Pairs Of Somites Research Articles

Portuguese contributions to the discovery and characterization of the embryonic molecular clock

Embryonic development is strictly regulated both in time and in space. This extraordinary control is clearly evidenced during the process of somitogenesis. In this process, pairs of somites are formed periodically, such that the time required to form a new somite pair is constant and species specific. The tight temporal control underlying somitogenesis has been shown to depend upon a molecular clock, manifested by the cyclic expression of an increasing number of genes in the unsegmented paraxial mesoderm. Portuguese researchers have been intimately connected to the achievements that have been made in this new field of research: the somitogenesis molecular clock. This article intends to report the Portuguese contributions to the discovery and characterization of the molecular clock underlying somite formation and possibly other embryonic processes. This work inspired many scientists around the world and it has been followed in Portugal by teams that keep on pursuing the characterization of the machinery of this molecular oscillator and its function in the acquisition of both temporal and positional information during development.

Impaired embryonic haematopoiesis yet normal arterial development in the absence of the Notch ligand Jagged1

Specific deletion of Notch1 and RBPjkappa in the mouse results in abrogation of definitive haematopoiesis concomitant with the loss of arterial identity at embryonic stage. As prior arterial determination is likely to be required for the generation of embryonic haematopoiesis, it is difficult to establish the specific haematopoietic role of Notch in these mutants. By analysing different Notch-ligand-null embryos, we now show that Jagged1 is not required for the establishment of the arterial fate but it is required for the correct execution of the definitive haematopoietic programme, including expression of GATA2 in the dorsal aorta. Moreover, successful haematopoietic rescue of the Jagged1-null AGM cells was obtained by culturing them with Jagged1-expressing stromal cells or by lentiviral-mediated transduction of the GATA2 gene. Taken together, our results indicate that Jagged1-mediated activation of Notch1 is responsible for regulating GATA2 expression in the AGM, which in turn is essential for definitive haematopoiesis in the mouse.

Expression of avian prickle genes during early development and organogenesis

Chicken homologues of prickle-1 (pk-1) and prickle-2 (pk-2) were isolated to gain insight into the extent of planar cell polarity signaling during avian embryogenesis. Bioinformatics analyses demonstrated homology and showed that pk-1 and pk-2 exhibited conserved synteny with ADAMTS20 and ADAMTS9, GON-related zinc metalloproteases. Expression of pk-1 and pk-2 was established during embryogenesis and early organogenesis, using in situ hybridization and sections of chicken embryos. At early stages, pk-1 was expressed in Hensen's node, primitive streak, ventral neural tube, and foregut. In older embryos, pk-1 transcripts were detected in dorsolateral epithelial somites, dorsomedial lip of dermomyotomes, and differentiating myotomes. Furthermore, pk-1 expression was seen in lateral body folds, limb buds, and ventral metencephalon. pk-2 was expressed in Hensen's node and neural ectoderm at early stages. In older embryos, pk-2 expression was restricted to ventromedial epithelial somites, except in the most recently formed somite pair, and limb bud mesenchyme.

Chick Hairy1 protein interacts with Sap18, a component of the Sin3/HDAC transcriptional repressor complex

