Early Step In Development Research Articles

Developmental regulation of V(D)J recombination and lymphocyte differentiation

Recent insights into the mechanism of V(D)J recombination have clarified the direct role of the products of the recombination-activating genes Rag-1 and Rag-2 in site-specific DNA cleavage at recombination signal sequences and have identified components of the general DNA double-strand break repair pathway that participate in the rejoining of the Rag-1 and Rag-2-cut receptor gene segments. The V(D)J reaction is restricted to particular antigen receptor loci in a lineage-specific and stage-specific manner. This specificity appears to involve cis-regulatory elements, some of which also regulate transcription of the germline antigen receptor loci. Early developmental steps in the T and B lineages — including phenotypic differentiation, expansion of precursors, and selection processes — are effected in a stepwise fashion by signals generated, at least in part, by the products of the functionally rearranged antigen receptor genes themselves.

Cell death in the midbrain of the murine mutation weaver

The midbrain of the adult homozygous weaver (wv/wv) mouse is notable for a reduction in the numbers of dopamine-containing cells in the substantia nigra (A9) and the retrorubral nucleus (A8). We have determined that the reduction in tyrosine hydroxylase (TH)-positive neurons in the ventral midbrain of the weaver is attributable to the loss of neurons after postnatal day 7 (P7). Because the number and spatial distribution of TH-positive mesencephalic neurons in wv/wv, heterozygous weavers (+/wv), and wild-type mice are not significantly different on P7, we conclude that the early developmental steps of proliferation and migration have taken place normally in the mutant. Although numbers and distribution of cells are normal in the wv/wv on P7, the appearance of the TH-stained ventral midbrain is abnormal because of the paucity of TH-stained dendritic processes. The ventrally extending TH-positive dendrites are largely absent in the young wv/wv. The wv/wv also can be distinguished from both homozygous normal (+/+) and wv/wv littermates on P7 based on the appearance of dendrites that are more numerous than in the wv/wv but thin, disorganized, and sparse compared with +/+. Most cell death seems to take place in wv/wv before P21. However, at least one subset of dopamine-containing neurons disappears later. The zone of densely packed TH-positive neurons in the substantia nigra that is likely to be the origin of innervation to striosomes in the caudoputamen disappears between P21 and adulthood. Despite the early pathology evident in the mesencephalic dopamine-producing neurons of the +/wv, no evidence for cell death was observed there even in the oldest +/wv weavers studied.

Distribution and possible role of perinuclear theca proteins during bovine spermiogenesis

The perinuclear theca (PT) is a unique cytoskeletal element that encapsulates the mammalian sperm nucleus. It is divided into subacrosomal and postacrosomal regions. The objective of this study was to analyze and stage the intracellular distribution of several prominent bull PT proteins during spermatogenesis. For this purpose, polyclonal antibodies raised and affinity-purified against the 15.5-, 25-, 28-, 32-, 36-, and 60-kDa bull PT polypeptides were used as probes on sections of aldehyde-fixed testes. Immunoperoxidase staining revealed that the PT polypeptides first appeared early in spermiogenesis, concomitant with early steps of development of the acrosomic system. Immunogold labeling further showed that these polypeptides were peripherally associated with the entire acrosomal membrane, before and during the attachment of the acrosomic vesicle onto the spermatid's nucleus. Once the acrosome had capped the nucleus the labeling resided mainly in the subacrosomal region of the spermatid, between the inner acrosomal membrane and nuclear envelope. Later, during the elongation of the spermatid's nucleus, the labeling with all antibodies except the anti-15.5-kDa antibody extended caudally over the assembling postacrosomal sheath. This study suggests that the perinuclear theca proteins play an instrumental role in the attachment, spreading, and binding of the acrosome onto the nucleus of spermatids.

Anterior Duplication of the Sonic hedgehog Expression Pattern in the Pectoral Fin Buds of Zebrafish Treated with Retinoic Acid

The Sonic hedgehog gene has been identified as a candidate for the signal mediating the function of the zone of polarizing activity (ZPA) during limb development in tetrapods. To better understand the early steps of development of paired fin buds in fish, we have analyzed the regulation of the zebrafish Sonic hedgehog gene ( shh/vhh-1) in response to retinoic acid. Systemic administration of retinoic acid (RA) to zebrafish embryos during the initial stages of pectoral fin bud development resulted in the induction of ectopic expression of shh/vhh-1 on the anterior margin of the bud under the apical ectodermal ridge and in abnormal pectoral fin bud morphology. RA treatment also resulted in ectopic shh/vhh-1 expression in floor plate cells at the caudal end of the neural keel. These results suggest that the control of ZPA function during the initial stages of development of paired appendages has been conserved between fish and tetrapods.

Flow cytometric analysis of early steps in development of adriamycin resistance in a human gastric cancer cell line.

