Mechanisms of primordium formation during adventitious root development from walnut cotyledon explants

 In walnut (Juglans regia L.), an otherwise difficult-to-root species, explants of cotyledons have been shown to generate complete roots in the absence of exogenous growth regulators. In the present study, this process of root formation was shown to follow a pattern of adventitious, rather than primary or lateral, ontogeny: (i) the arrangement of vascular bundles in the region of root formation was of the petiole type; (ii) a typical root primordium was formed at the side of the procambium within a meristematic ring of actively dividing cells located around each vascular bundle; (iii) the developing root apical meristem was connected in a lateral way with the vascular bundle of the petiole. This adventitious root formation occurred in three main stages of cell division, primordium formation and organization of apical meristem. These stages were characterized by expression of LATERAL ROOT PRIMORDIUM-1 and CHALCONE SYNTHASE genes, which were found to be sequentially expressed during the formation of the primordium. Activation of genes related to root cell differentiation started at the early stage of primordium formation prior to organization of the root apical meristem. The systematic development of adventitious root primordia at a precise site gave indications on the positional and biochemical cues that are necessary for adventitious root formation. Received: 30 July 1999 / Accepted: 16 February 2000     

Direct and indirect requirements of Shh/Gli signaling in early pituitary development

Induction of early pituitary progenitors is achieved through combined activities of signals from adjacent embryonic tissues. Previous studies have identified a requirement for oral ectoderm derived Sonic Hedgehog (Shh) in specification and/or proliferation of early pituitary progenitors, however how different Gli genes mediate Shh signaling to control pituitary progenitor development has not yet been determined. Here we show that Gli2, which encodes a major Gli activator, is required for proliferation of specific groups of pituitary progenitors but not for initial dorsoventral patterning. We further show that the action of Gli2 occurs prior to the closure of Rathke' pouch. Lastly, we show that Shh/Gli2 signaling controls the diencephalic expression of Bone morphogenetic protein 4 (Bmp4) and Fibroblast growth factor 8 (Fgf8), two genes that are known to play critical roles in patterning and growth of Rathke's pouch. Our results therefore suggest both cell-autonomous and non-cell-autonomous requirements for Gli2 in regulation of pituitary progenitor specification, proliferation and differentiation.     

Transforming growth factor beta inhibits proliferation of somatic cells without influencing germ cell number in the chicken embryonic ovary

The gonadal development of chicken embryo is regulated by hormones and growth factors. Transforming growth factor beta (TGF-β) isoforms may play a critical role in the regulation of growth in chicken gonads. We have investigated the effect of the TGF-β isoforms on the number of germ and somatic cells in the ovary of the chicken embryo. Ovaries were obtained from chicken embryos at 9 days of incubation. They were organ-cultured for 72 h in groups treated with TGF-β1, TGF-β2, soluble betaglycan, TGF-β1 plus soluble betaglycan, or TGF-β2 plus soluble betaglycan, and untreated (control). TGF-β1 and TGF-β2 diminished the somatic cell number in the ovary of the chicken embryo at this age by inhibiting the proliferation of the somatic cells without increasing apoptosis. On the other hand, TGF-β1 and TGF-β2 did not affect the number of germ cells in the cultured ovary. The capacity of TGF-β1 and TGF-β2 to diminish the number of somatic cells in the ovary was blocked with soluble betaglycan, a natural TGF-β antagonist. However, changes in the location of germ cells within the ovary suggested that TGF-β promoted the migration of the germ cells from the ovarian cortex to the medulla. Thus, TGF-β affects germ and somatic cells in the ovary of the 9-day-old chicken embryo and inhibits the proliferation of somatic cells.This work was supported by DGAPA-UNAM (IN214403) and CONACYT (45030).     

Proteomic analysis of egg white proteins during the early phase of embryonic development

Avian egg albumen participates in embryonic development by providing essential nutrients as well as antimicrobial protection. Although various biological functions of egg white proteins were suggested during embryogenesis, global changes of these proteins under incubation conditions remained uninvestigated. This study presents a proteomic analysis on the change of egg white proteins during the first week of embryonic development. By using 2-DE, together with MALDI-TOF MS/MS, thirty protein spots representing eight proteins were identified showing significant changes in abundance during incubation. An accelerating degradation of ovalbumin was observed in a wide range of molecular weight. In addition, four protein complexes were predicted according to the detected molecular weight increase. Among these speculated protein complexes, an ovalbumin spot coupled with RNA-binding protein was detected. The absence of these protein complexes before incubation, followed by the constant increase in abundance during incubation indicates conceivable pivotal roles in embryonic development. To better understand the function of the proteins identified in this study, discrepancies of egg white protein changes between fertilized and unfertilized chicken eggs were additionally demonstrated. These findings will provide insight into the embryogenesis process to improve our knowledge of egg white proteins in regulating and supporting early embryonic development.     

