Second Half Of Embryogenesis Research Articles

Ontogenetic features of redox reactions in the myocardium of geese

Under conditions of physiological stress of different aetiologies, cells of any organism undergo compensatory metabolic changes. The specificity of such changes primarily depends on functional and histochemical features of the tissues of the body. The purpose of this work was to describe the functioning of the main components of the oxidation-reduction system, (ORS, the Krebs cycle, and the antioxidant system) in the myocardium of geese under conditions of the physiological stress occurring during the transition from the embryonic to postnatal development. It has been experimentally established that in the second half of geese embryogenesis, the content of short-chain fatty acids is significantly increased and the content of long-chain polyunsaturated fatty acids in their myocardium decreases. During the first two weeks of the postnatal period, there are reversible changes in the fatty acid composition of this tissue. The second half of the embryogenesis is characterized by the activation of dehydrogenases of the Krebs cycle in the low variability of antioxidant enzyme activity. At the same time, the experiment demonstrated an increase in the antioxidant activity coefficient, with the exception of its significant reduction on the first day of postnatal ontogenesis. The functioning of the ORS in the myocardium of geese is characterized by the presence of correlations between the nature of the changes of 19 investigated components (dehydrogenases of the Krebs cycle, antioxidant enzymes, lipid peroxidation products, antioxidant activity coefficient, fatty acids, their total content and unsaturation) and structuring of the system into two clusters.

Reactivity of Microvessels in the Cerebral Hemispheres and Skeletal Muscles in Chicks during the Second Half of Embryogenesis

At Leghorn hens in the second half of embryogenesis and in 4-day-old chicks are studied reaction volume flow velocity (VF) in the superficial layers of the cerebral hemispheres and in skeletal muscle (Lazer Doppler FIowmetry) after the local influence of norepinephrine and sodium nitroprusside. It is shown that the response to these substances begins to manifest itself in the hemisphere in the last quarter of embryogenesis and authentically expressed by the end of it and in the chickens. It is noted that the response to these substances skeletal muscle VF (according to the new and previously published data on gastrocnemius and pectoral muscle) is also clearly manifested by the end of embryogenesis.

Tissue oxygen partial pressure in organs of chickens in the second half of embryogenesis and first days after hatching

The aim of this study is to measure the oxygen partial pressure (pO2) in developing chicken tissues, namely, in the cerebral hemispheres, liver, m. pectoralis, and m. gastrocnemius, and to estimate the correlation of pO2 with the earlier measured values (laser Doppler flowmetry) of volume blood flow (BF) in these organs. We have studied 10-, 15-, and 19-day-old embryos and 4-day-old chickens anesthetized with urethane. The pO2 has been measured in the surface layers of organs with a membrane amperometric Clark-type O2 electrode (cathode diameter of approximately 50 μm) placed in the center of the sensor unit (outer diameter of 3.4 mm). Noticeable distinctions between both the tissue pO2 values in different organs and the dynamics of their changes during the observation time have been recorded. The following differences are the most important: (1) the lowest pO2} {cm(and BF) is observed in the brain and, especially, in the liver of 10-day-old embryos; (2) in the subsequent period of embryogenesis, the pO2 in the brain increases 1.9-fold (BF also increases), falls 1.7-fold in m. pectoralis, and displays minor changes in the liver and m. gastrocnemius on the background of constant BF value in the liver and both muscles; and (3) after hatching, pO2 in the liver and m. pectoralis increases severalfold (BF increases too) but does not change in a statistically significant manner in the brain and m. gastrocnemius despite an increase in BF (more pronouncedly in the muscle). Two possible mechanisms underlying the changes in the tissue pO2 in developing chicken organs have been proposed: one is determined by the specific features of intracardiac blood flows and the other is associated with the oxyhemoglobin dissociation pattern in the blood capillary circulation in the organs, determined by the specific features in its oxidative metabolism.

Visualizing late insect embryogenesis: extraembryonic and mesodermal enhancer trap expression in the beetle Tribolium castaneum.

