Pairs Of Somites Research Articles

Peptide-Mediated Protection from Ethanol-Induced Neural Tube Defects

Ethanol inhibition of L1-mediated cell adhesion may contribute to the spectrum of neurological, behavioral and morphological abnormalities associated with prenatal ethanol exposure. We showed previously that the neuroprotective peptides NAPVSIPQ (NAP) and SALLRSIPA (SAL) antagonize ethanol inhibition of L1 adhesion and prevent ethanol-induced growth retardation in mouse whole embryo culture. Here we ask whether NAP and SAL also prevent ethanol-induced major malformations of the nervous system. Gestational day 8.0 (3–5 somites) C57BL/6J mouse embryos were grown for 6 h in control medium, 100 mM ethanol and 10^–10M peptides and then maintained for an additional 20 h in control medium. At the end of the culture period, only embryos having 18–19 somite pairs were examined and compared for the degree of neural tube closure. Ethanol exposure resulted in neural tube defects (NTDs) consistent with total dysraphia and anencephaly. Co-incubation with ethanol and L-NAP (all L-amino acids), D-NAP (all D-amino acids) or SAL significantly increased the percentage of embryos that had begun to close their neural folds at the level of the forebrain/midbrain junction or that had progressed beyond this stage of closure. P7A-NAP (NAPVSIAQ), which lacks neuroprotective activity, but retains activity as an antagonist of ethanol inhibition of L1 adhesion, was effective in preventing ethanol-induced NTDs. In contrast, I6A-NAP (NAPVSAPQ), which shows reduced efficacy as an ethanol antagonist but retains its neuroprotective efficacy, did not significantly diminish the induction of NTDs by ethanol. These findings demonstrate the ability of NAP and SAL to prevent ethanol-induced NTDs and support the hypothesis that ethanol teratogenesis is caused in part by ethanol inhibition of L1-mediated cell adhesion.

Heparin-binding EGF-like growth factor shows transient left–right asymmetrical expression in mouse myotome pairs

Heparin-binding EGF-like growth factor (HB-EGF) is a potent mitogen and chemoattractant for diverse cell types including, keratinocytes, fibroblasts and vascular smooth muscle cells. In adult mice, skeletal muscle and endothelial cells prominently express HB-EGF, although analysis of embryonic expression has been limited to studies of heart and kidney development. Here we survey HB-EGF mRNA expression in E7.5–E15 mouse embryos and show that HB-EGF is expressed in branchial arches, limb buds and, transiently, in mature somites between E9.25 and E11. This somitic expression is restricted to the myotomal compartment. Intriguingly, within myotome pairs, the expression of HB-EGF is stronger on the left side of the body, whilst cognate receptors, ErbB1 and ErbB4, are symmetrically expressed in left and right somite pairs. In iv/iv mutant embryos, with inverted left–right body axis, the expression of HB-EGF was also inverted, now being stronger in myotomes on the right side of the body. Thus, the expression of HB-EGF in myotome pairs is regulated by global cues that define the left–right body axis.