BackgroundThe vertebrate adult axial skeleton, trunk and limb skeletal muscles and dermis of the back all arise from early embryonic structures called somites. Somites are symmetrically positioned flanking the embryo axial structures (neural tube and notochord) and are periodically formed in a anterior-posterior direction from the presomitic mesoderm. The time required to form a somite pair is constant and species-specific. This extraordinary periodicity is proposed to depend on an underlying somitogenesis molecular clock, firstly evidenced by the cyclic expression of the chick hairy1 gene in the unsegmented presomitic mesoderm with a 90 min periodicity, corresponding to the time required to form a somite pair in the chick embryo. The number of hairy1 oscillations at any given moment is proposed to provide the cell with both temporal and positional information along the embryo's anterior-posterior axis. Nevertheless, how this is accomplished and what biological processes are involved is still unknown. Aiming at understanding the molecular events triggered by the somitogenesis clock Hairy1 protein, we have employed the yeast two-hybrid system to identify Hairy1 interaction partners.ResultsSap18, an adaptor molecule of the Sin3/HDAC transcriptional repressor complex, was found to interact with the C-terminal portion of the Hairy1 protein in a yeast two-hybrid assay and the Hairy1/Sap18 interaction was independently confirmed by co-immunoprecipitation experiments. We have characterized the expression patterns of both sap18 and sin3a genes during chick embryo development, using in situ hybridization experiments. We found that both sap18 and sin3a expression patterns co-localize in vivo with hairy1 expression domains in chick rostral presomitic mesoderm and caudal region of somites.ConclusionHairy1 belongs to the hairy-enhancer-of-split family of transcriptional repressor proteins. Our results indicate that during chick somitogenesis Hairy1 may mediate gene transcriptional repression by recruiting the Sin3/HDAC complex, through a direct interaction with the Sap18 adaptor molecule. Moreover, since sap18 and sin3a are not expressed in the PSM territory where hairy1 presents cyclic expression, our study strongly points to different roles for Hairy1 throughout the PSM and in the prospective somite and caudal region of already formed somites.

Watch-ing out for chick limb development

Time control is a crucial issue during embryonic development. Nevertheless, little is known about how embryonic cells measure time. Until recently, the only molecular clock known to operate during vertebrate embryonic development was the somitogenesis clock, exclusively functioning in coordinating the precise timing of each new pair of somites formed from the presomitic mesoderm. We have recently evidenced that a similar molecular clock also underlies the timing at which autopod chondrogenic precursors are laid down to form a skeletal limb element. In addition, we herein suggest that the molecular clock is not the only parallelism that can be established between somitogenesis and limb-bud development. In an evolutionary perspective, we support the previously proposed idea that the molecular mechanisms involved in the segmentation of the body axis may have been partially reused in the mesoderm of the lateral plate, thereby allowing the emergence of paired appendages.

An Nkx2-5/Bmp2/Smad1 Negative Feedback Loop Controls Heart Progenitor Specification and Proliferation

During heart development the second heart field (SHF) provides progenitor cells for most cardiomyocytes and expresses the homeodomain factor Nkx2-5. We now show that feedback repression of Bmp2/Smad1 signaling by Nkx2-5 critically regulates SHF proliferation and outflow tract (OFT) morphology. In the cardiac fields of Nkx2-5 mutants, genes controlling cardiac specification (including Bmp2) and maintenance of the progenitor state were upregulated, leading initially to progenitor overspecification, but subsequently to failed SHF proliferation and OFT truncation. In Smad1 mutants, SHF proliferation and deployment to the OFT were increased, while Smad1 deletion in Nkx2-5 mutants rescued SHF proliferation and OFT development. In Nkx2-5 hypomorphic mice, which recapitulate human congenital heart disease (CHD), OFT anomalies were also rescued by Smad1 deletion. Our findings demonstrate that Nkx2-5 orchestrates the transition between periods of cardiac induction, progenitor proliferation, and OFT morphogenesis via a Smad1-dependent negative feedback loop, which may be a frequent molecular target in CHD.

Colonisation of the gut by neural crest‐derived enteric nervous system precursors: migration pathways and developmental potentials

The neurons and glia of the enteric nervous system (ENS) are derived from vagal and sacral neural crest cells (NCC). Vagal (hindbrain) NCC migrate from the dorsal neural tube, accumulate in the caudal branchial arches, enter the foregut and colonise the entire gut in a rostro-caudal direction, reaching the terminal hindgut of the chick at embryonic day 7.5. Sacral NCC, which arise caudal to the 28th pair of somites, initially form the nerve of Remak in the chick, then enter the gut after it has been colonised by vagal cells. The contribution of sacral cells to the ENS is restricted to the post-umbilical gut, and mainly the hindgut. It therefore appears that vagal and sacral NCC have different ENS developmental potentials, since vagal cells form most of the ENS along the entire gut, whereas sacral NCC provide a limited number of cells to the hindgut. This idea is further supported by heterotopic grafting experiments: transplanting vagal NCC into the sacral region results in rapid and extensive gut colonisation, whereas transplanting sacral cells into the vagal region results in diminished colonisation, compared with vagal NCC. To better understand the differences in ENS developmental potentials between these two populations, we analysed the expression of ENS signalling molecules in vagal and sacral NCC, and show that increasing the expression of the receptor tyrosine kinase RET in sacral NCC leads to increased gut colonisation.

