further investigate the role of EMB506 gene expression in Arabidopsis we have generated green fluorescent protein (GFP) marked emb506 mosaic sectors at temporally defined stages during embryogenesis and additionally during various stages of vegetative growth, in otherwise phenotypically wild-type plants. We confirm the essential requirement for EMB506 gene expression in chloroplast biogenesis as reflected by the decreased chlorophyll content in emb506 mosaic sectors. We also show that the influence of EMB506 gene expression as it impinges on chloroplast biogenesis is first relevant at an intermediate stage in embryogenesis and that the role of EMB506 gene expression in chloroplast biogenesis is distinct from the essential role of EMB506 gene expression during early embryo development. By inducing emb506 mosaicism after the essential requirement for EMB506 gene expression in embryogenesis and also during vegetative growth we reveal that EMB506 gene expression additionally is required for correct cotyledon-, true leaf- and cauline leaf margin development. The strategy that we describe can be tailored to the mosaic analysis of any cloned EMB gene for which a corresponding mutant exists and can be applied to the mosaic analysis of mutant lethal genes in general.     

Mosaic aneuploidy in early fetal losses

Chromosomal mosaicism is the presence of 2 or more cell lines with different karyotypes in the same individual. Mosaic karyotypes are a remarkable feature of early stages of human embryo development. They result from mitotic errors in chromosome segregation and demonstrate the clearest example of somatic mutagenesis in human beings. This review is devoted to the classification of chromosomal mosaicism and the analysis of its underlying mechanisms, incidence and phenotypic effects during embryo development. A model for tissue-specific aneuploid cell line compartmentalization in spontaneous abortions is introduced.     

Stylochus tauricus,a predator of the barnacle Balanus improvisus in the Black Sea

The flatworm Stylochus tauricus Jacubova has been found associated with the barnacle Balanus improvisus Darwin, on which it feeds. The predation rate (the number of barnacles eaten by one polyclad in a month) ranges between 5–10. Inside the empty shells of B. improvisus some egg-plates of S. tauricus were observed. Pelagic Götte's larvae aged 2–3 days possess 4 lobes while those aged 7–8 days have 5 lobes. Flatworms can prey on the young of another species Balanus eburneus Gould, whereas predation on the mussels Mytilus galloprovincialis Lam. is rare. There is a direct correlation between predator abundance and prey ingested.     

Pattern duplications in larvae of the polyembryonic wasp Copidosoma floridanum

 Copidosoma floridanum is a polyembryonic wasp that undergoes total cleavage of the egg followed by proliferation of blastomeres to produce up to 2,000 embryos from a single egg. This unusual mode of development raises several questions about how axial polarity is established in individual embryonic primordia. By examining embryonic development of larvae with duplicated structures (conjoined larvae), we determined that conjoined larvae form by mislocalization of two embryonic primordia to a common chamber of the extraembryonic membrane that surrounds individual embryos. Analysis of an anterior marker, Distalless, in mislocalized early embryos indicated that anterior structures form independently of one another. This suggests each embryonic primordium has some intrinsic polarity. However, during germband extension embryos usually fuse in register with each other, resulting in conjoined larvae with heads facing each other. Analysis of the posterior segmental marker, Engrailed, in conjoined embryos suggested that fusion in register initiates during germband extension. Thus, even though embryonic primordia initially have a random axial orientation, conjoined larvae usually possess a common orientation due to reorientation during germband extension. These observations suggest that differential cellular affinities during segmentation play an important role in embryo fusion. Received: 13 June 1996 / Accepted: 15 August 1996     

Influence of homoeotic genes on the aldehyde oxidase pattern in imaginal discs of Drosophila melanogaster

In a study of the regulation of enzyme patterns in imaginal discs the aldehyde oxidase pattern was determined for some homoeotic mutations of D. melanogaster. Earlier indications that suggested that this pattern follows the determinitive state of compartments within imaginal discs were confirmed by the aldehyde oxidase (AO) pattern of both the wing and haltere discs from en¹; bx³, en¹; pbx, and en¹; bx³ pbx larvae and the antennal discs from Antp^73b and ss^a larvae. We additionally analyzed whether AO activity depended on the determinative state of an entire compartment or was expressed autonomously in clones. Homozygous engrailed clones were induced by mitotic recombination. From the AO clones found in normally negative areas of the posterior compartment it was concluded that enzyme activity depended upon the determinative state of the cells and was not a function of the compartment as a whole. The results are described with reference to a scheme in which compartmental and subcompartmental selector genes are thought to determine a binary code on which AO patterns depend.     

