New perspectives on the angiosperm female gametophyte

This review builds upon previous classifications of angiosperm female gametophytes but offers two new perspectives. Firstly, the course of development is compared to an algorithm: a predetermined set of rules that produces a mature female gametophyte. This analogy allows hypotheses to be developed as to what changes in the “developmental program” are responsible for variant forms of development. Secondly, the review recognizes that the four meiotic products of a megaspore mother cell have different genetic constitutions and may have conflicting interests. In most cases, only one member of a megaspore tetrad gives rise to a functional egg. This megaspore is called the germinal spore. The other members of the tetrad are called somatic spores. Somatic spores do not give rise to functional eggs and, therefore, cannot leave direct genetic descendants. Non-monosporic embryo sacs are genetic chimeras containing derivatives of more than one megaspore nucleus. Conflict may arise within such embryo sacs between the derivatives of the germinal megaspore nucleus and the derivatives of somatic megaspore nuclei. “Antipodal eggs” and chalazal “strike” are interpreted as evidence of this conflict. The behavior of somatic spores and their derivatives is often variable for different embryo sacs produced by the same sporophyte. This has created difficulties for existing classifications of embryo sac “types” because more than one type is sometimes recognized within a species. A new classification of developmental algorithms is presented that emphasizes the fate of the germinal spore and its derivatives.     

Increased female fertility in aquaporin 8-deficient mice

Aquaporin-8 (AQP8) is a water channel expressed extensively in male and female reproductive systems. But its physiological functions are largely unknown. In the present study, we first found significantly increased number of offspring delivered by AQP8(-/-) mothers compared with wild-type mothers in cross-mating experiments. Comparison of ovulation in the two genotypes demonstrated that AQP8(-/-) ovaries released more oocytes (9.5 ± 1.9 vs. 7.1 ± 2.1 in normal ovulation and 37.8 ± 6.7 vs. 27.9 ± 5.7 in superovulation). Histological analysis showed increased number of corpus luteums in mature AQP8(-/-) ovaries, suggesting increased maturation and ovulation of follicles. By RT-PCR, western blot and immunohistochemistry analyses, we determined the expression of AQP8 in mouse ovarian granulosa cells. Granulosa cells isolated from AQP8(-/-) mice showed 45% of decreased membrane water permeability than wild-type mice. As the atresia of ovarian follicles is primarily due to apoptosis of granulosa cells, we analyzed the apoptosis of isolated granulosa cells from wild-type and AQP8(-/-) mice. The results indicated significantly lower apoptosis rate in AQP8(-/-) granulosa cells (21.3 ± 3.6% vs. 32.6 ± 4.3% in AQP8(+/+) granulosa cells). Taken together, we conclude that AQP8 deficiency increases the number of mature follicles by reducing the apoptosis of granulosa cells, thus increasing the fertility of female mice. This discovery may offer new insight of improving female fertility by reducing granulosa cell apoptosis through AQP8 inhibition.     

Trans-generational radiation-induced chromosomal instability in the female enhances the action of chemical mutagens

Genomic instability can be produced by ionising radiation, so-called radiation-induced genomic instability, and chemical mutagens. Radiation-induced genomic instability occurs in both germinal and somatic cells and also in the offspring of irradiated individuals, and it is characterised by genetic changes including chromosomal rearrangements. The majority of studies of trans-generational, radiation-induced genomic instability have been described in the male germ line, whereas the authors who have chosen the female as a model are scarce. The aim of this work is to find out the radiation-induced effects in the foetal offspring of X-ray-treated female rats and, at the same time, the possible impact of this radiation-induced genomic instability on the action of a chemical mutagen. In order to achieve both goals, the quantity and quality of chromosomal damage were analysed.

In order to detect trans-generational genomic instability, a total of 4806 metaphases from foetal tissues from the foetal offspring of X-irradiated female rats (5 Gy, acute dose) were analysed. The study's results showed that there is radiation-induced genomic instability: the number of aberrant metaphases and the breaks per total metaphases studied increased and were found to be statistically significant (p ≤ 0.05), with regard to the control group.

In order to identify how this trans-generational, radiation-induced chromosomal instability could influence the chromosomal behaviour of the offspring of irradiated rat females in front of a chemical agent (aphidicolin), a total of 2481 metaphases were studied. The observed results showed that there is an enhancement of the action of the chemical agent: chromosomal breaks per aberrant metaphases show significant differences (p ≤ 0.05) in the X-ray- and aphidicolin-treated group as regards the aphidicolin-treated group.