Practical Lessons from Theoretical Models about the Somitogenesis

Vertebrae and other mammalian repetitive structures are formed from embryonic organs called somites. Somites arise sequentially from the unsegmented presomitic mesoderm (PSM). In mice, a new bilateral pair of somites arise every two hours from the rostral PSM. On the other hand, cells are added to the caudal side of the PSM due to cell proliferation of the tail bud. Somite formation correlates with cycles of cell-autonomous expression in the PSM of genes like Hes7. Because the somitogenesis is a highly dynamic and coordinated process, this event has been subjected to extensive theoretical modeling. Here, we describe the current understanding about the somitogenesis in mouse embryos with an emphasis on insights gained from computer simulations. It is worth noting that the combination of experiments and computer simulations has uncovered dynamical properties of the somitogenesis clock such as the transcription/translation delays, the half-life and the synchronization mechanism across the PSM. Theoretical models have also been useful to provide predictions and rigorous hypothesis about poorly understood processes such as the mechanisms by which the temporal PSM oscillations are arrested and converted into an spatial pattern. We aim at reviewing this theoretical literature in such a way that experimentalists might appreciate the resulting conclusions.

Ultradian Oscillators in Somite Segmentation and Other Biological Events

Somite formation occurs every 2 hours in mouse embryos by periodic segmentation of the anterior ends of the presomitic mesoderm, and this process is controlled by a biological clock called the segmentation clock. During this process, the basic helix-loop-helix gene Hes7 is cyclically expressed, and each cycle leads to generation of a bilateral pair of somites. Both sustained expression and loss of expression of Hes7 result in severe somite fusion, indicating that Hes7 constitutes an essential component of the segmentation clock. Interestingly, expression of the related gene Hes1 also oscillates with a periodicity of about 2 hours in many cell types. Both sustained Hes1 expression and loss of Hes1 activity lead to retardation of the G(1) phase of the cell cycle, suggesting that Hes1 oscillation with an ultradian rhythm is required for efficient cell proliferation. Both Hes1 and Hes7 oscillations are regulated by negative feedback and rapid degradation of their gene products. Strikingly, expression of other factors such as Stat-Socs and Smad signaling molecules also display ultradian rhythms. All of these data suggest that ultradian oscillations are more general responses than were previously thought and that oscillatory and sustained gene expression results in different biological outcomes.

18 Circulation Plays an Essential Role in Distributing Hematopoietic Progenitors from the Yolk Sac to the Embryo Proper; Lessons from the Ncx1-Null Mouse.

The yolk sac is the lone site of primitive hematopoiesis. However, the role of the yolk sac in generating definitive hematopoietic progenitors has remained controversial since the onset of circulation alters the localization of hematopoietic progenitors. The Ncx1 knockout mouse fails to initiate a heartbeat and thus permits an analysis of the temporal and spatial distribution of definitive hematopoietic progenitor cells in an embryonic environment lacking circulation. Embryos were harvested from timed Ncx1 heterozygote crosses beginning at the onset of circulation at embryonic day 8.5 (E8.5) and ending 36 hours later (E10). The developmental stage of each embryo was determined by counting somite pairs. All embryos were carefully separated from their yolk sac (YS) and the paraaortic splanchnopleura (PSp) was dissected from the embryo proper. The tissues were digested and plated in methylcellulose. Colonies were counted after seven days. At E8.5, definitive hematopoietic progenitors are enriched in the YS more than 28-fold compared to the PSp in both wild type and Ncx1-null embryos. This ratio drops to 3-fold enrichment in the YS following progenitor redistribution by a functional circulation (E10) in wild type embryos, as reported previously (McGrath, et al. Blood 2003). The YS of Ncx1-null embryos, which lack a functional circulation, contain as many definitive progenitors as wild type YS and PSp combined. Few hematopoietic progenitors are found in the Ncx1-null PSp as late as E10, resulting in a 72-fold enrichment of progenitors in the Ncx1-null YS. We conclude that the PSp does not appear to function as a site of active hematopoiesis prior to E10. Furthermore, our findings support a model in which large numbers of definitive hematopoietic progenitors are generated in the YS between E8.5 and E10 and are redistributed to the embryo proper of the mouse embryo once cardiac function initiates a functional circulation.