125MORPHOLOGICAL CHARACTERIZATION OF DAY 14 AND DAY 21 IVP AND CLONED BOVINE EMBRYO DEVELOPMENT

The establishment of viable embryonic development following in vitro production (IVP) or nuclear transfer by SUZI (subzonal injection) and handmade cloning (HMC) methods is pivotal for practical implementation of embryo technology. The development of cloned embryos is currently impeded by the high rate of pre- and postnatal losses. This project is aimed at developing screening methods using both ultra-structural and molecular markers of development of post-hatching/pre-implantation stage embryos to enable selection of embryos with greater developmental competence. Initially twenty embryos derived by IVP or nuclear transfer (SUZI or HMC) were transferred to six recipient cows on Day 7 (n=120). Embryos were collected on Days 14 and 21 by flushing the uterine tracts of slaughtered cows. Embryos were fixed in 4% paraformaldehyde and processed for paraffin embedding. Serial sections were stained with haematoxylin and eosin and evaluated by light microscopy. On each day of collection, certain characteristics were expected to be observed according to normal in vivo development (Maddox-Hyttel et al., 2003, Reproduction 125, 607–623). On Day 14, IVP embryos (n=6) were ovoid to filamentous in shape. Three embryos had extensive foldings of trophoblast in the process of forming an amniotic cavity. NT-SUZI embryos (n=4) had a mainly round uneven shape. No NT-HMC embryos were recovered. On Day 21, IVP embryos (n=4) more closely resembled in vivo embryos than any other group and formed either a neural groove or a neural groove with somites. NT-SUZI embryos (n=6) were mainly filamentous, with one embryo showing complete development of the neural tube and 5 somite pairs. NT-HMC (n=17) development ranged from ovoid to multi-protrusions of the yolk sac. One embryo displayed the formation of a neural tube. Embryos, irrespective of production method, which had an abnormal morphology, presented only an incomplete hypoblast and severe degeneration of the epiblast. Those embryos that were morphologically comparable to in vivo embryos also displayed developmental stages which ranged from presentation of a primitive streak and formation of a neural tube to having a neural tube, differentiation of mesoderm and somites. At present, the low number of embryos recovered as well as their abnormal development clearly reflect the low success rate to term of live calves. It is possible that no Day 14 NT-HMC embryos were recovered due to collection efficiency not being optimal. It is apparent that IVP and cloned embryos have different developmental time lines as compared to that of in vivo embryos.

Mouse placenta is a major hematopoietic organ.

Placenta and yolk sac from 8- to 17-day-old (E8-E17) mouse embryos/fetuses were investigated for the presence of in vitro clonogenic progenitors. At E8-E9, the embryonic body from the umbilicus caudalwards was also analysed. Fetal liver was analysed beginning on E10. At E8, between five and nine somite pairs (sp), placenta, yolk sac and embryonic body yielded no progenitors. The first progenitors appeared at E8.5 at the stage of 15 sp in the yolk sac, 18 sp in the embryonic body, 20 sp in the placenta and only at E12 in the fetal liver (absent at E10, at E11 not determined). Progenitors with a high proliferation potential that could be replated for two months, as well as the whole range of myeloid progenitors, were found at all stages in all organs. However, the earliest of these progenitors (these yielding large, multilineage colonies) were 2-4 times more frequent in the placenta than in the yolk sac or fetal liver. In the fetal liver, late progenitors were more frequent and the cellularity increased steeply with developmental age. Thus, the fetal liver, which is a recognized site for amplification and commitment, has a very different hematopoietic developmental profile from placenta or yolk sac. Placentas were obtained from GFP transgenic embryos in which only the embryonic contribution expressed the transgene. 80% of the colonies derived from these placental cells were GFP+, and so originated from the fetal component of the placenta. These data point to the placenta as a major hematopoietic organ that is active during most of pregnancy.

Mitochondrial benzodiazepine receptors regulate oxygen homeostasis in the early mouse embryo

The peripheral benzodiazepine receptor (Bzrp) has been implicated in the control of several processes, including mitochondrial biogenesis and embryo development. The present study examined the impact that specific Bzrp ligands have on oxygen homeostasis in the early mouse embryo. Day 9 embryos at the 16–18 somite pair stage were exposed to standard (21% oxygen) and suboptimal (5% oxygen) oxygen tensions in whole embryo culture. Analysis of gene expression used relative PCR to monitor changes in nuclear respiratory factor-1 ( Nrf1), mitochondrial 16S ribosomal RNA (16S rRNA), and genes for several glycolytic enzymes. Ocular development was highly sensitive to periods of hypoxia through a mechanism blocked with the potent Bzrp ligand PK11195. Hypoxia led to a decline of Nrf1 and 16S rRNA levels also through a mechanism blocked with PK11195. Similar activity was observed for FGIN-1-27 whereas Ro5-4864 had contradictory effects. Morpholino-based gene knockdown of Nrf1 (anti-NRF1) produced a sequence-specific decrease in 16S rRNA insensitive to PK11195. These functional relationships suggest that Bzrp-dependent signals regulate the Nrf1 → Tfam1 → mtDNA → 16S rRNA pathway in response to oxygen levels. The activity of PK11195 most likely has a pharmacodynamic basis with regards to specific embryonic precursor target cell populations, transducing a mitochondrial signal to an Nrf1 response analogous to retrograde regulation in yeast for mitochondria-to-nucleus signaling.