We have established a low‐level adriamycin (ADM)‐resistant human gastric cancer cell line (MKN45R) from the parental cell line (MKN45) by exposure to step wise increases of ADM concentration (final concentration, 0.026 μg/ml). The purpose of this study was to identify the early steps in the development of ADM resistance in MKN45R by flow cytometric (FCM) analysis. Comparison of the concentration required for 50% growth inhibition, determined by a tetrazolium‐based colorimetric assay, showed that MKN45R was about 2.6‐fold more resistant to ADM than MKN45. However, the inhibition index values were 89.5% for MKN45 and 86.4% for MKN4SR, respectively, showing that ADM was judged to be “effective” against both cell lines. On the other hand, cell kinetic analysis by FCM revealed that the increase of the ratio of G2M accumulation induced by ADM treatment was significantly lower (p0.01) in MKN45R. Moreover, the efflux of ADM estimated by FCM analysis was significantly increased (P<0.05) in MKN45R even though there was no significant increase of P‐glycoprotein expression. These results suggest that although ADM was still effective based on a standard drug sensitivity test, the cancer cells were already acquiring resistance to ADM as judged from FCM analysis. Moreover, the mechanism of this ADM resistance is considered to be independent of P‐glycoprotein expression. Thus, FCM analysis is useful for detecting the early steps in the development of drug resistance of cancer cells.

Dynamic expression of the murine Achaete-Scute homologue Mash-1 in the developing nervous system

The Drosophila Achaete-Scute Complex genes encode transcriptional regulators belonging to the basic-helix-loop-helix family which control early steps of development of the central and peripheral nervous systems. We have isolated two mouse homologues of Achaete-Scute Complex genes, Mash-1 and Mash-2, by using the conservation of the basic-helix-loop-helix domain in this family. In this article, we analyse the expression of Mash-1 from its onset during neurulation to adult stages by RNA in situ hybridization on whole mounts and sections. As was observed for the rat Mash-1 protein, mouse Mash-1 RNA expression is restricted to cells of the developing central and peripheral nervous systems. We have observed three successive phases in the distribution of Mash-1 transcripts in the developing central nervous system. Initially, between embryonic day 8.5 and 10.5, Mash-1 transcripts are found in restricted domains in the neuroepithelium of the midbrain and ventral forebrain, as well as in the spinal cord. Between embryonic day 10.5 and 12.5, Mash-1 expression pattern changes from a restricted to a widespread one. Mash-1 transcripts are then found at variable levels in the ventricular zone in all regions of the brain. From embryonic day 12.5 to post-natal stages, Mash-1 is also expressed in cells outside of the ventricular zone throughout the brain. In addition, Mash-1 is expressed during development of the olfactory epithelium and neural retina. Overall, its expression pattern suggest that Mash-1 plays a role at early stages of development of specific neural lineages in most regions of the central nervous system and of several lineages in the peripheral nervous system. We have also compared the expression of Mash-1 and mouse Notch because their Drosophila homologues have been shown to interact genetically. The two genes show very similar expression patterns, both spatially and temporally, in the early developing brain and in the retina, suggesting that both genes may participate in the development of the same neural lineages.

Sulfation is required for mobility of veliger larvae of Concholepas concholepas (Mollusca; Gastropoda; Muricidae)

AbstractThe sulfation reaction seems to be a critical biochemical process during early steps of development. We have evaluated the effect of sulfation on the mobility of veliger larvae of the gastropod Concholepas concholepas. It was found that incubation of larvae in low‐sulfate artificial sea water had strong inhibitory effect on mobility. The use of sodium chlorate, a specific inhibitor of sulfation, also resulted in a strong inhibition of larval mobility. At the biochemical level, the synthesis of proteoglycans (PGs) and detergent‐soluble sulfoproteins and sulfolipids was specifically inhibited by chlorate, without affecting either total protein synthesis or phosphorylation. Intracellular levels of the sulfate donor 3′‐phosphoadenosine 5′‐phosphosulfate (PAPS) were decreased to 4% by chlorate treatment, indicating that this molecule is also involved in sulfation of marine invertebrates. Both effects of chlorate, the inhibition of sulfation and the larval mobility, were reversible. It is therefore concluded that sulfation is required for larval mobility in the mollusc C. concholepas. © 1992 Wiley‐Liss, Inc.