Hematopoietic potential of the pre-fusion allantois

We previously showed that the fetal component of the placenta has a vigorous hematopoietic activity. Whether this organ is an environmental niche where hematopoietic stem cells (HSC) proliferate and become committed to various lineages, or whether it is also a site for HSC emergence, was left open. This issue can be addressed only if the components that will give rise to the placenta are tested prior to vascularization. The fetal part of the placenta forms through the fusion of the allantois and the chorionic plate around the stage of 7 somite pairs. The allantois, a mesodermal rudiment that provides fetal blood vessels to the placenta, was retrieved before fusion. We found in this rudiment expression of CD41, a known marker of early embryonic hematopoietic progenitors. c-Kit encoding a progenitor specific receptor was also expressed. Significantly, as early as the 1–2 somite stage, the allantois yielded erythroid, myeloid and multipotent clonogenic progenitors, when pre-cultured in toto prior to seeding in a semisolid medium. These results provide evidence that the allantois has hematopoietic potential per se. Whether this potential also involves the ability to produce HSC is still to be determined.

The Role of Phospholipid Hydroperoxide Glutathione Peroxidase Isoforms in Murine Embryogenesis

Phospholipid hydroperoxide glutathione peroxidase (GPx4) is a selenocysteine-containing enzyme, and three different isoforms (cytosolic, mitochondrial, and nuclear) originate from the GPx4 gene. Homozygous GPx4-deficient mice die in utero at midgestation, since they fail to initiate gastrulation and do not develop embryonic cavities. To investigate the biological basis for embryonic lethality, we first explored expression of the GPx4 in adult murine brain and found expression of the protein in cerebral neurons. Next, we profiled mRNA expression during the time course of embryogenesis (embryonic days 6.5-17.5 (E6.5-17.5)) and detected mitochondrial and cytosolic mRNA species at high concentrations. In contrast, the nuclear isoform was only expressed in small amounts. Cytosolic GPx4 mRNA was present at constant levels (about 100 copies per 1000 copies of glyceraldehyde-3-phosphate dehydrogenase mRNA), whereas nuclear and mitochondrial isoforms were down-regulated between E14.5 and E17.5. In situ hybridization indicated expression of GPx4 isoforms in all developing germ layers during gastrulation and in the somite stage in the developing central nervous system and in the heart. When we silenced expression of GPx4 isoforms during in vitro embryogenesis using short interfering RNA technology, we observed that knockdown of mitochondrial GPx4 strongly impaired segmentation of rhombomeres 5 and 6 during hindbrain development and induced cerebral apoptosis. In contrast, silencing expression of the nuclear isoform led to retardations in atrium formation. Taken together, our data indicate specific expression of GPx4 isoforms in embryonic brain and heart and strongly suggest a role of this enzyme in organogenesis. These findings may explain in part intrauterine lethality of GPx4 knock-out mice.

Embryonic development of the European smelt Osmerus eperlanus eperlanus (L.) (Neva population)

The embryonic development of the European smelt Osmerus eperlanus eperlanus has been described. The whole period of the development has been subdivided into 6 subperiods. More than 80 stages may be described for all of these periods. The development of the European smelt embryo was studied in the 5 different regimes of a constant temperature in the range 9.5–18.3°C. The duration of different stages under different temperature regimes was expressed in the relative units of τ0 (the time of a single cleavage) and τs (the time of formation of a single pair of somites). It was established that the τs value had proportionally changed by embryogenesis. Due to this fact, the duration of the stages was expressed only as τs. The dependency of the speed of the European smelt embryogenesis on temperature has been shown. This dependence was expressed using the standard interval τs and described using the equation log τs(t) = 3.22665−0.13876t + 0.00297t2, where t is the temperature.