In conclusion, our findings indicate that there is trans-generational, radiation-induced chromosomal instability in the foetal cells from X-ray-treated female rats and that this RIGI enhances the chromosomal damage caused by the chemical agent aphidicolin.     

Involvement of the gonadal germinal epithelium during sex reversal and seasonal testicular cycling in the protogynous swamp eel,Synbranchus marmoratus Bloch 1795 (Teleostei,Synbranchidae)

The swamp eel, Synbranchus marmoratus, is a protogynous, diandric species. During sex reversal, the ovarian germinal epithelium, which forms follicles containing an oocyte and encompassing follicle cells during the female portion of the life cycle, produces numerous invaginations, or acini, into the ovarian stroma. Within the acini, the gonia that formerly produced oocytes become spermatogonia, enter meiosis, and produce sperm. The acini are bounded by the basement membrane of the germinal epithelium. Epithelial cells of the female germinal epithelium, which formerly became follicle (granulosa) cells, now become Sertoli cells in the developing testis. Subsequently, lobules and testicular ducts form. The swamp eel testis has a lobular germinal compartment in both primary and secondary males, although the germinal compartment in testes of secondary males resides within the former ovarian lamellae. The germinal compartment, supported by a basement membrane, is composed of Sertoli and germ cells that give rise to sperm. Histological and immunohistochemical techniques were used to describe the five reproductive classes that were observed to occur during the annual reproductive cycle: regressed, early maturation, mid-maturation, late maturation, and regression. These classes are differentiated by the presence of continuous or discontinuous germinal epithelia and by the types of germ cells present. Synbranchus marmoratus has a permanent germinal epithelium. Differences between the germinal compartment of the testes of primary and secondary males were not observed.     

Differentiation of mouse ectopic germinal cells in intra- and perigonadal locations

Four hundred and thirteen ectopic germinal cells in the testicular and extratesticular stroma and in the rete testis of mouse fetuses from day 13 of uterine development to term were studied together with 161 ectopic germinal cells in the rete ovarii and periovarian stroma of female fetuses at days 17 and 18 of intrauterine life. The morphology and the differentiation of these ectopic germinal cells were compared to those of germinal cells within seminiferous and ovigerous cords. While the ectopic germinal cells in the testis and in the rete testis followed patterns of differentiation identical with those in the seminiferous cords throughout the period included in the study, those in the extratesticular stroma behaved like entopic germinal cells only through day 17, since at days 18 and 19 many of them entered meiotic prophase just like XX germinal cells in the ovigerous cords. No differences were noted between ectopic and entopic ovarian germinal cells. The results of this study show that the factors responsible for the male differentiation of XY germinal cells are not limited to the seminiferous cords but operate throughout the testicular territory, and confirm that outside the testis, XY germinal cells differentiate as female; our study also corroborates the thesis that the differentiation of XX germinal cells is an autonomous and ubiquitous process.     

New insights on the mechanism of testis differentiation from the morphogenesis of experimentally induced testes in genetically female chick embryos

Embryonic testes grafted in the extraembryonic coelom of 3-day-old genetically female chick embryos may induce total and definitive reversal of gonadal sex differentiation. In this experimental condition, the left gonad becomes a testis instead of an ovary. This makes it possible to compare testicular and ovarian morphogenesis in animals having the same genetic sex and to discount what is due to differences in the genetic determination between male and female. The morphogenesis of such testes is marked by a disappearance of the cortical germinal epithelium. The medullary sex cords keep a narrow lumen instead of becoming large lacunae. The germ cells remain few in the sex cords and do not become meiotic. Furthermore, interstitial cell development is known to be very slow. As a consequence the gross size of the gonad is much smaller than that of an ovary. All these morphogenetic phenomena are unlike those observed during normal ovarian differentiation and evidence an inhibiting influence of the grafted testes. Since inhibition and masculinization are concomitant, inhibition appears to be the mechanism responsible for gonadal sex reversal. The extraembryonic situation of the grafted testes and their relation with the embryo only via the blood stream demonstrates the role of a secreted substance or substances still to be exactly identified. Previous data suggest that this could be the anti-Müllerian-hormone (AMH). Furthermore, previous and present results show that testis differentiation can be actively induced in a bird. This does not agree with the hypothesis that the gonads of the homogametic sex, i.e., the testes in birds, do not need any inducer in order to differentiate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conserved form and function of the germinal epithelium through 500 million years of vertebrate evolution

