Cell Migration and Induction in the Development of the Surface Ectodermal Pattern of the Xenopus laevis Tadpole

The surface of the Xenopus tadpole contains three specialized, transient cell types; the ciliated, hatching gland, and cement gland cells. To distinguish whether the appearance of these cell types on the surface is due to induction of surface cells or due to migration of deep ectodermal cells into the surface, we transplanted labelled surface or deep cells to unlabelled hosts at early to mid-gastrulae. After raising the host to a tadpole (Stage 28), we examined the embryo's surface for ciliated, hatching gland, and cement gland cells, and observed which cells were labelled. We find that all ciliated cells move into the surface from the deep ectodermal layer along with other cells of unknown function. Hatching gland cells arise by induction of surface cells as do the majority of cement gland cells. A few deep cells give rise to cement gland cells. Therefore, migration of deep cells to the surface and localized induction of surface cells contribute to the final surface patterning of the Xenopus tadpole.     

Homoiogenetic regulation through the ectoderm on localized expression of the hatching gland phenotype in the head area of Xenopus embryos

Ectoderm pieces explanted from embryos of Xenopus laevis were cultured and examined for differentiation of hatching gland cells, using immunoreactivity against anti-XHE (Xenopus hatching enzyme) as a marker. The anterio-dorsal ectoderm excised from stage 12-13 (mid-late gastrula) embryos developed hatching gland cells. Meanwhile, the posterio-, but not the anterio-dorsal ectoderm from stage 11 (early gastrula) embryos developed these cells, although it is not fated to do so during normogenesis. This hatching gland cell differentiation from stage 11 posterior ectoderm was not affected by conjugated sandwich culture with the mesoderm but was suppressed when explants contained an anterior portion of the ectoderm. Conjugated cultures of anterior and posterior portions of the ectoderm in various combinations indicated that differentiation of hatching gland cells from stage 11 posterior and stage 12 anterior portions was suppressed specifically by stage 11 anterior ectoderm. Northern blot analyses of cultured explants showed that XHE was expressed in association with XA-1, suggesting its dependence on the anteriorized state. These results indicate that the planar signal(s) emanating from stage 11 anterior ectoderm participates in suppression of the expression of the anteriorized phenotype so that an ordered differentiation along the anteroposterior axis of the surface ectoderm is accomplished.     

Expression of estrogen induced gene 121-like (EIG121L) during early Xenopus development

Estrogen induced gene 121 (EIG121) and EIG121-like (EIG121L) are evolutionarily conserved genes. But, their function is still unknown. Here, we report the expression pattern of Xenopus EIG121-like (xEIG121L) during early development. Its expression was first detected at stage 9 after mid-blastula transition, attained its maximal level at the gastrula stage, and remained constant until the tadpole stage. Whole-mount in situ hybridization revealed that xEIG121L was expressed strongly in the ventral ectoderm at the gastrula stage, and in the anterior ectoderm surrounding the neural plate at the neurula stage. xEIG121L expression was especially high in the presumptive hatching gland and cement gland regions in the neurula. At the tailbud stage, xEIG121L expression was limited to the hatching gland; an inverted Y type staining, characteristic of the hatching gland, was observed. However, at the tadpole stage, xEIG121L was expressed broadly in the head, heart and fin.     

Expression pattern of BXR suggests a role for benzoate ligand-mediated signalling in hatching gland function

The Xenopus laevis nuclear receptor BXR has recently been shown to be activated by a class of endogenous benzoate metabolites, indicating the presence of a novel and unsuspected benzoate ligand-dependent signalling pathway. The receptor is expressed ubiquitously in blastula and gastrula stage embryos, and its expression declines during neurula stages. In order to examine further this novel vertebrate signalling system, we have examined the expression of the BXR gene in tailbud stage embryos and adults. We show here that in Xenopus tailbud stage embryos expression is restricted to the hatching gland, suggesting a role in hatching gland function. Neither BXR nor a BXR-VP16 fusion is sufficient to specify hatching gland in neurally-induced tissue. In adults, BXR expression is abundant in the brain and gonads. This expression pattern in adults is distinct from any of the putative mammalian homologues. A nuclear receptor that mediates benzoate signalling has yet to be found in mammals.     