Interaction between X-Delta-2 and Hox genes regulates segmentation and patterning of the anteroposterior axis

In vertebrates, the paraxial mesoderm already exhibits a complex Hox gene pattern by the time that segmentation occurs and somites are formed. The anterior boundaries of the Hox genes are always maintained at the same somite number, suggesting coordination between somite formation and Hox expression. To study this interaction, we used morpholinos to knockdown either the somitogenesis gene X-Delta-2 or the complete Hox paralogous group 1 (PG1) in Xenopus laevis. When X-Delta-2 is knocked down, Hox genes from different paralogous groups are downregulated from the beginning of their expression at gastrula stages. This effect is not via the canonical Notch pathway, as it is independent of the Notch effector Su(H). We also reveal for the first time a clear role for Hox genes in somitogenesis, as loss of PG1 gene function results in the perturbation of somite formation and downregulation of the X-Delta-2 expression in the PSM. This effect on X-Delta-2 expression is also observed during neurula stages, before the somites are formed. These results show that somitogenesis and patterning of the anteroposterior axis are closely linked via a feedback loop involving Hox genes and X-Delta-2, suggesting the existence of a coordination mechanism between somite formation and anteroposterior patterning. Such a mechanism is likely to be functional during gastrulation, before the formation of the first pair of somites, as suggested by the early X-Delta-2 regulation of the Hox genes.

Short-term exposures to dihaloacetic acids produce dysmorphogenesis in mouse conceptuses in vitro

The haloacetic acids (HAAs) are a family of xenobiotics found in tap water as a result of drinking water disinfection. Administration of HAAs to rats produces a variety of adverse effects, including developmental toxicity. The dysmorphogenic potencies of all nine bromo/chloro-acetic acids have been determined in rodent whole embryo culture using standard 26-h exposure. Since the half-lives of the HAAs in vivo are typically <8 h, the developmental effects of short-term exposures to dihaloacetates were evaluated. Gestation day 8 (3–6 somite pairs) CD-1 mouse conceptuses were exposed to 11,000 μM dichloroacetic acid (DCA), 300 μM dibromoacetic acid (DBA) or 300 μM bromochloroacetic acid (BCA) for culture periods of 1, 3, 6 or 26 h. Following 1, 3 or 6 h of exposure to HAAs, conceptuses were transferred to control medium to complete a 26-h culture period. The amounts of HAAs present in embryos after 1, 3 and 6 h of exposure were determined. Increased incidences of dysmorphic embryos were produced by 6 or 26-h exposures to DCA; a 26-h exposure to DBA; or 3, 6 or 26-h exposures to BCA. The dysmorphology produced was dependent upon the length of exposure and chemical. The embryonic concentration of each HAA (104.5, 2.5 and 2.6 pmol/μg protein for DCA, DBA and BCA, respectively) was reached by 1 h of exposure and did not change at the subsequent time points examined. The current studies demonstrate that BCA is more potent than DBA or DCA at disrupting embryogenesis since shorter exposures alter morphogenesis. Since the embryonic HAA concentrations were the same at the three time points measured, the time-dependence in dysmorphogenesis does not appear to be a simple function of increasing embryonic concentration of these chemicals. These studies demonstrate that for these dihaloacetic acids relatively high concentrations and long exposures are needed to alter rodent development in vitro.