The beetle Tribolium castaneum has increasingly become a powerful model for comparative research on insect development. One recent resource is a collection of piggyBac transposon-based enhancer trap lines. Here, we provide a detailed analysis of three selected lines and demonstrate their value for investigations in the second half of embryogenesis, which has thus far lagged behind research on early stages. Two lines, G12424 and KT650, show enhanced green fluorescent protein (EGFP) expression throughout the extraembryonic serosal tissue and in a few discrete embryonic domains. Intriguingly, both lines show for the first time a degree of regionalization within the mature serosa. However, their expression profiles illuminate distinct aspects of serosal biology: G12424 tracks the tissue’s rapid maturation while KT650 expression likely reflects ongoing physiological processes. The third line, G04609, is stably expressed in mesodermal domains, including segmental muscles and the heart. Genomic mapping followed by in situ hybridization for genes near to the G04609 insertion site suggests that the transposon has trapped enhancer information for the Tribolium orthologue of midline (Tc-mid). Altogether, our analyses provide the first live imaging, long-term characterizations of enhancer traps from this collection. We show that EGFP expression is readily detected, including in heterozygote crosses that permit the simultaneous visualization of multiple tissue types. The tissue specificity provides live, endogenous marker gene expression at key developmental stages that are inaccessible for whole mount staining. Furthermore, the nonlocalized EGFP in these lines illuminates both the nucleus and cytoplasm, providing cellular resolution for morphogenesis research on processes such as dorsal closure and heart formation. In future work, identification of regulatory regions driving these enhancer traps will deepen our understanding of late developmental control, including in the extraembryonic domain, which is a hallmark of insect development but which is not yet well understood.

Aorta, pulmonary artery, and blood flows on them in chickens in the second half of embryogenesis and after hatching

The aim of the study was to determine changes in the blood flow in arterial trunks (coming out of the heart of chickens) by changes of the lumen of these arteries during embryogenesis (on the 10th, 15th, and 19th days) and 6 days after the hatching. For this purpose, posthumous morphometry of aorta, pulmonary arteries, and arterial (Botallo's) ducts (AD) from their exit from the heart until final extraorgan branching was conducted. It was demonstrated that, in this period, (1) initial lumens of aorta and pulmonary arteries are equal to each other and are equally increased (with temporary stop in last quarter of embryogenesis) with an increase of the body weight (BW); (2) the portion of the right ventricle in a total blood circulation minute volume (BCMV) is somewhat smaller than the portion of the left ventricle, but it approaches equality to it by the end of embryogenesis; (3) with the growth of embryos, the portion of total BCMV flowing through the anterior (before the inflow of AD into the aorta) part of the body decreases; that in the average part increases (from 17 to 31%); that in the posterior part (after bifurcation of aorta), where chorioallantoic membrane (CAM) is located, remains almost unchanged; (4) after the hatching (and disappearance of CAM), BCMV of the left ventricle multiply increases due to the junction of two blood flows from the heart (through the ascending aorta and AD) into a single flow, which flows sequentially by lesser and greater circulations, resulting in multiple increase in the organ blood flow.

Localization of apoptotic and proliferating cells and mRNA expression of caspases and Bcl-2 in gonads of chicken embryos