Ontogeny of estrogen receptor (ER) alpha and its co-localization with pituitary hormones in the pituitary gland of chick embryos

Estrogen is involved in regulating the development and hormone secretion of the anterior pituitary gland following its binding to estrogen receptors (ERs) expressed on pituitary cells. However, the pituitary is comprised of several cell types, and to date, there is no data about the specific cell types expressing ERs in embyonic chick pituitary. We therefore followed, by immunohistochemistry, the ontogeny of the pituitary ER alpha (ER), and the cell types expressing ER throughout chick embryo development. ER immunoreacitivity was restricted to the nuclei of pituitary cells. ER-immunopositive (ER⁺) cells were first detected at embryonic day 6.5 (E6.5), after which ER⁺ cells were consistently detected throughout the anterior pituitary gland, although the density of ER⁺ cells in the caudal lobe of the pars distalis was higher than that in the cephalic lobe. The proportion of ER⁺ cells in the pituitary was about 6% at E8.5; expression increased to 22% by E18.5 of gestation, with no additional change until hatching. Double-labeling of ER and pituitary hormones showed that the dominant cell types expressing ER were gonadotrophs immunopositive for luteinizing hormone (LH); the proportion of ER⁺ cells expressing LH increased throughout gestation and reached approximately 57% at hatching. About 2%–6% of thyroid-stimulating-hormone-immunopositive and 1%–2% prolactin-immunopositive cells expressed ER at later stages of embryonic development, but no growth-hormone-positive or adrenocorticotropic-hormone-positive cells expressed ER during the embryonic period. Thus, gonadotrophs are the main cell population expressing ER in the anterior pituitary gland of chick embryo, and ER is involved in regulating the development of the pituitary gland and the maturation of the hormone-secreting function.This work was supported by grants from the Natural Science Foundation for Outstanding Young Scientists of China (30325034) and the Natural Science Foundation of China (30170693, 30471264).     

Teratogenicity of 3-hydroxynorvaline in chicken and mouse embryos

Summary. 3-Hydroxynorvaline (HNV; 2-amino-3-hydroxypentanoic acid), a microbial L-threonine analogue, is toxic to mammalian cells and displays antiviral properties. In view of this, we investigated the toxicity and/or potential teratogenicity of HNV in developing chicken and mouse embryos. HNV was administered to chicken embryos (in ovo; dose 75–300 μmole/egg; 48 h post-incubation) and pregnant Hanover NMRI mice (per os; total dose 900–1800 mg/kg body mass; gestation days 7–9). Control animals received sterile saline solutions. Harvested embryos (chicken embryos, 10 days post-incubation; mouse embryos; gestation day 18) were fixed in glutaraldehyde and stereomicroscopically inspected for signs of dysmorphogenesis. Body mass, body and toe length and mortality of chicken embryos, and the body mass and mortality of mouse embryos were recorded. HNV exposure significantly increased the incidence of embryotoxic (growth retardation, toxic mortality) and congenital defects in both chicken and mouse embryos. All the observed effects were dose-dependent. In conclusion, HNV is an embryotoxic and teratogenic compound, which caused significant developmental delay and congenital defects in developing chicken and mouse embryos.     

Distribution of MAP1A,MAP1B,and MAP2A&;B during layer formation in the optic tectum of developing chick embryos

The expression patterns of three microtubule-associated proteins (MAP1A, MAP1B, and MAP2A&B) were investigated in the developing optic tectum. Expression of MAP1B and middle-molecular-weight peptide of neurofilament (NF-M) was first observed in the same mesencephalic cells on day 3 of incubation, indicating that neuroblasts had been produced. At day 5, MAP1A and MAP2A&B expression appeared in the cellular layer containing the first neuroblasts that differentiate into large multipolar cells. The NF-M⁺ neurites in the striatum album centrale (SAC) and the striatum opticum (SO) were MAP1B⁺ up to day 19, but the intensity of MAP1B immunoreactivity decreased with development. All three MAPs were expressed in large multipolar neurons in the developing stratum griseum centrale from the beginning of maturation. Stratum griseum et fibrosum centrale cellular layers, containing radially arranged piriform neurons, were MAP1A^–/MAP2A&B^– on day 11 but became MAP1A⁺/MAP2A&B⁺ during later stages. These results suggest that the timing of MAP expression in neuronal maturation of large multipolar cells differs from that of piriform cells. The expression of MAPs has revealed specific cellular events in the developing optic tectum. Based on our observations, the development of the optic tectum can be divided into four periods.     