Vascular endothelial growth factor: a regulator of vascular morphogenesis in the Japanese quail embryo.

Experiments in mouse embryos indicate that a critical level of VEGF is required for normal vascular development, as mice lacking a single VEGF allele die at midgestation. Thus VEGF concentration may be a determinant of the size and location of major blood vessels during formation of the primary capillary plexus. Ectopic VEGF delivery was used to examine the effect of VEGF concentration on early vascular patterning in the quail embryo. VEGF was delivered by implanting VEGF-soaked heparin chromatography beads at three rostral-caudal locations in embryos with six somite pairs, which allowed us to study the effect of VEGF on different cellular activities. Ectopic VEGF resulted in significant changes in the vascular pattern at three rostral-caudal levels. Quantitation demonstrated an increased vascularity in the area of the implanted VEGF bead compared to the vascular pattern of embryos with control beads. Areas lateral to the dorsal aortae that are normally avascular became vascularized, and there was an apparent fusion between the dorsal aorta and lateral capillary plexus.

The myth of ventrally emigrating neural tube (VENT) cells and their contribution to the developing cardiovascular system.

The cardiac neural crest cells are a group of cells that emigrate from the dorsal side of the neural tube during a specific time window and contribute to the pharyngeal arch arteries and the aorticopulmonary septum of the heart. Recent publications have suggested that another group of cells emigrating from the ventral side of the neural tube also contributes to the developing cardiovascular system. The first aim of our study was to define the specific time window of cardiac neural crest cell migration by injecting a retrovirus containing a lacZ reporter gene into a chick embryo at different stages during development. The second aim was to study the contribution of the supposed ventrally emigrating neural tube cells to the cardiovascular system using three approaches. One approach was to inject a lacZ retrovirus into the lumen of the chick hindbrain. Secondly, we injected the retrovirus into the neural tube at the position of the 10-12 somite pair. Finally, we used the chimera technique in which we transplanted a quail neural tube segment into a chick embryo. Cardiac neural crest cells were shown to emigrate from the dorsal side of the neural tube between HH9 and HH13(-). The HH13(+) neural tube has ceased to produce cardiac neural crest cells between the level of the otic placode and the fourth pair of somites. Retroviral injection directly into the chick hindbrain at HH14 resulted in 50% of the embryos with minimal labeling of the hindbrain and intense labeling of the adjacent mesenchyme, suggesting that virus was spilled. This implies that this technique is not useful for confirming the existence of ventrally emigrating cells. Both retroviral injections into the neural tube lumen at HH14 at the position of the 10-12 somite pair and the chimeras showed no signs of ventrally emigrating neural tube cells. We conclude that there is no contribution of ventral neural tube cells to the developing cardiovascular system.

Cloning of rainbow trout (Oncorhynchus mykiss) alpha-actin, myosin regulatory light chain genes and the 5'-flanking region of alpha-tropomyosin. Functional assessment of promoters.