The aim of the present study was to analyze participation of apoptosis and proliferation in gonadal development in the chicken embryo by: (1) localization of apoptotic (TUNEL) and proliferating (PCNA immunoassay) cells in male and female gonads and (2) examination of mRNA expression (RT-PCR) of caspase-3, caspase-6 and Bcl-2 in the ovary and testis during the second half of embryogenesis and in newly hatched chickens. Apoptotic cells were found in gonads of both sexes. At E18 the percentage of apoptotic cells (the apoptotic index, AI) in the ovarian medulla and the testis was lower (p<0.05) than in the ovarian cortex. In the ovarian medulla, the AI at E18 was lower (p<0.05) than on E12. In the testis, the AI was significantly lower (p<0.05) at E18 than at E15 and 1D. The percentage of proliferating cells (the proliferation index: PI) within the ovary significantly increased from E15 to 1D in the cortex, while proliferating cells in the medulla were detected only at E15. In the testis, the PI gradually increased from E12 to 1D. The mRNA expression of caspase-3 and -6 as well as Bcl-2 was detected in male and female gonads at days 12 (E12), 15 (E15) and 18 (E18) of embryogenesis and the day after hatching (1D). The expression of all analyzed genes on E12 was significantly higher (p<0.05) in female than in male gonads. This difference was also observed at E15 and E18, but only for the caspase-6. The results obtained showed tissue- and sex-dependent differences in the number of apoptotic and proliferating cells as well as mRNA expression of caspase-3, -6 and Bcl-2 genes in the gonads of chicken embryos. Significant increase in the number of proliferating cells in the ovarian cortex and lack of these cells in the ovarian medulla (stages E12, E18, 1D) simultaneous with decrease in the intensity of apoptosis only in the medulla indicates that proliferation is the dominant process involved in the cortical development, which constitutes the majority of the functional structure of the fully developed ovary. No pronounced changes in the expression of apoptosis-related genes found during embryogenesis suggest that they cannot be considered as important indicators of gonad development. The molecular mechanisms of the regulation of balance between apoptosis and proliferation in developing avian gonads need to be further investigated.

Course of hatch and developmental changes in thyroid hormone concentration in blood of chicken embryo following in ovo riboflavin supplementation

The influence of riboflavin on the function of the hypothalamo-pituitary-thyroid axis during chicken embryogenesis is poorly understood. Therefore, examination of the effects of in ovo riboflavin supplementation on the possible linkage between the thyroid gland function and chick embryonic development seems to be interesting. Eggs on the sixth day of incubation were injected with 0 (control), 60, or 600 µg of riboflavin/egg. Blood samples were collected on the 12th, 15th, 18th, and 20th days of embryogenesis. Thyroxine (T4) and triiodothyronine (T3) concentrations in plasma samples were determined by radioimmunoassay method. The time of external pipping and hatching of each chick as well as the body weights of sampled and newly hatched chicks were recorded. Generally, riboflavin supplied in ovo did not affect chicken embryo mortality and hatchability; however, a dose of 600 µg of riboflavin/egg had a tendency to reduce embryo body weight. Chicks exposed to 60 µg of riboflavin/egg hatched 3.7 h earlier in comparison with controls and were characterized by a higher synchronization degree of hatching. Both applied doses of riboflavin significantly elevated T4 concentrations in blood plasma of the chicken embryos; however, on day 20 of embryogenesis, both applied doses of riboflavin decreased T3 levels in blood circulation. The data presented here suggest that riboflavin supplementation at the early stages of embryogenesis markedly affects embryonic development and influences thyroid hormone metabolism during the second half of embryogenesis.

The role of the formin gene fhod-1 in C. elegans embryonic morphogenesis.

During the second half of embryogenesis, the ellipsoidal Caenorhabditis elegans embryo elongates into a long, thin worm. This elongation requires a highly organized cytoskeleton composed of actin microfilaments, microtubules and intermediate filaments throughout the epidermis of the embryo. This architecture allows the embryonic epidermal cells to undergo a smooth muscle-like actin/myosin-based contraction that is redundantly controlled by LET- 502/Rho kinase and MEL-11/myosin phosphatase in one pathway and FEM-2/PP2c phosphatase and PAK-1/p21-activated kinase in a parallel pathway(s). Although actin microfilaments surround the embryo, the force for contraction is generated mainly in the lateral (seam) epidermal cells whose actin microfilaments appear qualitatively different from those in their dorsal/ventral neighbors. We have identified FHOD-1, a formin family actin nucleator, which acts in the lateral epidermis. fhod-1 mutants show microfilament defects in the embryonic lateral epidermal cells and FHOD-1 protein is detected only in those cells. fhod-1 genetic interactions with let-502, mel-11, fem-2 and pak-1 indicate that fhod-1 preferentially regulates those microfilaments acting with let-502 and mel-11, and in parallel to fem-2 and pak-1. Thus, FHOD-1 may contribute to the qualitative differences in microfilaments found in the contractile lateral epidermal cells and their non-contractile dorsal and ventral neighbors. Different microfilament populations may be involved in the different contractile pathways.