Adult differentiation from partialDrosophila embryos after egg ligation during stages of nuclear multiplication and cellular blastoderm

When embryos are ligated during different stages of nuclear multiplication and cellular blastoderm they develop into partial larvae which never hatch. The partial larvae were injected into adult females for further development. During this in vivo culture the imaginal disk cells divide and achieve competence to differentiate into adult structures. We find that independent of the fragment size anterior embryonic tissues give rise to cranial adult structures and posterior fragments to adult caudal structures. This indicates that during the first nuclear divisions cranial versus caudal development is already determined. The two complementary fragments do not add up to a total embryo when separated very early; however, if separation of the two parts occurs at cellular blastoderm stage all adult structures of the fly can be found.     

Embryonic development and newly hatched larvae of the little dragon sculpinBlepsias cirrhosus

This paper describes both embryonic development and newly hatched larval morphology of the little dragon sculpinBlepsias cirrhosus. The eggs ofB. cirrhosus are almost spherical, 3.0–3.2 mm in diameter, and have a yolk color of burnt orange. Development is very slow, being especially sluggish once the embryo appears. The embryo begins forming from the 10th day. In size, the early embryo is less than 1/6 of the yolk’s circumference. Incubation at 10°C takes about 200 days, 50 days shorter than the incubation period in a natural environment, with a mean water temperature of 11°C. The notochord length of newly-hatched larvae averages 11.1 mm. The larvae are developed so fully that the notochord is already flexing and the caudal and pectoral rays are forming.     

Mosaicism diminishes the value of pre-implantation embryo biopsies for detecting CRISPR/Cas9 induced mutations in sheep

The production of knock-out (KO) livestock models is both expensive and time consuming due to their long gestational interval and low number of offspring. One alternative to increase efficiency is performing a genetic screening to select pre-implantation embryos that have incorporated the desired mutation. Here we report the use of sheep embryo biopsies for detecting CRISPR/Cas9-induced mutations targeting the gene PDX1 prior to embryo transfer. PDX1 is a critical gene for pancreas development and the target gene required for the creation of pancreatogenesis-disabled sheep. We evaluated the viability of biopsied embryos in vitro and in vivo, and we determined the mutation efficiency using PCR combined with gel electrophoresis and digital droplet PCR (ddPCR). Next, we determined the presence of mosaicism in?~?50% of the recovered fetuses employing a clonal sequencing methodology. While the use of biopsies did not compromise embryo viability, the presence of mosaicism diminished the diagnostic value of the technique. If mosaicism could be overcome, pre-implantation embryo biopsies for mutation screening represents a powerful approach that will streamline the creation of KO animals.     

Visualization of skeletons and intervertebral disks in live fish larvae by fluorescent calcein staining and disk specific GFP expression

Zebrafish and medaka have become popular models for studying skeletal development because of high fecundity, shorter generation period, and transparency of fish embryo. The first step to study skeletal development is visualizing bone and cartilage. Live animal staining with fluorescent calcein have several advantages over the standard skeletal staining protocol by using alizarin red and alcian blue for bone and cartilage. However, there is no detailed study examining skeletal development of live marine fish larvae by calcein staining. Here we applied calcein staining to examine skeletal development in red sea bream larvae. In addition, green fluorescent protein (GFP) reporter zebrafish was employed to trace lineage analysis of intervertebral disk cells in live fish larvae. Calcein staining of red sea bream larvae successfully visualized development of craniofacial skeletons as well as urinary calculus. Histochemical detection of alkaline phosphatase (ALP) activity revealed that abnormal segmentation of notochord induced by RA during vertebral development in zebrafish. Immunohistochemistry clearly revealed that GFP‐positive cells in intervertebral space was nucleus polposus like cell in twhh‐GFP transgenic zebrafish. It was demonstrated usefulness of calcein and ALP staining and twhh‐GFP transgenic zebrafish for studying skeletal development in live fish larvae.     

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.     

Development of the embryo nest ofVespa affinis (Hymenoptera: Vespidae) in Southern Japan

Summary The aerial nests ofVespa affinis on Ishigaki Island, Japan, were found mainly within 1 m of the ground, attached to a twig roughly 4 mm in diameter. These are probably adaptations to survive adverse weather conditions. The favoured nesting habitat was in open farmland areas rather than in the natural forest. The entire envelope and more than half of the comb of the embryo nest is constructed during the first 10–12 days. The first workers emerge around 34 days after nest initiation. The duration of larval development was found to be linked to the number of larvae present in the nest, with the earlier progeny developing faster. The changes in the ovarian index of the mother queen during the early stages of nesting are described.