We report PCR cloning of rainbow trout alpha-actin (alpha-OnmyAct), myosin regulatory light chain (OnmyMLC2) genes and the 5'-flanking region of alpha-tropomyosin (alpha-OnmyTM). Being expressed in skeletal and cardiac muscle, alpha-OnmyAct was a predominant isoform in trunk muscle of adult rainbow trout. Exon structure of this gene was identical to all known vertebrate skeletal and to some of the cardiac alpha-Act genes. Two distinct OnmyMLC2 promoters were cloned and both included transposon-like sequences. The coding part of OnmyMLC2 consisted of seven exons whose length was typical for vertebrate MLC2 genes. The upstream regions of alpha-OnmyAct and OnmyMLC2 included a TATA box and a number of putative regulatory motifs (E-boxes in all three sequences and CArG-boxes in alpha-OnmyAct), whereas there were no canonical motifs in the alpha-OnmyTM promoter. LacZ reporter gene was fused with the 5'-flanking regions of alpha-OnmyAct, two OnmyMLC2 genes and alpha-OnmyTM promoters. These constructs were transferred into rainbow trout eggs. At the stage of 39 somite pairs, LacZ reporter was detected in the myotomes, neural plate and neural crest, brain and yolk syncytial layer of all analysed embryos. alpha-OnmyTMLacZ was also expressed in the heart. Functionality of promoters and the alpha-OnmyAct terminator was confirmed in rainbow trout primary embryonic cell cultures. We cloned rainbow trout glucose transporter type I (OnmyGLUT1) into vectors including the alpha-OnmyAct and OnmyMLC2 promoters and the alpha-SkAct terminator. Recombinant OnmyGLUT1 transcripts were detected in rainbow trout embryos during somitogenesis.

Inhibition of trophoblast stem cell potential in chorionic ectoderm coincides with occlusion of the ectoplacental cavity in the mouse.

At the blastocyst stage of pre-implantation mouse development, close contact of polar trophectoderm with the inner cell mass (ICM) promotes proliferation of undifferentiated diploid trophoblast. However, ICM/polar trophectoderm intimacy is not maintained during post-implantation development, raising the question of how growth of undifferentiated trophoblast is controlled during this time. The search for the cellular basis of trophoblast proliferation in post-implantation development was addressed with an in vitro spatial and temporal analysis of fibroblast growth factor 4-dependent trophoblast stem cell potential. Two post-implantation derivatives of the polar trophectoderm - early-streak extra-embryonic ectoderm and late-streak chorionic ectoderm - were microdissected into fractions along their proximodistal axis and thoroughly dissociated for trophoblast stem cell culture. Results indicated that cells with trophoblast stem cell potential were distributed throughout the extra-embryonic/chorionic ectoderm, an observation that is probably attributable to non-coherent growth patterns exhibited by single extra-embryonic ectoderm cells at the onset of gastrulation. Furthermore, the frequency of cells with trophoblast stem cell potential increased steadily in extra-embryonic/chorionic ectoderm until the first somite pairs formed, decreasing thereafter in a manner independent of proximity to the allantois. Coincident with occlusion of the ectoplacental cavity via union between chorionic ectoderm and the ectoplacental cone, a decline in the frequency of mitotic chorionic ectoderm cells in vivo, and of trophoblast stem cell potential in vitro, was observed. These findings suggest that the ectoplacental cavity may participate in maintaining proliferation throughout the developing chorionic ectoderm and, thus, in supporting its stem cell potential. Together with previous observations, we discuss the possibility that fluid-filled cavities may play a general role in the development of tissues that border them.

Patterning of angiogenesis in the zebrafish embryo.