Effects of nonsteroidal ecdysone agonist RH-5992 and chitin biosynthesis inhibitor lufenuron on Spodoptera littoralis (Boisduval, 1833)

Abstract Comparative studies of the effects of two compounds, tebufenozide (an ecdysone agonist) and lufenuron (an insect growth regulator inhibiting chitin synthesis), were conducted on Spodoptera littoralis (Boisduval, 1833). The compounds, orally administered, caused larval mortality proportional to the concentrations in the food source. Tebufenozide initiated precocious molting, and lufenuron, and inhibited chitin synthesis. In both cases, larvae were unable to complete the molting process and died in the old larval cuticle. Larvae contaminated by sublethal doses completed their development to adulthood. Lufenuron is more active than is tebufenozide. LD-50 for lufenuron is 0.0001ppm and for tebufenozide 0.001ppm. Topical application of the test compounds to eggs caused dose- and agedependent inhibition of embryonic development. Application of tebufenozide in the second half of embryogenesis caused precocious molting of eclosed larvae of the 1st instar. Some morphological changes in the process of larval-pupal transformation were also observed. Tested compounds also reduced reproduction in adult individuals that had been treated by the tested compounds in the larval stage.

Effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on steroid concentrations in blood and gonads of chicken embryo

The aim of the present study was to examine the effect of TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) injected at the early stage of incubation on hormonal activity of the chicken ovary and testis during the second half of embryogenesis measured by steroid concentrations in blood plasma and gonads. The effect of TCDD on hatchability was also evaluated. In the first experiment TCDD was administrated to chicken eggs on day 7 of embryogenesis (E7) at doses of 0 (control), 2.5, 5 and 10ng/egg dissolved in 50μl of peanut oil. Blood plasma for estradiol (E2) and testosterone (T) determination was collected from embryos at E14, E18, E20 and at hatching (D1). In the second experiment TCDD at doses of 0 (control), 2.5, 5 and 10ng/egg dissolved in 50μl of DMSO was injected on E6. Blood plasma and gonads were collected on the day of hatching for progesterone (P4), T and E2 concentrations measured by means of the RIA method. It was found that TCDD injection: (1) increased the level of T and decreased the level of E2 in the plasma of female chicken embryos during the second half of embryogenesis, while on the other hand in male embryos the effect of TCDD was opposite, (2) increased T and E2 concentrations in plasma of newly hatched female chickens with a concomitant decrease of these hormones in the ovary; P4 in the ovary was elevated by TCDD, (3) decreased P4 and T in plasma and testes, whereas increased E2 concentration in the plasma of newly hatched male embryos (E2 concentration in the testes was below the sensitivity of the applied RIA method), and (4) decreased hatchability at all examined doses. The results obtained clearly demonstrate that exposure to TCDD at an early stage of embryogenesis affects steroid production and secretion by chicken gonads. The effect of TCDD depends on (1) the applied dose, (2) the day of embryogenesis, and (3) the sex. It is suggested that dioxins are potent modulators of the process of steroidogenesis in the chicken gonads. Moreover, the data obtained indicate that dioxins exert an effect on embryo development and the hatching process.

Ontogenic characterization of gene expression in the developing neuroendocrine system of the chick

The neuroendocrine system consists of five major hypothalamic-pituitary hormone axes that regulate several important metabolic processes, and it develops in all vertebrates during embryogenesis. In order to define initiation and establishment of these five axes, mRNA expression profiles of hypothalamic releasing and release-inhibiting factors, their pituitary receptors, and pituitary hormones were characterized during the second half of embryogenesis and first week post-hatch in the chick. Axis initiation was defined as the age when pituitary hormone mRNA levels began to increase substantially, and establishment was defined as the age when mRNA for all components had reached maximum expression levels. The adrenocorticotropic axis appears established by e12, as there were no major increases in gene expression after that age. Hypothalamic thyrotropin-releasing hormone and pituitary thyroid-stimulating hormone β-subunit increased between e10 and e18, indicating establishment of the thyrotropic axis during this period. Pituitary growth hormone substantially increased on e16, and hypothalamic growth hormone-releasing hormone did not increase until e20, indicating that somatotropic axis activity is established late in embryonic development. Lactotropic axis initiation is evident just prior to hatch, as pituitary prolactin and vasoactive intestinal peptide receptor 1 did not increase until e18 and e20, respectively. Hypothalamic gonadotropin-releasing hormone 1 increased after hatch, and pituitary luteinizing hormone β-subunit expression remained low until d3, indicating the gonadotropic axis is not fully functional until after hatching. This study is the first to characterize major hypothalamic and pituitary components of all five neuroendocrine axes simultaneously and considerably increases our understanding of neuroendocrine system establishment during development.