Regulation of somitogenesis by Ena/VASP proteins and FAK duringXenopusdevelopment

The metameric organization of the vertebrate body plan is established during somitogenesis as somite pairs sequentially form along the anteroposterior axis. Coordinated regulation of cell shape, motility and adhesion are crucial for directing the morphological segmentation of somites. We show that members of the Ena/VASP family of actin regulatory proteins are required for somitogenesis in Xenopus. Xenopus Ena (Xena) localizes to the cell periphery in the presomitic mesoderm (PSM), and is enriched at intersomitic junctions and at myotendinous junctions in somites and the myotome, where it co-localizes with beta1-integrin, vinculin and FAK. Inhibition of Ena/VASP function with dominant-negative mutants results in abnormal somite formation that correlates with later defects in intermyotomal junctions. Neutralization of Ena/VASP activity disrupts cell rearrangements during somite rotation and leads to defects in the fibronectin (FN) matrix surrounding somites. Furthermore, inhibition of Ena/VASP function impairs FN matrix assembly, spreading of somitic cells on FN and autophosphorylation of FAK, suggesting a role for Ena/VASP proteins in the modulation of integrin-mediated processes. We also show that inhibition of FAK results in defects in somite formation, blocks FN matrix deposition and alters Xena localization. Together, these results provide evidence that Ena/VASP proteins and FAK are required for somite formation in Xenopus and support the idea that Ena/VASP and FAK function in a common pathway to regulate integrin-dependent migration and adhesion during somitogenesis.

Myogenin in model pufferfish species: Comparative genomic analysis and thermal plasticity of expression during early development

Myogenin (Myog) is a muscle-specific basic helix-loop-helix transcription factor that plays an essential role in the specification and differentiation of myoblasts. The myogenin genes from the tiger pufferfish, Takifugu rubripes, and green-spotted pufferfish, Tetraodon nigroviridis, were cloned and a comparative genomic analysis performed. The gene encoding myogenin is composed of three exons and has a relatively similar genomic structure in T. rubripes, T. nigroviridis and human. Introns 1 and 2 were approximately 2-fold and 8-fold longer respectively in human than pufferfish. Myogenin is located in a 100 kb region of conserved synteny between these organisms, corresponding to chromosome 1 in human, chromosome 11 in T. nigroviridis and scaffold 208 in T. rubripes. Pufferfish myogenin contained a serine-rich region at the carboxyl terminus that is highly conserved amongst teleosts. During embryonic development of T. rubripes, myogenin was expressed in a rostral–caudal gradient in the developing somites and subsequently during the pharyngula period in the pectoral fin bud primordia, jaw muscles and extraocular muscle precursors. In T. rubripes, the time required to form a somite pair during the linear phase of somitogenesis (≡ somite-interval) was 122 min, 97 min and 50 min in embryos incubated at 15, 18 and 21 °C, respectively. Myogenin mRNA transcripts were quantified using qPCR and normalised to the highest level of expression. Peak myogenin expression occurred later with respect to developmental stage (standardised using somite-intervals) and was over 2-fold higher at 21 °C than at either 18 or 15 °C. Changes in the relative timing and intensity of myogenin expression are a potential mechanism for explaining thermal plasticity of muscle phenotype in larvae via effects on the differentiation programme.

Circulation Plays an Essential Role in Distributing Mammalian Yolk Sac Definitive Hematopoietic Progenitor Cells to the Embryo Proper; Using the Ncx1 Knockout Mouse Model To Prevent Circulation.

The yolk sac is the lone site of primitive hematopoiesis. The role of the yolk sac in generating definitive hematopoietic progenitors, however, has remained controversial. One complicating factor preventing an accurate investigation of this subject has been the onset of early circulation which alters the localization of hematopoietic progenitors. An Ncx1 knockout mouse (which fails to initiate a heartbeat) is used here to investigate the temporal and spatial distribution of definitive hematopoietic progenitor cells (HPCs: adult type BFU-E, CFU-GM, CFU-GEMM) in an environment lacking circulation. Embryos were harvested from timed pregnancies from Ncx1 heterozygote crosses beginning at the onset of circulation 8.5 days post conception (E8.5) and ending 36 hours later (E10). Developmental age was determined by somite pair number. All embryos were carefully separated from their yolk sacs and both samples were digested and plated in methylcellulose using a previously published definitive hematopoietic progenitor colony assay. Colonies were counted at seven days and then collected for genotyping. In embryos of all genotypes at E8.5, definitive HPCs were enriched more than 28 fold in the yolk sac compared to the embryo proper (EP), but following redistribution by a functional circulation in E10 wild type and heterozygous embryos, the ratio drops to a 3 fold enrichment in the yolk sac (similar to the 3–5 fold enrichment previously reported at this age; KM & JP, Blood 2003). Ncx1 null embryos lacking circulation produced few HPCs in the EP and never redistribute the HPCs as late as E10 resulting in a 72 fold enrichment in the yolk sac even at E10. Whole mount ζ-hemoglobin mRNA staining was carried out to visualize the distribution of blood cells in the yolk sac and embryo proper in the presence and absence of circulation. The staining pattern in null embryos confirms that cells from the yolk sac blood band remain in the yolk sac and are not found in the EP. Our findings support a model in which the primitive and definitive hematopoietic progenitors are generated in the yolk sac and are only redistributed to the EP upon the onset of circulation. Not only is the yolk sac an important source of definitive HPCs, it is the developing embryo's primary source through E10.