The spatial and temporal expression of delta-like protein 1 in the rat pituitary gland during development

An analysis of secreted proteins by the signal sequence trap method using a cDNA library of the rat pituitary anlage at embryonic days (E) 13.5 revealed the abundant expression of delta-like protein 1 (Dlk1) in the pituitary gland. Dlk1, an epidermal growth factor-like repeat protein in preadipocytes, functions in maintaining the preadipose state. Expression of Dlk1 mRNA in the pituitary at E13.5 and in the adult pituitary was confirmed by in situ hybridization. The expression pattern of Dlk1 during pituitary development was also studied by immunohistochemistry. Dlk1 protein first appeared in Rathke’s pouch and the infundibulum at E11.5; as development proceeded, expression became restricted to the pars distalis and pars tuberalis (PT). Dlk1 was expressed in most ACTH cells during the embryonic stages, but its expression was limited to only a few ACTH cells in the adult pituitary. It was also expressed in a small population of TSH, GTH, and PRL cells throughout development, whereas it was present in the cytoplasm of most GH cells at all developmental stages. Similarly, Dlk1 was localized in the cytoplasm of PT cells during development. These findings provide new insights into the mechanism of Dlk1 regarding its regulation of pituitary hormone-secreting cells during development.     

Distribution of GABAergic perikarya and terminals in the centers of the higher auditory pathway of the chicken

Summary The distribution of presumed GABAergic neurons and axon terminals in nuclei of the higher auditory pathway of the chicken was investigated by immunocytochemical methods employing antisera to the rate-limiting enzyme of GABA synthesis, glutamic acid decarboxylase, and to GABA. In the mesencephalic auditory center (MLD) about 20% of the cells reveal immunoreactivity. In contrast, the thalamic relay station nucleus ovoidalis is devoid of immunostained somata. This nucleus contains a high density of punctate immunoreactive structures presumed to be GABAergic axon terminals. In the auditory forebrain center field L and the auditory portions of the hyperstriatum ventrale, up to 8% of the cells were immunopositive. These neurons were significantly smaller than estimated from measurements of the overall cell population in these nuclei. From the two-dimensional arrangement of immunopositive neurons it is suggested that the GABAergic system in the avian auditory telencephalon consists of two separate groups of neurons: one subgroup mediating local inhibitory interactions, the other responsible for lateral inhibition between different frequency representations.This work was supported by the Deutsche Forschungsgemeinschaft (SFB 45)     

Organization of atrial natriuretic factor-like immunoreactive system in the brain of the frog Rana esculenta during development

Immunocytochemical distribution of the atrial natriuretic factor (ANF) has been studied in the brain and pituitary of the anuran Rana esculenta during development and in juvenile animals. Using human ANF and rat α-ANF antisera, immunoreactive cell bodies and nerve fibers were revealed in stage II–III tadpoles and in successive larval stages. Soon after hatching, stages II–III, the ANF-like-immunoreactive elements were confined to the preoptic area-median eminence complex. During successive stages of development, new groups of ANF-immunoreactive cell bodies appeared. In larval stage VI, immunoreactive perikarya were found in the rostral part of the anteroventral area of the thalamus and numerous ANF-like-immunoreactive cells appeared in the pars distalis of the pituitary. In larval stages XIV and XVIII, the distribution of ANF immunoreactivity was virtually similar. The ANF-immunoreactive cells in the preoptic nucleus and in the pituitary pars distalis were comparatively more abundant than in stage VI. During the metamorphic climax (stages XXI–XXII), a new group of ANF-immunoreactive cell bodies appeared in the rostral part of the ventrolateral area of the thalamus. During this stage, ANF-immunoreactive fiber projections were found in the pars intermedia for the first time. However, the pars distalis cells were very weakly immunofluorescent. The pattern of ANF immunoreactivity in the brain of juvenile animals was very similar to that described for stages XXI and XXII, whereas the pars distalis cells showed no immunoreactivity. It is conceivable that, early during development, ANF-related peptides may be involved in the regulation of pituitary secretion by means of autocrine mechanisms or may act as a classic pituitary hormone. Received: 28 July 1997 / Accepted: 8 December 1997     