Circulatory system in chicken skeletal muscle in the second half of embryogenesis: Shape, blood flow, and vascular reactivity

A restructuring of the capillary bed-from the embryonic structure with a three-dimensional network of wide and long protocapillaries to the mature structure with high density of thin and short capillaries along the fibers-has been demonstrated in the chick skeletal muscle on embryonic days 10-19 by morphometric analysis. In this case, the volumetric flow rate and capillary luminal area per cm3 of the muscle remained unaltered, while the blood volume in it significantly dropped. The response of muscle circulation to nitroprusside (increase) and noradrenaline (decrease) appeared in 19-day-old embryos, but this response could develop only under conditions of initially low or high bloodstream, respectively. We propose that the arterial trunk lumen area to the total capillary lumen area remains constant as the intraorganic circulation is formed, which provides for the required linear blood velocity in capillaries.

Effect of hypoxia on amnion rhythmic contractions and heart rate in the embryonic European pond turtle Emys orbicularis (Reptilia: Emydidae)

The effect of acute hypoxia (10% O2 for 30 min) on the rate of amnion rhythmic contractions and heart rate (HR) was studied in two age groups of European pond turtle (Emys orbicularis) embryos, on days 19-27 and 37-43 of incubation (30-40 and 60-70% of the period until hatching). Under the control conditions, the two age groups of embryos did not differ from each other in either parameter. Hypoxia did not affect significantly the amnion contraction rate but decreased the HR. The time course of the HR during hypoxia depended on the embryo age. The mean HR in the first group of embryos was 8% decreased by minutes 10-14 of hypoxia and did not change afterwards; in the second group, it was 18% decreased by minutes 3-8 and then partly or completely restored before the end of hypoxic exposure. It has been assumed that the capacity of European pond turtle embryos for restoring the normal HR when exposed to acute hypoxia during the second half of embryogenesis is related to the development of neurohumoral control mechanisms.

On the content of protein and nucleic acids in organs of chick embryos of different age

In 10-, 14-and 18-days-old chick embryos, concentrations of total protein and nucleic acids (NA) were measured using spectrophotometry in liver, brain, red gastrocnemial and white pectoral muscles and in chorioallantois. It was shown that the initial protein and NA concentration was the highest in liver and decreased markedly in the above-mentioned organs. At the second half of embryogenesis, concentration of organ protein increased 4 times in muscles, 2 times in brain, and 1.4 times in liver. This indicates a rise of cell density in the organs or of the intracellular protein concentration. The protein/NA ratio reflecting the cell size or concentration of their protein practically did not change in liver, but increased 2.3 and 2.5 times in pectoral and gastrocnemial muscles, respectively, and 2 times in brain. In chorioallantois the maximal protein concentration was observed in then 14-day old embryos, with the protein/NA ratio remaining practically unchanged.