A homologue of cysteine-rich secretory proteins induces premature degradation of vitelline envelopes and hatching of Xenopus laevis embryos

We cloned Xenopus laevis CRISP, XCRISP, a homologue of the mammalian family of cysteine-rich secretory proteins (CRISPs), which has been previously identified as a Wnt3a/noggin responsive gene in an expression screen [Mech. Dev. 87 (1999) 21]. We detected XCRISP expression exclusively in the hatching gland. XCRISP enters the secretory pathway and accumulates on the surface of presumptive hatching gland cells. Overexpression studies of XCRISP and XCRISP-mutants show that XCRISP induces premature hatching of embryos preceded by degradation of the vitelline envelope. A deletion mutant that lacks a 35 amino acid domain even accelerates hatching, while further deletion of the carboxy-terminus reverses these effects. From our studies, we conclude that XCRISP is sufficient to induce degradation of vitelline envelopes and that this activity maps to the most C-terminal amino acids, while the adjacent domain regulates XCRISP activity.     

Xenopus galectin-VIa shows highly specific expression in cement glands and is regulated by canonical Wnt signaling

Anterior-posterior neural patterning of Xenopus embryo is determined during gastrulation and then followed by differentiation of neural structures including brain and eye. The cement gland is a mucus-secreting neural organ located in the anterior end of the neural plate. This study analyzed expression patterns of Xenopus galectin-VIa (Xgalectin-VIa) by whole-mount in situ hybridization, and found highly restricted expression of this gene in the cement gland region. These patterns were similar to those of XAG-1 and XCG, known cement gland-specific genes. In addition, Xgalectin-VIa was expressed in the dorsal edge of eye vesicles, the otic vesicle, and in part of the hatching gland at the tadpole stage. Although the spatial expression pattern was similar, the temporal expression of Xgalectin-VIa differed from that of XAG-1 and XCG. RT-PCR analysis showed only weak Xgalectin-VIa expression in early neurula embryos, whereas both XAG-1 and CGS were strongly expressed at that stage. We also showed that Xgalectin-VIa expression is repressed by enhancement of Wnt signaling and increased by its inhibition. Furthermore, Xgalectin-VIa expression was activated by neural-gene inducer Xotx2, as is the case for XAG-1 and CGS. Together, these results indicated that Xgalectin-VIa possesses different features from other cement gland genes and is a novel and useful marker of the cement gland in developing embryos.     

卤虫(Artemia salina)孵化腺细胞分化时相的免疫细胞化学研究(简报)

动物孵化酶(hatching enzyme,HE)是早期胚胎在特定发育阶段由孵化腺细胞产生和分泌的,在动物早期胚胎孵化中具有关键性作用。孵化腺细胞(hatching gland cell,HGC)一般为单细胞腺体,是从胚胎发育到特定阶段(孵化前)出现、至胚胎孵出后的特定时期消失的一时性细胞(transient type ofcells)。完全分化的HGC内充满了低电子密度的酶原颗粒(孵化酶原颗粒),在鱼胚中的分布因物种而异。在大多数鱼中,HGC分布在胚体的外表面和/或卵黄囊中,一般为外胚层来源。如在虹蹲鱼HGC分布在胚体的前表面、卵黄囊、咽部、鳃的内表面及外表面,属于外胚层来源。而日本鳉鱼HGC     

Localization of specific mRNAs in Xenopus embryos by whole-mount in situ hybridization.

We have adapted a non-radioactive technique to detect localized mRNAs in whole-mount Xenopus embryos. Synthetic antisense RNA transcribed in the presence of digoxygenin-UTP is used as a probe and is detected via an anti-digoxygenin antibody. We show that localized mRNAs can be detected from late gastrula to tadpole stages and that high as well as low abundance RNAs can be detected. The method was tested on muscle actin and alpha-globin RNAs, whose localization has previously been characterized. In addition, we used the method to determine the distribution of XA-1 RNA, an anterior ectoderm-specific RNA, which we show is expressed in the periphery of the cement gland as well as in the region of the hatching gland. The sequence of an XA-1 cDNA predicts a protein rich in proline and histidine.     