The expression of chicken NOV, a member of the CCN gene family, in early stage development

The nephroblastoma overexpressed gene, NOV, is a member of the CCN gene family. We investigated the NOV gene expression pattern in the chicken during early stage embryogenesis. Several embryonic structures showed a distinct expression pattern. The initial expression was detected in Hensen's node (Hamburger and Hamilton stage (HH) 5). The expression was noted in the presumptive notochord and floor plate forming cells. The expression on the left side was more elongated posteriorly, a type of left-right asymmetry. Chicken NOV gene expression in the forming notochord and floor plate was observed until HH 18. The expression was also detected in the ventral area of the mesencephalon and isthmus at HH 14-16.     

Induction from posterior hypothalamus is essential for the development of the pituitary proopiomelacortin (POMC) cells of the toad (Bufo japonicus)

The role of the posterior hypothalamus in the development of the epithelial hypophysis was studied in Bufo embryos. In animals from which the central part of the neural plate (NP) had been surgically removed at the open neurula stage, the infundibulum did not develop, and the epithelial hypophysis was formed away from the normal site without morphological connection with the brain. Immunoreactive MSH cells and ACTH cells, i.e, the pituitary POMC cells, were not detected in any of the surgically treated animals while other types of secretory cells (PRL, GH, TSH and GTH cells) were invariably present. In view of the fact that POMC cells originate in the anterior neural ridge, and not in the neural plate, the embryonic brain seems to exert an inductive influence upon the primordial pituitary POMC cells. Since these cells differentiate in a tail graft, isolated from the brain at a later stage (tail-bud stage), the inductive stimuli must be conveyed from/via the posterior hypothalamus to the pituitary anlage between the open neurula and the tail-bud stages.Abbreviations ACTH Adrenocorticotropic hormone - ANR anterior neural ridge - GH growth hormone - GTH gonadotropic hormone - MSH metanocyte stimulating hormone - NP central part of anterior neural plate - POMC proopiomelanocortin - PRL prolactin - TSH thyroid-stimulating hormone     

Carbendazim-induced abnormal development of the acrosome during early phases of spermiogenesis in the rat testis

Effects of a single, high dose of orally administered carbendazim (100 mg/kg) on acrosome formation in the early phases of spermiogenesis were examined by electron microscopy and immunocytochemistry up to day 7.5 post-treatment. No obvious abnormality of acrosome development was noted in the Golgi phase spermatids on day 1.5 post-treatment. On day 3, step 1 spermatids were seen in stage III seminiferous tubules. In stage V tubules at this post-treatment interval, direct connections between the trans-side saccules of the Golgi stacks and the outer acrosomic membranes were observed in step 5 spermatids. Similar direct connections between these two organelles were also observed in the advanced round spermatids in later stages at days 4.5 and 7.5. On day 4.5, step 1 and 3 spermatids were seen in stage V tubules. On day 7.5, round spermatids with various abnormalities of acrosome development were observed in stage VII tubules, in addition to the discontinuous and granular acrosomes reported previously. These features were not observed in testes of control animals. In the immunocytochemical analysis using an antibody mMN7 that recognizes a protein delivered from the Golgi apparatus to the acrosome, spermatids exposed to carbendazim showed various abnormal immunostaining patterns in the acrosomes. On the other hand, strong immunoreactivity was observed in the Golgi saccules connecting to the acrosomes. These results suggest that in testis treated with carbendazim acrosome development is impaired during the early phases of spermiogenesis, and material supply from the Golgi apparatus to the acrosome is perturbed, which is a possible cause of the abnormal development. Received: 31 March 1998 / Accepted: 28 May 1998     

The early stages of ommatidial development in the flour beetle Tribolium castaneum (Coleoptera; Tenebrionidae)