Effect of Temperature on the Rate of Oxygen Consumption during the Second Half of Embryonic and Early Postembryonic Development of European Pond Turtle Emys orbicularis (Reptilia: Emydidae)

Oxygen consumption by eggs of European pond turtle was determined at two constant incubation temperatures of 25 and 28 degrees C during the second half of embryogenesis. During development at both temperatures, the rate of oxygen consumption initially increased to remain constant during the last quarter of embryogenesis. The difference between the rates of oxygen consumption at these temperatures decreased during the studied period. The coefficient Q10 for the rate of oxygen consumption decreased from 9 to 1.7. At an incubation temperature of 28 degrees C, the changes in the rate of oxygen consumption in response to a short-term temperature decrease to 25 degrees C or increase to 30 degrees C depended on the developmental stage and were most pronounced at the beginning of the studied period. During late embryonic and first 2.5 months of postembryonic development, the rate of oxygen consumption did not significantly differ after such temperature changes. The regulatory mechanisms formed during embryonic development are proposed to maintain the level of oxygen consumption during temperature changes.

Embryonic expression pattern of the Drosophila Kallmann syndrome gene kal-1

The role of kal-1, the gene responsible for the X chromosome-linked form of Kallmann syndrome, is not well definite. In Drosophila, the kal-1 gene encodes a putative protein with the characteristic kal-1 topology but with only two Fibronectin-like type III (FnIII) domains. We studied the embryonic expression pattern of kal-1 using whole mount in situ hybridization. This gene is expressed in the second half of embryogenesis showing a complex and dynamic pattern. kal-1 is expressed during important morphogenetic processes such as germ band retraction, dorsal closure and head involution. We found expression in cells associated with different sensory organs, such as the antennal organ, which has an olfactory function, the chordotonal organ, the Keilin's organ and the dorsal pharyngeal organ. Expression of kal-1 in the head also regards some ectodermal cells of the gnathal lobes. By studying the expression in Dfd and cnc homeotic mutants, we found that these ectodermal cells derive from the anterior and posterior mandibular segment, whose determination depends on cnc, and that the expression in the posterior mandibular segment requires Dfd activity. kal-1 is also expressed in the posterior part of the male gonads in a specific subset of the somatic cells called male-specific somatic gonadal precursors (msSGPs). This is the first time that the expression of a kal-1 ortholog has been demonstrated to be sex specific making the kal-1 transcript a useful tool for the study of sex determination in the gonad.

Changes in the activities of aldolase and other enzymes for carbohydrate metabolism during embryonic and postembryonic development ofBombyx mori

Eggs at the early stages of embryogenesis and the larval fat body in Bombyx mori were confirmed to have an aldolase (ALD) isozyme type S. Its activity ratio with substrates fructose 1,6-bisphosphate (FBP) and fructose 1-phosphate (F1P) was 3. This isozyme was considered to be in favor of rather efficient utilization of F1P, since eggs in early stages of embryogenesis and the fat body had high activities of NADP-sorbitol dehydrogenase (NADP-SDH) and NAD-sorbitol dehydrogenase (NAD-SDH) responsible for the polyol pathway generating F1P. On the other hand, eggs at the second half of embryogenesis and the larval and adult muscle (plus epidermal cells and cuticle) possessed an ALD isozyme type F, whose FBP/F1P activity ratio was 10, suggesting that F1P utilization is less effective. This is in agreement with the fact that the NADP-SDH and NAD-SDH activities were low and the phosphofructokinase (PFK) activity was high in eggs at these stages and in muscle. Arch. Insect Biochem. Physiol. 36:139–148, 1997. © 1997 Wiley-Liss, Inc.

Developmental variability of metallothionein Mtn gene expression in the species of the Drosophila melanogaster subgroup

Developmental expression of the Drosophila melanogaster metallothionein Mtn gene has been analysed. Transcripts of this gene accumulate during the vitellogenic phase of oogenesis in a ring of follicular cells at the oocyte-nurse cell margin and in the follicular cells surrounding the oocyte. There is also strong expression of the Mtn gene during the second half of embryogenesis in hemocytes, the endoderm midgut, and Malpighian tubules. A banded expression pattern is observed transiently in the midgut at stage 13. The two Mtn alleles, Mtn and Mtn, show quantitative differences in their expression patterns. Copper intoxication of flies does not induce ectopic expression of the Mtn gene, but rather leads to over-expression of the gene in the structures where it is normally transcribed. Mtn transcription is not altered in homozygous mutants of four genes (lab, wg, dpp, bap) known to be involved in midgut morphogenesis. Expression of Mtn has been also studied in six other species of the melanogaster subgroup. This analysis demonstrates that regulation of Mtn gene transcription has changed during evolution of the Drosophila lineage. For example, Mtn is expressed specifically in the Malpighian tubules of D. melanogaster, while in D. mauritiana and D. sechellia the amnioserosa is a specific location of expression. Nonetheless, expression of Mtn in the midgut is common to the seven species, suggesting a basic role for the MTN protein during embryogenesis in this organ, possibly in the release of metallic ions from vitellogenins. In contrast, two genes also expressed in the embryonic midgut, lab and dFRA, display identical patterns in all species of the melanogaster subgroup. The diversity of Mtn patterns in closely related Drosophila species exemplifies the rapid evolution of a gene regulatory system.