Little is known about how vascular patterns are generated in the embryo. The vasculature of the zebrafish trunk has an extremely regular pattern. One intersegmental vessel (ISV) sprouts from the aorta, runs between each pair of somites, and connects to the dorsal longitudinal anastomotic vessel (DLAV). We now define the cellular origins, migratory paths and cell fates that generate these metameric vessels of the trunk. Additionally, by a genetic screen we define one gene, out of bounds (obd), that constrains this angiogenic growth to a specific path. We have performed lineage analysis, using laser activation of a caged dye and mosaic construction to determine the origin of cells that constitute the ISV. Individual angioblasts destined for the ISVs arise from the lateral posterior mesoderm (LPM), and migrate to the dorsal aorta, from where they migrate between somites to their final position in the ISVs and dorsal longitudinal anastomotic vessel (DLAV). Cells of each ISV leave the aorta only between the ventral regions of two adjacent somites, and migrate dorsally to assume one of three ISV cell fates. Most dorsal is a T-shaped cell, based in the DLAV and branching ventrally; the second constitutes a connecting cell; and the third an inverted T-shaped cell, based in the aorta and branching dorsally. The ISV remains between somites during its ventral course, but changes to run mid-somite dorsally. This suggests that the pattern of ISV growth ventrally and dorsally is guided by different cues. We have also performed an ENU mutagenesis screen of 750 mutagenized genomes and identified one mutation, obd that disrupts this pattern. In obd mutant embryos, ISVs sprout precociously at abnormal sites and migrate anomalously in the vicinity of ventral somite. The dorsal extent of the ISV is less perturbed. Precocious sprouting can be inhibited in a VEGF morphant, but the anomalous site of origin of obd ISVs remains. In mosaic embryos, obd somite causes adjacent wild-type endothelial cells to assume the anomalous ISV pattern of obd embryos. Thus, the launching position of the new sprout and its initial trajectory are directed by inhibitory signals from ventral somites. Zebrafish ISVs are a tractable system for defining the origins and fates of vessels, and for dissecting elements that govern patterns of vessel growth.

Cloning and expression of Ventrhoid, a novel vertebrate homologue of the Drosophila EGF pathway gene rhomboid

In Drosophila melanogaster, the seven-pass transmembrane protein Rhomboid (Rho) is a crucial positive modulator of EGF signaling playing a substantial role in patterning of the ventral neuroectoderm and fate specification of neuroblasts. Here, we describe the cloning and expression pattern of Ventrhoid ( Vrho), the novel evolutionarily conserved vertebrate cDNA related to fruit fly rho. Most importantly, like rho in Drosophila, Vrho is also expressed in a spatially restricted manner. Vrho expression is most prominent along the developing ventral neural tube, and is also detectable in the ventral forebrain, prospective diencephalon, otic vesicles, mandibular arches, cranial sensory placodes, last formed pair of somites and hindgut in midgestational mouse embryos.

Signals That Instruct Somite and Myotome Formation Persist in Xenopus laevis Early Tailbud Stage Embryos

Somite formation is a lengthy process that begins at gastrulation and continues through tailbud stages to form approximately 50 pairs of somites in the frog, Xenopus laevis. In Xenopus, the somite primarily gives rise to myotome. We sought to determine whether the formation of somites and myotome requires a transient signal active during gastrulation or a constitutive signal active throughout development to instruct dorsal mesodermal cells to form the posterior somites. Previous work from our lab revealed that cells from the neural ectoderm are capable of responding to mesoderm-inducing signals [Domingo and Keller: Dev Biol 2000;225:226–240]. Thus, to test for the presence of somite-inducing signals, we performed a series of grafting experiments in which we used gastrula cells from the anterior neural ectoderm (ANE). Fluorescently labeled ANE cells were grafted to the posterior paraxial mesoderm of progressively older host embryos between stages 11 (mid gastrula) and 23 (early tailbud). Our results showed that signals within the paraxial mesoderm can instruct prospective ANE cells, which normally give rise to head structures, to instead differentiate into myotome cells. We found that the grafted cells adopted the local paraxial mesoderm cell behaviors, which consists of mediolateral intercalation, segmentation, somite cell rotation, and differentiation to myotome. In addition, we show that the grafted ANE cells that adopt a myotome morphology also express the muscle-specific marker, 12/101. Through a series of heterochronic grafts, we determined that the duration of somite-inducing signals extends from the early gastrula (stage 11) through the early tailbud (stage 23) stage embryos. These results demonstrate that somite induction is not a transient event that occurs during gastrulation, but that it is instead a continuous event that can occur as new somites are added to the posterior axis.