The germinal epithelium, i.e., the site of germ cell production in males and females, has maintained a constant form and function throughout 500 million years of vertebrate evolution. The distinguishing characteristic of germinal epithelia among all vertebrates, males, and females, is the presence of germ cells among somatic epithelial cells. The somatic epithelial cells, Sertoli cells in males or follicle (granulosa) cells in females, encompass and isolate germ cells. Morphology of all vertebrate germinal epithelia conforms to the standard definition of an epithelium: epithelial cells are interconnected, border a body surface or lumen, are avascular and are supported by a basement membrane. Variation in morphology of gonads, which develop from the germinal epithelium, is correlated with the evolution of reproductive modes. In hagfishes, lampreys, and elasmobranchs, the germinal epithelia of males produce spermatocysts. A major rearrangement of testis morphology diagnoses osteichthyans: the spermatocysts are arranged in tubules or lobules. In protogynous (female to male) sex reversal in teleost fishes, female germinal epithelial cells (prefollicle cells) and oogonia transform into the first male somatic cells (Sertoli cells) and spermatogonia in the developing testis lobules. This common origin of cell types from the germinal epithelium in fishes with protogynous sex reversal supports the homology of Sertoli cells and follicle cells. Spermatogenesis in amphibians develops within spermatocysts in testis lobules. In amniotes vertebrates, the testis is composed of seminiferous tubules wherein spermatogenesis occurs radially. Emerging research indicates that some mammals do not have lifetime determinate fecundity. The fact emerged that germinal epithelia occur in the gonads of all vertebrates examined herein of both sexes and has the same form and function across all vertebrate taxa. Continued study of the form and function of the germinal epithelium in vertebrates will increasingly clarify our understanding of vertebrate reproduction. J. Morphol. 277:1014–1044, 2016. © 2016 Wiley Periodicals, Inc.     

Modelling two possible mechanisms for the regulation of the germinal center dynamics

Research on the germinal center has tried to unravel the mechanisms that control its dynamics. In this study we focus on the termination of the germinal center reaction, which is still an open problem. We propose two hypothetical biological mechanisms that may be responsible for the control of germinal center dynamics and analyze them through mathematical models. The first one is based on the differentiation of follicular dendritic cells and/or T cells. Interaction of these cells in the differentiated state with germinal center B cells would promote B cell differentiation into memory B cells and Ab-forming cells, ending the germinal center reaction. The second mechanism applies only to a scenario without recycling and consists of the decay of a hypothetical proliferation signal for centroblasts that limits the number of cell divisions. Each of the models makes predictions that can be experimentally tested.     

Morphological and histochemical examination of male and female gonads in Homarus gammarus (L. 1758)

The reproductive organs of both male and female European lobsters (Homarus gammarus) are H-shaped gonads that lie dorsal to the gut on the large hepatopancreas. The ovary consists of a pair of tubular, parallel lobules with a connecting bridge. The germarium of the ovary containing oogonia is concentrated in the center of the ovarian lobe. As oogonesis proceeds, the oocytes move to the peripheral regions of the ovary. The follicle cells begin to surround the oocytes in the previtellogenic stage, and the mature oocytes are completely surrounded by the follicle cells. Carbohydrates exist in both early and late vitellogenic oocytes that give PAS positive reaction. However, their rising protein content in late vitellogenic oocytes makes them stain with Bromophenol blue. Testes show convoluted lobules with a germinal epithelium and a central collecting duct, and the paired vasa deferentia have three distinct parts. Spermatophores are nonpedunculate and tubular, which extrude as a continuous column and consist of a sperm mass covered with primary and secondary layers. The primary layer stains with Bromophenol Blue and gives a PAS positive reaction. But the secondary layer only weakly stains with Bromophenol Blue. The histochemical results may indicate that the function of the two layers is different.     