卤虫（Artemia salina）孵化腺细胞分化时相的免疫细胞化学研究（简报）

动物孵化酶(hatching enzyme HE)是早期胚胎在特定发育阶段由孵化腺细胞产生和分泌的,在动物早期胚胎孵化中具有关键性作用^[4]。孵化腺细胞(hatching gland cell,HGC)一般为单细胞腺体,是从胚胎发育到特定阶段(孵化前)出现、至胚胎孵出后的特定时期消失的一时性细胞(transient type of     

HATCHING GLANDS IN THE TELEOSTS, BRACHYDANIO RERIO. DANIO MALABARICUS, MOENKHAUSIA OLIGOLEPIS AND BARBUS SCHUBERTI

Many teleost embryos produce an enzyme within specialized glands, which facilitate hatching. The enzyme attacks the chorion which becomes so weak that it may be ruptured easily by a blow of the tail.
The embryos of Brachydanio rerio, Danio malabaricus, Moenkhausia oligolepis and Barbus schuberti show some morphological differences in the distribution of the hatching gland cells. More specificity can be found in the ultrastructure of hatching gland cells, which are loaded with enzyme granules prior to hatching. In all four species the nucleus is located near the basis of the cell. The hatching enzyme is contained within granules, which arise from the Golgi body.     

Amphibian hatching gland cells: pattern and distribution in anurans

The hatching gland (HG) is a transient organ, found in most anuran embryos and early larvae, and located on the dorsal side of the head. The enzymes secreted by hatching gland cells (HGCs) aid the embryos to escape from their enveloping coats. Analysis of HG morphology and distribution in 20 anuran species from six families using scanning electron microscopy revealed small differences in the shape and pattern of the gland particularly in the length and width of the posterior mid-dorsal extension of the gland. The four species of foam-nest making leptodactylids examined had HGs of a somewhat different shape to the others, but otherwise, there was little sign of a relationship between HG shape and taxonomic position. In the single Eleutherodactylus species examined, cells with the appearance and location of HGCs were transiently present long before the active stage of hatching. No sign of HGCs was seen on the head surface of one species, Phyllomedusa trinitatis. It seems possible that in this species, hatching is achieved by a mechanical rather than an enzymatic mechanism. The microvilli characteristic of the surfaces of HGCs were quite variable in density and length from species to species, and at different stages. HGCs remained at the surface of the embryo for some time after hatching and the possibility of a post-hatching function is briefly discussed.     

Analysis of the origin and development of hatching gland cells by transplantation of the embryonic shield in the fish, Oryzias latipes

Hatching gland cells of the medaka, Oryzias latipes, have been observed to differentiate from the anterior end of the hypoblast, which seems to first involute at the onset of gastrulation. These results suggest that the hatching gland cells of medaka originate from the embryonic shield, the putative organizer of this fish. The present study investigated whether hatching gland cells really originate from the embryonic shield in the medaka. Transplantation experiments with embryonic shield and in situ hybridization detection of hatching enzyme gene expression as a sign of terminal differentiation of the gland cells were carried out. The analysis was performed according to the following processes. First, identification and functional characterization of the embryonic shield region were made by determining the expression of medaka goosecoid gene and its organizer activity. Second, it was confirmed that the embryonic shield had an organizer activity, inducing a secondary embryo, and that the developmental patterns of hatching gland cells in primary and secondary embryos were identical. Finally, the hatching gland cells as identified by hatching enzyme gene expression were found to coincide with the dye-labeled progeny cells of the transplanted embryonic shield. In conclusion, it was determined that hatching gland cells were derived from the embryonic shield that functioned as the organizer in medaka.     

Hatching in the pike Esox lucius L.: Evidence for a single hatching enzyme and its immunocytochemical localization in specialized hatching gland cells

Antibodies against purified hatching enzyme (HE) from the pike, Esox lucius L., have been used to examine different aspects of the presence of the enzyme in the ontogeny of this teleostean fish. Immunochemical analysis indicates that the two proteolytic enzymes which occur in the hatching medium arise from a single protease, HE itself. The second proteolytic fraction found in gel filtration of hatching medium could be a heterogeneous population of complexes of HE with digestion fragments of its natural substrate, the zona radiata. Immunofluorescence microscopy by means of anti-HE antibodies demonstrates that HE is localized in the so-called hatching gland cells (HGCs). The HGCs in pike appear as oval to round cells 10–15 μm in diameter containing granules of 1.5–2.3 μm. They are found interspersed between the periderm and the presumptive epidermis. The number of HGCs and their granule content increase significantly until the 35-somite stage to reach about 1200 and 30, respectively. From then on these numbers do not change until hatching in the 66-somite stage. The distribution of the HGCs over the embryo also changes, probably since HGC precursors in the yolk sac differentiate to HGCs later than their counterparts in the head region. The immunocytochemical procedure further shows that HE can be detected from the 10-somite stage on. Discrete hatching gland remnant bodies, phagocytized by epidermal cells, are observed in larval stages until 3–7 days after emergence of the embryo.     