Angiogenesis in association with the migration of gonadotropic hormone-releasing hormone (GnRH) systems in embryonic mice, early human embryos and in a fetus with Kallmann's syndrome

This chapter examines the theoretical notion that the development of the vasculature in the head, and in particular in the nasal mesenchyme, may play a role in the migration and, ultimately, in the function of the gonadotropin-releasing hormone (GnRH) or luteinizing hormone-releasing hormone (LHRH) systems in mammals. The GnRH immunoreactive neurons in mice are first detected by immunocyotchemical procedures and antibodies to GnRH at about 11 to 11.5 days of embryogenesis (more than 45 pairs of somites are present at this age). The GnRH immunoreactive neurons are found in the epithelium of the medial part of the olfactory pit, either side of midline. GnRH neurons are widely separated from each other throughout the placodal epithelium, and as they emerge into the nasal mesenchyme they form cords along, or surrounded by, N-CAM-immunoreactive axons of the terminalis and vomeronasal nerves and perforating branches of the nasal arteries and veins. The GnRH cells are never found independent of these axons in the nasal mesenchyme. The chapter further describes the formation of the meninges in mice and development of the vasculature and observations of the development of the GnRH systems and meninges in human embryos.

The identity and distribution of neural cells expressing the mesodermal determinant spadetail

BackgroundThe spadetail (spt) gene of zebrafish is expressed in presomitic mesoderm and in neural cells previously suggested to be Rohon-Beard neurons. The mechanism(s) generating the apparently irregular rostrocaudal distribution of spt-expressing cells in the developing CNS is unknown.Resultsspt-expressing neural cells co-express huC, a marker of neurons. These cells also co-express the genes islet-1, -2 and -3 but not valentino. The islet-1 gene expression, irregular distribution and dorsolateral position of spt-expressing cells in the developing CNS are characteristic of dorsal longitudinal ascending (DoLA) interneurons. Shortly after their birth, these neurons extend processes rostrally into which spt mRNA is transported. At 24 hours post fertilisation(hpf), spt-expressing neurons occur most frequently at rostral levels caudal of the 5th-formed somite pair. There is no apparent bias in the number of spt-expressing cells on the left or right sides of embryos. Extended staining for spt-transcription reveals expression in the dorsocaudal cells of somites at the same dorsoventral level as the spt-expressing neurons. There is frequent juxtaposition of spt-expression in newly formed somites and in neurons. This suggests that both types of spt-expressing cell respond to a common positional cue or that neurons expressing spt are patterned irregularly by flanking somitic mesoderm.Conclusionsspt-expressing cells in the developing CNS appear to be DoLA interneurons. The irregular distribution of these cells along the rostrocaudal axis of the spinal cord may be due to "inefficient" patterning of neural spt expression by a signal(s) from flanking, regularly distributed somites also expressing spt.

Development of Lumbosacral Spina bifida: Three-Dimensional Computer Graphic Study of Human Embryos at Carnegie Stage Twelve

There is still some controversy as to whether sacral spina bifida in humans is the result of a defect of the primary or secondary neural tube. As somites are related to the development of vertebrae and the primary neural tube is related to the development of the spinal cord in embryos, it is very important to determine the number of somites in normal human embryos at the time of closure of the primary neural tube to understand the contribution of primary neural tube defects to the development of spina bifida. However, in the literature, the number of somites in stage 12 human embryos is still controversial. The aim of this study is to find the number of somites in human embryos at Carnegie stage 12. Four human embryos at Carnegie stage 12 were selected from the laboratory of the Congenital Anomaly Research Center in Japan. The neural tube and somites were reconstructed from their slices by a three-dimensional computer graphic reconstruction technique. The reconstructed embryos were examined from multidirectional magnified images. Thirty-three pairs of somites were present in all these reconstructed embryos. As the 33rd pair of somites corresponds to the fifth sacral segment, the presence of 33 pairs of somites at Carnegie stage 12 suggests that spina bifida develops from defects of the primary neural tube.