Structure, expression and duplication of genes which encode phosphoglyceromutase of Drosophila melanogaster.

We report here the isolation and characterization of genes from Drosophila that encode the glycolytic enzyme phosphoglyceromutase (PGLYM). Two genomic regions have been isolated that have potential to encode PGLYM. Their cytogenetic localizations have been determined by in situ hybridization to salivary gland chromosomes. One gene, Pglym78, is found at 78A/B and the other, Pglym87, at 87B4,5 of the Drosophila polytene map. Pglym78 transcription follows a developmental pattern similar to other glycolytic genes in Drosophila, i.e., substantial maternal transcript deposited during oogenesis; a decline in abundance in the first half of embryogenesis; a subsequent increase in the second half of embryogenesis which continues throughout larval life; a decline in pupae and a second increase to a plateau in adults. This transcript has been mapped by cDNA and genomic sequence comparison, RNase protection, and primer extension. Using similar analyses transcripts of Pglym87 could not be detected. Pglym78 has two introns which interrupt the coding region, while the Pglym87 gene lacks introns. This and other features support a model of retrotransposition mediated gene duplication for the origin of Pglym87. The apparent absence of a complete, intact coding frame and transcript suggest that Pglym87 is a pseudogene. However, retention of reading frame and codon bias suggests that Pglym87 may retain coding function, or may have been inactivated recently, substantially after the time of duplication, or that the molecular evolution of Pglym87 is unusual. Similarities of the unusual molecular evolution of Pglym87 and other proposed pseudogenes are discussed.

Transcriptional and translational regulation of the expression of the l(2) gl tumor suppressor gene of Drosophila melanogaster

By structural, biochemical and molecular genetic analyses, we have investigated the different mechanisms that control the expression of the lethal(2) giant larvae gene, a tumor suppressor gene of Drosophila melanogaster. Transcription of the l(2) gl gene is controlled by two highly identical promoters that result from the duplication of the 2.8 kb proximal portion of the gene. These two repeats are 96% homologous. Reverse genetic analysis has shown that each promoter can drive gene expression. In addition to the promoters, both repeats express two or three exons according to the pattern of splicing. The most distal exon in the second repeat is required because it contains the ATG initiating codon at the beginning of the open reading frame. The 3′ untranslated region appears to contain motifs that specifically destabilize the transcript. Deletion of this region results in the formation of more stable mRNAs. The l(2) gl gene is characterized by an unusual codon usage that may reflect an enhanced translation efficiency by moderating the strength of pairing between codons and anticodons and may therefore increase the expressivity of this gene. Analysis of the spatio-temporal expression of the l(2) gl transcripts and proteins has shown that transcripts and proteins are produced ubiquitously during early embryogenesis, at a time when expression of the gene is required for preventing tumorigenesis. In the second half of embryogenesis, l(2) gl expression becomes restricted to tissues that do not show any phenotypic alteration in mutant animals. The l(2) gl protein exhibits two distinct intracellular localizations. It is preferentially found free in the cytoplasm but can become associated with the inner face of the plasma membrane where it is restricted to domains facing contiguous cells. In particular, the l(2) gl protein is absent from the basal and apical domains of the plasma membrane. The aim of the current research is directed towards understanding the functional relevance of the l(2) gl protein binding to the plasma membrane and its role in the control of cell proliferation and differentiation.