Morphofunctional changes of female germinal epithelium to support spermatozoa along the annual reproductive cycle in an inseminating catfish (Trachelyopterus galeatus,Auchenipteridae)

The reproductive system of some fish species presents elaborate mechanisms by which the females store spermatozoa inside their ovaries, keeping them viable for fertilization for an extended period of time. However, as intriguing as this sperm storage is, it is not yet understood how the sperm can remain viable in the ovary. Aiming to understand this phenomenon, the epithelium covering the ovarian lamellae, that is, the germinal epithelium, of the Cangati (Trachelyopterus galeatus), an inseminating catfish, was evaluated taking into account the different stages of the annual reproductive cycle. The germinal epithelium morphology changed during the annual reproductive cycle, presumably in preparation to receive the spermatozoa and keep them viable until fertilization. There was a progressive increase of the epithelium height. Also the number of intercellular junctions, desmosomes, and extended tight junctions, apparently increased forming chains that could be regarded as a barrier to isolate the sperm from the female immune system. Synthetic organelles were active releasing cytoplasmic granules and secretion in the epithelial enfolds in which the spermatozoa were deeply embedded. Concomitantly, oogonium nests were formed in the germinal epithelium during early folliculogenesis. J. Morphol. 275:65–75, 2014. © 2013 Wiley Periodicals, Inc.     

The multiple roles of mps1 in Drosophila female meiosis

The Drosophila gene ald encodes the fly ortholog of mps1, a conserved kinetochore-associated protein kinase required for the meiotic and mitotic spindle assembly checkpoints. Using live imaging, we demonstrate that oocytes lacking Ald/Mps1 (hereafter referred to as Ald) protein enter anaphase I immediately upon completing spindle formation, in a fashion that does not allow sufficient time for nonexchange homologs to complete their normal partitioning to opposite half spindles. This observation can explain the heightened sensitivity of nonexchange chromosomes to the meiotic effects of hypomorphic ald alleles. In one of the first studies of the female meiotic kinetochore, we show that Ald localizes to the outer edge of meiotic kinetochores after germinal vesicle breakdown, where it is often observed to be extended well away from the chromosomes. Ald also localizes to numerous filaments throughout the oocyte. These filaments, which are not observed in mitotic cells, also contain the outer kinetochore protein kinase Polo, but not the inner kinetochore proteins Incenp or Aurora-B. These filaments polymerize during early germinal vesicle breakdown, perhaps as a means of storing excess outer kinetochore kinases during early embryonic development.     

Ultrastructure of 3 beta HSDH (delta 5-3 beta hydroxysteroid dehydrogenase)-containing cells in ducks. III. Hybrid ducks (Anas platyrhynchos male X Cairina moschata female) during egg laying]

All the cells of the ovary of the hybrid duck (Peking duck X Barbary duck) which contain an active 3 beta HSDH enzyme show ultrastructural characteristics of steroid cells: a smooth, developed reticulum, tubularcrested mitochondria, a considerable "charge" in lipid inclusion. These cells are found not only in the internal theca of follicles as with the female gender, but also in the cortical zones void of germinal elements, a tendancy particular to the female.     

Development of the germinal epithelium and early folliculogenesis during ovarian morphogenesis in the cichlid fish Cichlasoma dimerus (Teleostei,Perciformes)

Formation of the germinal epithelium and folliculogenesis during ovarian development in Cichlasoma dimerus were described at the light‐ and electron‐microscopic levels. Prior to gonadal differentiation, germ cells and enveloping support cells reside within an inpocketing of the coelomic epithelium. Separation of the germinal and interstitial compartments of the gonad by a basement membrane is apparent from early gonadal development. Upon ovarian differentiation, oogonia undergo cyst‐forming divisions leading to the formation of clusters of interconnected cystocytes that synchronously enter meiosis, becoming oocytes. At the pachytene step, each oocyte becomes individualized by cytoplasmic extensions of prefollicle cells, thereby developing as an ovarian follicle. Subsequent somatic reorganization leads to the formation of the ovarian lumen in a cephalo‐caudal gradient. As a result, the germinal epithelium becomes internalized and lines the ovarian lumen. As defined by its origin from the germinal epithelium, the ovarian follicle is composed of an oocyte and the surrounding follicle cells. Thecal cells derived from the stroma encompass the basement membrane outside the follicle, thus forming a follicle complex. A common basement membrane is shared by the germinal epithelium and the follicle complex along a small portion of its surface. This point of attachment represents the site at which the oocyte would be released to the ovarian lumen during ovulation.     