Antagonistic effects of Smad2 versus Smad7 are sensitive to their expression level during tooth development.

Members of the transforming growth factor-beta (TGF-beta) superfamily regulate cell proliferation, differentiation, and apoptosis, controlling the development and maintenance of most tissues. TGF-beta signal is transmitted through the phosphorylation of Smad proteins by TGF-beta receptor serine/threonine kinase. During early tooth development, TGF-beta inhibits proliferation of enamel organ epithelial cells but the underlying molecular mechanisms are largely unknown. Here we tested the hypothesis that antagonistic effects between Smad2 and Smad7 regulate TGF-beta signaling during tooth development. Attenuation of Smad2 gene expression resulted in significant advancement of embryonic tooth development with increased proliferation of enamel organ epithelial cells, while attenuation of Smad7 resulted in significant inhibition of embryonic tooth development with increased apoptotic activity within enamel organ epithelium. These findings suggest that different Smads may have differential activities in regulating TGF-beta-mediated cell proliferation and death. Furthermore, functional haploinsufficiency of Smad2, but not Smad3, altered TGF-beta-mediated tooth development. The results indicate that Smads are critical factors in orchestrating TGF-beta-mediated gene regulation during embryonic tooth development. The effectiveness of TGF-beta signaling is highly sensitive to the level of Smad gene expression.

Octanol antagonism of ethanol teratogenesis.

SPECIFIC AIMSAlcohol may cause birth defects in part by disrupting the developmentally critical L1 cell adhesion molecule. Because 1-octanol antagonizes ethanol inhibition of L1-mediated cell adhesion, we used mouse whole embryo culture to test whether 1-octanol also prevents ethanol teratogenicity.PRINCIPAL FINDINGS1. Low concentrations of 1-octanol are not toxic in whole embryo cultureTo determine the toxicity of 1-octanol, gestational day 8 (GD8) whole mouse embryos were cultured for 6 h in the absence and presence of 1-octanol and transferred to control medium for an additional 20 h. Somite pairs were counted after a total of 26 h in culture. Based on somite counts, treatment with 3 μM 1-octanol did not produce a delay in embryonic development, whereas 10 μM and higher concentrations of 1-octanol caused increasing dysmorphogenesis (Fig. 1a⤻ ). Figure 1.Effect of 1-octanol and ethanol on the morphology of cultured C57Bl/6J GD8 mouse embryos. a) Mean ± se number of somite pairs after treatment for 6 h w...

Growth in the pre-fusion murine allantois.

Prior to fusion with the chorion, the extraembryonic mesoderm of the murine (Mus musculus) allantois differentiates with distal-to-proximal polarity into at least two cell lineages: a chorio-adhesive cell lineage called mesothelium, and the endothelium of the umbilical vasculature. How the allantois grows is less clear, but cell proliferation and addition of mesoderm from the underlying primitive streak appear to play important roles. The aim of this study was to analyze growth in the murine allantois. Techniques of histology and microsurgery were used to examine pre-fusion allantoises at nine developmental timepoints that differed by approximately 2 h. Cell counts revealed that allantoic size increased over time. Two hours of exposure to colcemid enhanced mitotic figures, which were used to calculate the relative number of proliferating cells (mitotic index, MI) in pre-fusion allantoises at each developmental timepoint. Cell proliferation was highest in nascent allantoises and showed signs of slowing by two somite pairs. By five to six-somite pairs, when most allantoises are attaching to the chorion, the overall MI decreased significantly. No regional differences in the mitotic index were observed at any developmental stage. Total cell numbers and the mitotic index were then used to discover the extent of streak contribution to pre-fusion allantoises. Cell proliferation and streak activity were highest in nascent allantoises, after which growth occurred predominantly by cell proliferation. Formation of allantoic regenerates by microsurgical removal and culture in intact conceptuses provided independent confirmation that, as the allantois matured, the primitive streak ceased to be a major contributor to its growth. Thus, the allantois grows by both mitosis and addition of mesoderm from the streak. That the periods of highest cell proliferation and streak activity coincided raises intriguing questions concerning their interplay in the control of growth in the murine allantois.