 Using electron microscopy, the first stages of ommatidial development in the flour beetle Tribolium castaneum were analysed in relation to the cellular architecture of the adult compound eye and were compared to the corresponding patterning process in the fruit fly Drosophila melanogaster. The ommatidia of the slightly horse-shoe shaped beetle compound eye contain six peripheral and two central retinula cells. The rhabdomere of the posteriorly located central photoreceptor cell is restricted to the distal half of the rhabdom whilst that of the anterior one is restricted to its proximal half. The development of the compound eye takes place in an external eye imaginal disc. Most stages of ommatidial development, as known from Drosophila, i.e. arc-like cell groups, five-cell clusters, immature eight-cell clusters and symmetrical eight-cell clusters, are very precisely conserved between the two species. Two major differences exist: 1. In Tribolium, the cone cell precursor cells synchronously join to the immature eight-cell cluster. As a consequence, the symmetrical eight-cell cluster immediately transforms into a four-cone-cell cluster. 2. The maturing ommatidia do not undergo rotation in Tribolium. Overall, no morphological indiation for an equator in the adult Tribolium compound eye could be found. Considering the strong evolutionary conservation of early ommatidial development, homology of photoreceptor cells of distantly related insects is proposed to be inferred from their ontogenetic origin. Received: 6 November 1995 / Accepted: 9 April 1996     

Identification and evolutionary analysis of putative cytoplasmic mcpA-like protein in a bacterial strain living in symbiosis with a mycorrhizal fungus

In this paper we report the identification and characterization of a DNA region containing putative mcpA-like gene coding for a Methyl Accepting Chemotaxis Protein (MCP) and belonging to a Burkholderia endosymbiont of the arbuscular mycorrhizal fungus Gigaspora margarita. A genomic library of total DNA extracted from the fungal spores, representative of the bacterial genome, was used to investigate the prokaryotic genome. PCR experiments with primers designed on the Burkholderia mcpA-like gene and Southern blot analysis demonstrate that they actually belong to the genome of G. margarita endosymbiont. The expression of the mcpA-like gene in the fungal spores was demonstrated by RT-PCR experiments. The detailed comparative analysis of the bacterial MCPs available in databases allowed to draw a possible evolutionary pathway leading to the present-day mcpA genes. Accordingly, the ancestor of the mcpA-like genes was the result of a domain shuffling event involving two ancestral mini-genes encoding a PAS-PAC and a MA domains, respectively, followed by the elongation of the PAS-PAC moiety. The following evolutionary divergence involved not only point mutations, but also larger rearrangements (insertions and deletions) at the 3′ end of the gene.     

Methodology for the study of the effect of drugs on development and DNA replication in Drosophila melanogaster embryonic tissue

Methodology is described that will permit the study of the effect of various drugs on development and DNA replication in the cleavage nuclei of Drosophila eggs. It is shown that permeabilized eggs can be exposed to an aqueous incubation medium for up to 30 min without measureable effects on development and that such incubations can be performed with eggs that have a relatively sharp age distribution. The effect on development and viability of a variety of drugs has been examined as an aid for future studies directed toward achieving synchronous development in a population of eggs and electron microscopic studies of DNA replication in the presence of various drugs.     

Chemical ligation and recombination of DNA fragments through formation (exchange) of disulfide bonds located in the sugar-phosphate backbone

Effective methods of the directed introduction of diphosphoryl disulfide bridges into hairpin DNA duplexes in place of natural phosphodiester groups were developed using the H₂O₂-effected ligation of 3′- and 5′-thiophosphorylated oligonucleotides or by autoligation of a preactivated oligonucleotide derivative with a phosphorothioate-bearing oligomer. The postsynthetic recombination of the disulfide-linked oligonucleotide fragments was characterized. It was shown that, along with template-directed reactions, out-of-duplex formation and exchange of diphosphoryl disulfide bonds in the DNA sugar-phosphate backbone may occur. In modified hairpin DNA, a spontaneous exchange of disulfide-linked fragments virtually does not take place because of the intramolecular duplex formation.     

The effect of meteorological factors on the daily variation of airborne fungal spores in Granada (southern Spain)

A study was made of the link between climatic factors and the daily content of certain fungal spores in the atmosphere of the city of Granada in 1994. Sampling was carried out with a Burkard 7-day-recording spore trap. The spores analysed corresponded to the taxa Alternaria, Ustilago and Cladosporium, with two morphologically different spore types in the latter genus, cladosporioides and herbarum. These spores were selected both for their allergenic capacity and for the high level of their presence in the atmosphere, particularly during the spring and autumn. The spores of Cladosporium were the most abundant (93.82% of the total spores identified). The Spearman correlation coefficients between the spore concentrations studied and the meteorological parameters show different indices depending on the taxon being analysed. Alternaria and Cladosporium are significantly correlated with temperature and hours of sunlight, while Ustilago shows positive correlation indices with relative humidity and negative indices with wind speed. Received: 16 April 1998 / Revised: 27 September 1999 / Accepted: 27 October 1999