Bromochloro-haloacetic acids: Effects on mouse embryos in vitro and QSAR considerations

The haloacetic acids (HAA) are a family of chemicals that are drinking water disinfection by-products. We previously reported that haloacetic acids, including several bromo- and chloro-HAAs, alter embryonic development when mouse conceptuses are directly exposed to these xenobiotics in whole embryo culture. Craniofacial dysmorphogenesis was observed in exposed embryos and a quantitative structure activity relationship (QSAR) for induction of cranial neural tube dysmorphogenesis was established for a series of 10 HAAs, which also included fluoro- and iodo-HAA representatives. In the current study, we evaluate the effects of exposing neurulation staged (3–6 somite pairs) CD-1 mouse conceptuses to bromochloro- (BCA), dibromochloro- (DBCA) and bromodichloro-acetic (BDCA) acids in whole embryo culture at concentrations ranging from 50 to 2500 μM. Morphological development was assessed after a 26 h exposure period. Exposure of conceptuses to these HAAs produced dysmorphogenesis, including prosencephalic and pharyngeal arch hypoplasia as well as eye and heart tube abnormalities. Benchmark concentrations for induction of neural tube dysmorphogenesis were 63, 500 and 536 μM for BCA, DBCA and BDCA, respectively. Our previously developed HAA QSAR accurately predicted placement of these three chemicals in the larger context of the previously tested di- and tri-HAAs, also correctly predicting that BCA would be more potent than DBCA and BDCA, and that the latter two HAAs would be near equi-potent. This study describes the concentration-dependent induction of dysmorphogenesis in whole embryo culture by three mixed chloro/bromo-HAAs and demonstrates the ability of the HAA QSAR to predict relative potencies within this family of xenobiotics.

Effects of growth factors FGF4, TGFα, and TGFβ1 on development of parthenogenetic embryos of C57BL/6 mice

We studied the effects of three growth factors, fibroblast growth factor (FGF4), transforming growth factor α (TGFα), and transforming growth factor β1 (TGFβ1), on development of diploid parthenogenetic embryos of C57BL/6 mice, which are not capable of developing to somatic stages. Parthenogenetic embryos were treated with growth factors at optimal doses in vitro at the morula-blastocyst stages and transplanted in the uterus of pseudopregnant females. FGF4 and TGFα improved the development of parthenogenetic embryos at the preimplantation stages and the number of blastocysts increased under the influence of TGFα. All three growth factors improved the implantation of embryos in the uterus. When FGF4 or TGFβ1 2.4 were added to the nutrient medium, 2.4 or 1.6%, respectively, of parthenogenetic embryos reached the somatic stages in utero. No somitic embryos were observed in the control. The treatment of parthenogenetic embryos with two growth factors, FGF4 and TGFβ1 , simultaneously increased the amount of somatic embryos to 7.5%, while combination of three growth factors in creased the amount of such embryos to 16.7%. In the latter case, some parthenogenetic embryos reached the stage of 25–27 pairs of somites and were 2.0–2.5 mm long. The data we obtained suggest that, when combined, the growth factors FGF4, TGFα, and TGFβ1 possessed a synergistic effect leading to a significant improvement of the development of parthenogenetic C57BL/6 embryos.