Development of new methods in human gene mapping: selection for fragments of the human Y chromosome after chromosome-mediated gene transfer.

Chromosome-mediated gene transfer (CMGT) can be used to generate fragments of human chromosomes and chromosomal maps can be constructed using these fragments. In previous experiments CMGT techniques have been limited to those regions of the genome which encode biochemically selectable markers. We have extended the regions of the human genome which can be subjected to CMGT methods by employing a cell surface antigen as a selectable marker. These experiments have been facilitated by the discovery that co-transformation of chromosomes with a plasmid bearing a biochemically selectable marker followed by selection for the marker pre-selects for cells which have incorporated chromosomal fragments. The plasmid may also integrate into the donor chromosomes and this provides, in some cases, an additional selectable marker in the chromosome fragment of interest. Using these methods we have isolated for the first time cells containing varying portions of the human Y chromosome.     

Ultrastructural Studies on Development of the Tail Gland of a Cuttlefish, Sepiella japonica

Hatching glands in embryos of teleosts and amphibians have been reported to be indispensable for hatching of the embryos. The cephalopod has capsuled eggs, so we expected to find some exocrine organ in the embryos that functioned as a hatching gland. The tail gland (Hoyle's organ) has been suspected to be a hatching gland in the cephalopod, and therefore we examined it during the course of development of cuttlefish embryos. Cells in the tail gland appeared similar to the hatching gland cells (HGCs) of teleosts and amphibians, and contained a number of secretion granules that also resembled the hatching enzyme granules (HEGs) in HGCs of teleosts and amphibians in size, electron density and distribution in the cells. However, a few of these granules were discharged one after another from an early stages, whereas most of them were retained up to the stage just before hatching, and then discharged all at once. The former process of trickling discharge was similar to that in amphibians and the latter process of abrupt discharge resembled that in teleosts.     

鲤胚胎孵化腺细胞

鲤胚胎孵化腺为单细胞腺体,发生于外胚层,可特异地被PAS染色。最早可在眼色素期检验出孵化腺细胞(Hatching gland cell,HGC)它们主要分布在头部腹面及头部与卵黄囊连接处。开始,HGC位于表皮细胞下面,随发育迁移到胚胎表面。根据扫描和透射电镜观察,在分泌孵化酶的前后,HGC区表面细胞呈鸡冠花状和疣状两种突起。前者系HGC处于分泌孵化酶期间;后者系HGC业已完成分泌作用,由于相邻的表皮细胞活动而形成的。HGC内富有粗面内质网、线粒体、核糖体和高尔基体,并由后者合成酶原颗粒。HGC在完成分泌作用后,仍留在表皮中,以后逐渐退化,但在孵化后30h仍可见残留的HGC。     

Skin peptides in Xenopus laevis: morphological requirements for precursor processing in developing and regenerating granular skin glands

The biosynthesis of the peptides caerulein and PGLa in granular skin glands of Xenopus laevis proceeds through a pathway that involves discrete morphological rearrangements of the entire secretory compartment. Immunocytochemical localization of these peptides during gland development indicates that biosynthetic precursors are synthesized in intact secretory cells, whereas posttranslational processing requires morphological reorganization to a vacuolated stage. The bulk of the processed secretory material is then stored in vacuolae- derived storage granules. In the mature gland, storage granules are still formed at a low level. However, in this case processing takes place in a distinct cytoplasmic structure, the multicored body, which we suggest to be functionally equivalent to vacuolae. When granular glands regenerate after having lost all their storage granules upon strong stimuli, another morphological pathway is used. 2 wk after gland depletion, secretory cells become arranged in a monolayer that covers the luminal surface of the gland. Storage granules are formed continuously within these intact secretory cells. Here, precursor processing does not require a vacuolated stage as in newly generated glands but occurs in multicored bodies. Most storage granules seem to be formed in the third week of regeneration. The high biosynthetic activity is also reflected by the high activity of the putative processing enzyme dipeptidyl aminopeptidase during this period of regeneration.