Interspecific hybridization between an anural and urodele ascidian: Differential expression of urodele features suggests multiple mechanisms control anural development

Anural development in the ascidian Molgula occulta was examined using tissue-specific markers and interspecific hybridization. Unlike most ascidians, which develop into a swimming tadpole larva (urodele development), M. occulta eggs develop into a tailless slug-like larva (anural development) which metamorphoses into an adult. M. occulta embryos show conventional early cleavage patterns, gastrulation, and neurulation, but then diverge from the urodele developmental mode during larval morphogenesis. M. occulta larvae do not contain a pigmented sensory cell in their brain or form a tail with differentiated notochord and muscle cells. As shown by in situ hybridization with cloned probes and analysis of in vitro translation products, M. occulta embryos do not accumulate high levels of α actin or myosin heavy chain mRNA. In contrast, acetylcholinesterase is expressed in muscle lineage cells, indicating that various muscle cell features are differentially suppressed. M. occulta embryos also lack tyrosinase activity, suggesting that suppression of brain pigment cell differentiation occurs at an early step in development. M. occulta eggs fertilized with sperm from Molgula oculata (a closely related urodele species) develop into hybrid larvae exhibiting some of the missing urodele features. Some hybrid embryos develop tyrosinase activity and differentiate a brain pigment cell and a short row of notochord cells, and form a short tail. These urodele features appeared together or separately in different hybrid embryos suggesting that they develop by independent mechanisms. In contrast, α actin and myosin heavy chain mRNA accumulation was not enhanced in hybrid embryos. These results suggest that multiple mechanisms control anural development.

Expression of the homeobox Chick-en gene in chick/quail chimeras with inverted mes-metencephalic grafts

The homeobox gene Chick-en, sharing homologies to the engrailed gene of Drosophila, is expressed, during early steps of development, in a restricted area of the chick embryo including mes-metencephalic neuroepithelia. The expression of the Chick-en gene has been analyzed in chick/quail chimeric embryos in which a portion of the 2-day-old mes-metencephalic neuroepithelium has been transplanted in an inverted position. By means of a monoclonal antibody, “Mab 4D9,” recognizing engrailed proteins, it is shown that the expression of the Chick-en gene is regulated in the inverted neuroepithelium according to its new position in the host neural tube. The regulation takes place within 20 hr after transplantation. These results, together with previous data demonstrating that the phenotypic expression of the inverted neuroepithelium depends, also, on its new position in the host neural tube, strongly suggest that the engrailed protein could play an important role in the positional specification of the mes-metencephalic neuroepithelium.

Effects on ontogenesis of Carausius morosus hit by cosmic heavy ions.

Among the biological problems that arise in long duration spaceflights, the effects of weightlessness and ionizing radiation appear to be the two main risk factors. Eggs of the stick insect Carausius morosus were exposed to spaceflight conditions during the 12.56 day Biosatellite mission Cosmos 1887. Five different ages were used, representing different sensitivities to radiation and different capacities for regeneration. During spaceflight the eggs continued their development. Already, in the Spacelab D1 mission in 1985, it has been shown that microgravity leads to a reduced hatching rate of eggs exposed during the early steps of development. When the eggs were hit by a heavy ion, a further but not significant reduction of the hatching rate was observed. Hatching was normal for eggs which were exposed on a 1 g reference centrifuge in space. Heavy ion hits caused body anomalies. The combined action of heavy ions and microgravity resulted in an unexpectedly high rate of anomalies. In the experiment on Cosmos 1887 these results were confirmed. Studies on the embryonic development before hatching showed no major difference between flight and ground control specimen, neither in speed of development nor in morphological anomalies. Hatching therefore seems to be the critical point in insect ontogenesis.

The spoIIJ gene, which regulates early developmental steps in Bacillus subtilis, belongs to a class of environmentally responsive genes

The Bacillus subtilis spoIIJ locus is defined by a Tn917 insertion which leads to an oligosporogenous phenotype. Here we show that this mutation severely decreases transcription of spoIIA, spoIIE, and spoIIG, three operons involved in asymmetric septation, the earliest morphological event of sporulation. A 14.3-kilobase region overlapping the site of the spoIIJ::Tn917 insertion was cloned and the exact location of the spoIIJ gene was defined with various integrative plasmids carrying subfragments of that region. DNA sequencing established that spoIIJ is a monocistronic locus encoding a 606-amino-acid polypeptide which contains a canonical "transmitter" domain, indicating that spoIIJ is a new member of the "sensor" class of signal-transducing systems in bacteria. Thus, spoIIj, which is transcribed during vegetative growth, presumably under the control of sigma H, encodes a protein that could interact with major regulators of early sporulation stages, such as SpoOA and/or SpoOF.