The organization of germinal material and dynamics of mother sporocyst reproduction in the genus Echinostoma (Trematoda: Echinostomatidae)

The reproduction in the first parthenogenetic generation--mother sporocyst (MS) in two species of echinostomes (E. caproni, E. paraensei) is investigated. A group of densely packed cells, which noticeably differ from others, occupies the posterior part of the miracidium. They are characterized by large sizes and a large bubble-shaped nucleus with heterogeneous nucleolus and strong dispersed chromatin. The use of histological and electron microscopic methods has shown that with observed similarity these cells are classified in two tyoes and have a completely different origin. First of all, large secretory cells stand out. In E. caproni miracidia their number averages 6.8 +/- 0.2 and linear sizes is 10-12 microns. Secretory cells possess a large bubble-shaped nucleus. The caryoplasm looks optically empty because of strong dispersion of chromatin. A large nucleolus occupies a bit eccentric position. Eosinophilic cytoplasm contains poorly noticeable at light-optical level accumulation of small granules. The second group of cells is represented by typical germinal cells (GC). The number of GC does not exceed six. Their polymorphy is well above that of secretory cells. The sizes of GC vary from 5.4 to 9 microns. The largest cells (8.1-9 microns) occupy the front position and usually are located between secretory cells. Intensively basophilic cytoplasm surrounds bubble-shaped nucleus with a large nucleolus like border with uniforming thickness. The heterochromatin is evenly distributed over the caryoplasm. Its content of nuclei is more than that in nuclei of secretory cells. That is why they do not look optically empty. So, it is "mature" germinal cells. Four or five cells are located directly behind "mature" cells. Their sizes are gradually decrease towards the posterior of the miracidium (the diameter of the smallest cells reaches 5.4 microns). Nuclei with a centrally located nucleolus are characterized by larger amount and more condensation of the heterochromatin than those in "mature" cells. Meanwhile, they concern to nuclei of bubble-shaped type. In general, all cells of second type represent the primary germinal cells distinguished by the stage of their differentiation. Also, 2-3 undifferentiated cells occupy the most posterior part of the miracidium. Their sizes average 5.55 +/- 0.18 microns. The nucleus contains a lot of densely packed heterochromatin. On parasitic phase of MS development undifferentiated cells give rise to secondary GC. Electron microscopic data in details confirm the situation described above. The essentially similar results were received during the investigation of E. Paraensei miracidia. The differences are observed in parameterical characteristics of germinal material and in small variability of the extent of germinal material development. With E. paraensei, germinal material may be represented by not only GC and undifferentiated cells, but one germ as well. So, our investigation has shown that germinal material of echinostomes represents typical germinal mass. The germinal material condition does not change on parasitic phase of E. caproni MS development during the first day of post infection (PI). The activization of germinal material coincides in the time with the beginning of schizocoel formation in 2 Days PI. On the 3rd day of PI, the proliferation of undifferentiated cells begins and the first germs are free to float in enormous schizocoel. After 8 days of PI, MS release the first rediae. During the following 2-3 days the other rediae formed by primary GC left MS. The release of rediae derived from secondary GC was observed later. So, E. caproni MS give rise to 12-16 rediae which is much less than the number of GC formed in MS. The earlier release of the first E. paraensei rediae by MS is predetermined by the difference in the structure of germinal mass in E. paraensei miracidia. Therefore, Echinostomatidae is intermediate between two groups of trematodes. The first group has MS that completely realize reproductive function in the time of miracidial formation; but the second group includes higher trematodes characterized by the transfer of reproductive time on a parasitic phase of MS development. The question concerning to so-called "pedogenetic larvae of trematodes" is discussed.     

The effects of social environment on adult neurogenesis in the female prairie vole

In the mammalian brain, adult neurogenesis has been found to occur primarily in the subventricular zone (SVZ) and dentate gyrus of the hippocampus (DG) and to be influenced by both exogenous and endogenous factors. In the present study, we examined the effects of male exposure or social isolation on neurogenesis in adult female prairie voles (Microtus ochrogaster). Newly proliferated cells labeled by a cell proliferation marker, 5-bromo-2'-deoxyuridine (BrdU), were found in the SVZ and DG, as well as in other brain areas, such as the amygdala, hypothalamus, neocortex, and caudate/putamen. Two days of male exposure significantly increased the number of BrdU-labeled cells in the amygdala and hypothalamus in comparison to social isolation. Three weeks later, group differences in BrdU labeling generally persisted in the amygdala, whereas in the hypothalamus, the male-exposed animals had more BrdU-labeled cells than did the female-exposed animals. In the SVZ, 2 days of social isolation increased the number of BrdU-labeled cells compared to female exposure, but this difference was no longer present 3 weeks later. We have also found that the vast majority of the BrdU-labeled cells contained a neuronal marker, indicating neuronal phenotypes. Finally, group differences in the number of cells undergoing apoptosis were subtle and did not seem to account for the observed differences in BrdU labeling. Together, our data indicate that social environment affects neuron proliferation in a stimulus- and site-specific manner in adult female prairie voles.     