The developmental potentials of the caudalmost part of the neural crest are restricted to melanocytes and glia

The avian spinal cord is characterized by an absence of motor nerves and sensory nerves and ganglia at its caudalmost part. Since peripheral sensory neurons derive from neural crest cells, three basic mechanisms could account for this feature: (i) the caudalmost neural tube does not generate any neural crest cells; (ii) neural crest cells originating from the caudal part of the neural tube cannot give rise to dorsal root ganglia or (iii) the caudal environment is not permissive for the formation of dorsal root ganglia. To solve this problem, we have first studied the pattern of expression of ventral (HNF3β) and dorsal (slug) marker genes in the caudal region of the neural tube; in a second approach, we have recorded the emergence of neural crest cells using the HNK1 monoclonal antibody; and finally, we have analyzed the developmental potentials of neural crest cells arising from the caudalmost part of the neural tube in avian embryo in in vitro culture and by means of heterotopic transplantations in vivo. We show here that neural crest cells arising from the neural tube located at the level of somites 47–53 can differentiate both in vitro and in vivo into melanocytes and Schwann cells but not into neurons. Furthermore, the neural tube located caudally to the last pair of somites (i.e. the 53rd pair) does not give rise to neural crest cells in any of the situations tested. The specific anatomical aspect of the avian spinal cord can thus be accounted for by limited developmental potentials of neural crest cells arising from the most caudal part of the neural tube.

EMBO Workshop Report: Molecular genetics of muscle development and neuromuscular diseases Kloster Irsee, Germany, September 26-October 1, 1999.

The meeting held at the Irsee Convent in Southern Germany was organized by Hans‐Henning Arnold (Braunschweig), Renate Renkawitz‐Pohl (Marburg), Anna Starzinski‐Powitz (Frankfurt) and Bodo Christ (Freiburg). A total of 150 participants, 47 of whom were invited speakers, created an atmosphere of intense progress in a multifaceted field of research. This comprised somite patterning and determination of cell lineages as a paradigm of muscle precursor cell specification, cardiac myogenesis, signals involved in myogenesis, differentiation and the control of muscle‐specific gene expression, as well as the molecular genetics of myopathies and repair processes. Eric Olson (Dallas, TX) gave the keynote address on the transcriptional control of myogenesis. ### More signals and complexity: the somites The paraxial mesoderm is the source of cells that can differentiate into skeletal muscle. It is the primary structure in the vertebrate body showing segmentation when the somites form in a periodic sequence of cranio‐caudal subdivisions from the presomitic mesoderm in mice and the segmental plate in birds. Three years ago, Pourquie's group (Palmeirim et al ., 1997) presented the first experimental evidence for a periodic molecular event prefiguring the synchronous formation of subsequent somite pairs. They described the oscillating expression of c‐hairy1 in the segmental plate prior to somite formation. Such an oscillation had been postulated in theoretical models of somitogenesis. While an ortholog of c‐hairy1 in mouse has not been found yet, new oscillating genes have been described and attempts are being made to arrange them in a sequence of events. The most prominent gene with oscillating expression is lunatic fringe , an ortholog of Drosophila fringe , which modulates the Delta‐Notch pathway (O.Pourquie, Marseille, France; R.Johnson, Houston, TX). Mice homozygous for a targeted deletion in lunatic fringe lack caudal somites, and markers of a cranio‐caudal polarization such as uncx 4.1 are expressed irregularly (Evrard et al ., 1998; Pourquie, 1999). It appears that …