2,4-Dichlorophenoxyacetic acid (2,4-D) reduces acetylcholinesterase activity in rat muscle

A single dose (200 mg/kg body weight, i.p.) of 2,4-dichlorophenoxyacetic acid (2,4-D), commonly used as a herbicide, caused significant decreases in acetylcholinesterase (AChE) activity in diaphragm and other muscles of the rat. The 4S, 10S, and 16S forms of AChE were affected. The effect was maximal 15 to 24 h after injection. Choline acetyltransferase (CAT) activity was not affected. Neither AChE nor CAT activities changed in sciatic nerve from 2,4-D-treated animals. Spontaneous locomotor activity decreased dramatically 4 h after 2,4-D treatment. Myotonia that was present 1.5 h after 2,4-D injection became maximal at 2 to 6 h. Twenty-four hours after drug injection, when animals were recovering from myotonia, spontaneous locomotor activity was still depressed to 50% of control values. Prolonged distal motor latencies were observed 15 to 24 h after drug administration. AChE activity and spontaneous locomotor activity returned to control values at 48 h. Thus, 2,4-D causes a decrement of end-plate AChE, as well as behavioral and electrophysiologic changes. Decreased activity of AChE may be an early step in development of the myopathy that occurs after large dose 2,4-D.

Postnatal development of mevalonate phosphorylation in the chick brain and kidney.

The quantitative role of the brain and the kidney of the chick the phosphorylation of mevalonic acid was studied in the early steps of development. In both tissues no significant differences were found in the amount of phosphomevalonic acid formed until 4 days, but a sharp decrease between 4 and 6 days was observed. The amount of pyrophosphomevalonic acid formed showed some change with age, but this change was relatively small when compared with the changes in the phosphomevalonic acid formed. According to the function of the brain and the kidney in the sterol and nonsterol pathways in the metabolism of mevalonic acid during neonatal development of chick, an important role for the mevalonate kinase during myelination can be suggested.

Mevalonate phosphorylation in the neonatal chick liver

The phosphorylation of mevalonic acid by cell-free extracts from chick liver in the early steps of development was studied. A clear difference in the pH-activity profiles for phosphomevalonate and pyrophosphomevalonate formation has been observed. the amount of phosphomevalonate formed is quite similar at pH 7.5–9.5 whereas the pyrophosphomevalonate shows a clear maximum at pH 9.5. The pattern of mevalonate phosphorylation during the neonatal development shows no significant difference between 1–6 days after hatching, but a significant increase in the amount of phosphomevalonate formed at day 7 after hatching.

Mössbauer studies of ferro- and ferri-cyanide super complexes with 3d-transition elements

The homeobox gene Chick-en, sharing homologies to the engrailed gene of Drosophila, is expressed, during early steps of development, in a restricted area of the chick embryo including mes-metencephalic neuroepithelia. The expression of the Chick-en gene has been analyzed in chick/quail chimeric embryos in which a portion of the 2-day-old mes-metencephalic neuroepithelium has been transplanted in an inverted position. By means of a monoclonal antibody, “Mab 4D9,” recognizing engrailed proteins, it is shown that the expression of the Chick-en gene is regulated in the inverted neuroepithelium according to its new position in the host neural tube. The regulation takes place within 20 hr after transplantation. These results, together with previous data demonstrating that the phenotypic expression of the inverted neuroepithelium depends, also, on its new position in the host neural tube, strongly suggest that the engrailed protein could play an important role in the positional specification of the mes-metencephalic neuroepithelium.

Pressure vessel for elastic moduli determinations with results for fused quartz

The homeobox gene Chick-en, sharing homologies to the engrailed gene of Drosophila, is expressed, during early steps of development, in a restricted area of the chick embryo including mes-metencephalic neuroepithelia. The expression of the Chick-en gene has been analyzed in chick/quail chimeric embryos in which a portion of the 2-day-old mes-metencephalic neuroepithelium has been transplanted in an inverted position. By means of a monoclonal antibody, “Mab 4D9,” recognizing engrailed proteins, it is shown that the expression of the Chick-en gene is regulated in the inverted neuroepithelium according to its new position in the host neural tube. The regulation takes place within 20 hr after transplantation. These results, together with previous data demonstrating that the phenotypic expression of the inverted neuroepithelium depends, also, on its new position in the host neural tube, strongly suggest that the engrailed protein could play an important role in the positional specification of the mes-metencephalic neuroepithelium.

Branchial sources of the auditory ossicles in man. II. Observations of embryonic stages from 7 mm. to 28 mm. (CR length).

Introduction A companion article in the previous issue presented a historical review dealing with determination of origin of the auditory ossicles and important early developmental steps. The perplexing and controversial subject of ossicular embryology has been studied for over a century without evolution of a clear-cut, authoritative description. The large number of theories may be attributed partly to the wide variation in type and range of material studied, some investigators' tendencies to reconstruct in their own minds the gaps from one stage of development to another, and a willingness to accept as irrefutable the work of an influential author. As revealed by previous studies, determination of ossicular origin requires the examination of very young embryos, and the use of re— constructions or models. 1 The present study covers a full account of observations on the origin of the ossicles in man, as reflected in the 7 mm. to 28 mm.