Vascular smooth muscle cells of male and female rats in culture, migration, proliferation and chromosome number

The authors evaluated the migratory and proliferative properties and the chromosome number of cultivated male and female smooth muscle cells (SMC) obtained by the explanation method from the thoracic aorta of rats of a conventional and a specific pathogen-free (SPF) breed. It was found that male SMC, in most cases, began to migrate from the explants sooner than female SMC and that they migrated from a higher total number of explants. The time needed for the number of cells in the culture to double (doubling time) was practically the same for male and female SMC, but male SMC attained a higher maximum population density. Male SMC cultures (2nd passage) contained cells with a hyperploid chromosome number, whereas female SMC were diploid. It was also found that SMC from conventional rats, in which the presence of pathogens could be presumed, displayed higher migratory and proliferative capacity than the SMC of SPF rats. The capacity of the SMC of male rats for migration and proliferation could have been potentiated by the effect of a different composition of the intercellular matrix and a different chromosome number, and in conventional rats by the presence of pathogens.     

Effect of alpha-fetoprotein on isolated mouse oocytes

Data are presented which indicate a possible action of alpha-fetoprotein (AFP) on female germinal cells. The in vitro maturation of mature mice oocytes was significantly inhibited when mouse AFP replaced albumin in the culture medium. In addition, the degenerative aspect of oocytes cultured with AFP seemed to indicate that this me?otic inhibition was caused by a premature degeneration of oocytes rather than by a blockage at a specific stage of maturation. Thus AFP, perhaps through its ligands, may play a role in the reduction of germinal cells during fetal and immediate post-natal life rather than in the arrest of me?osis at the diplotene stage.     

Identification of genes expressed in the Arabidopsis female gametophyte

The angiosperm female gametophyte typically consists of one egg cell, two synergid cells, one central cell, and three antipodal cells. Each of these four cell types has unique structural features and performs unique functions that are essential for the reproductive process. The gene regulatory networks conferring these four phenotypic states are largely uncharacterized. As a first step towards dissecting the gene regulatory networks of the female gametophyte, we have identified a large collection of genes expressed in specific cells of the Arabidopsis thaliana female gametophyte. We identified these genes using a differential expression screen based on reduced expression in determinant infertile1 (dif1) ovules, which lack female gametophytes. We hybridized ovule RNA probes with Affymetrix ATH1 genome arrays and validated the identified genes using real-time RT-PCR. These assays identified 71 genes exhibiting reduced expression in dif1 ovules. We further validated 45 of these genes using promoter::GFP fusions and 43 were expressed in the female gametophyte. In the context of the ovule, 11 genes were expressed exclusively in the antipodal cells, 11 genes were expressed exclusively or predominantly in the central cell, 17 genes were expressed exclusively or predominantly in the synergid cells, one gene was expressed exclusively in the egg cell, and three genes were expressed strongly in multiple cells of the female gametophyte. These genes provide insights into the molecular processes functioning in the female gametophyte and can be used as starting points to dissect the gene regulatory networks functioning during differentiation of the four female gametophyte cell types.     

The production and elimination of supernumerary blast cells in the leech embryo

 Different species of leech vary greatly in body size but all have 32 body segments. It is unclear how the development of this precise number of segments is regulated, although it is known that the teloblasts of the early leech embryo initially produce more than the required numbers of segment founder cells (blast cells). We used fluorescent dextrans to show that the M teloblast of the Helobdella robusta embryo produces a variable number of additional (supernumerary) cells. These cells fail to enter the germinal band (which contains cells of all lineages and gives rise to the adult leech), but detach from its posterior end and disappear. Our observations suggest that some suffer an increase in membrane permeability while others fuse with the M teloblasts, but that they do not undergo apoptosis. The supernumerary cells of different lineages detach from the germinal band at different times, suggesting that detachment is not triggered by a global signal acting simultaneously on all lineages. We tested the hypothesis that the elimination of the supernumerary m blast cells results from a requirement of m blast cells for close interactions with cells of the other lineages for their survival, a condition that would not be achieved by the last-born m blast cells that fail to enter the germinal band. We cultured isolated M teloblasts and found that they do produce blast cells that themselves divide, indicating that cells of the M lineage can survive in the absence of any interactions with cells of the other lineages. Received: 17 August 1998 / Accepted: 20 November 1998