Embryonic Disc Research Articles

Klhl14 Antisense RNA is a Target of Key Skeletogenic Transcription Factors in the Developing Intervertebral Disc.

RNA in situ hybridization (RISH) allows for validation and characterization of the long noncoding (lnc) natural antisense RNA (NAT) Klhl14as in the embryonic murine intervertebral disc (IVD) in the context of loss-of-function mutants for key transcription factors (TFs) in axial skeleton development. Validation of Klhl14as in the developing murine IVD. The IVD is a focus of regenerative medicine; however, processes and signaling cascades resulting in the different cell types in a mature IVD still require clarification in most animals including humans. Technological advances increasingly point to implications of lnc NATs in transcription/translation regulation. Transcriptome data generation and analysis identified a protein encoding transcript and related noncoding antisense transcript as downregulated in embryos devoid of key TFs during axial skeleton development. Here, primarily, the antisense transcript is analyzed in this loss-of-function context. 4930426D05Rik and 6330403N15Rik were identified as Klhl14as and sense, respectively, two transcripts downregulated in the vertebral column of midgestation Pax1 and Pax9 mutant mouse embryos. RISH on wildtype and mutant embryos for the TF encoding genes Pax1/Pax9, Sox5/Sox6/Sox9, and Bapx1 was used to further analyze Klhl14as in the developing IVD. Klhl14as and Klhl14 were the top downregulated transcripts in Pax1; Pax9 E12.5 embryos. Our data demonstrate expression of Klhl14as and sense transcripts in the annulus fibrosus (AF) and notochord of the developing IVD. Klhl14as expression in the inner annulus fibrosus (iAF) seems dependent on the TFs Pax1/Pax9, Sox6, Sox9, and Bapx1. We are the first to suggest a role for the lncRNA Klhl14as in the developing IVD. Our data link Klhl14as to a previously established gene regulatory network during axial skeleton development and contribute further evidence that lnc NATs are involved in crucial gene regulatory networks in eukaryotic cells. N/A.

Characterisation of bovine embryos following prolonged culture in embryonic stem cell medium containing leukaemia inhibitory factor.

In order to help elucidate the process of epiblast and trophoblast cell differentiation in bovine embryos invitro, we attempted to develop a suitable culture medium to allow extended embryo culture. Day 7 bovine blastocysts developed in conventional medium were cultured further in embryonic stem cell medium with or without leukaemia inhibitory factor (LIF) until Day 23. At Day 14, the expression of octamer-binding transcription factor 3/4 (OCT3/4) and VIMENTIN was significantly higher in embryos cultured with than without LIF, but embryonic disc formation was not observed. Although expression of SRY (sex determining region Y)-box 17 (SOX17) mRNA was significantly lower in Day 14 embryos cultured with and without LIF than in invivo embryos, hypoblast cells formed just inside the trophoblast cells of the invitro-cultured embryos. On Day 23, expression of placental lactogen (PL) and prolactin-related protein 1 (PRP1) was not affected by LIF in invitro-cultured embryos, levels of both genes were significantly lower in the invitro than invivo embryos. Similar to invivo embryos, binucleate cell clusters seen in Day 23invitro-cultured embryos were composed of PL-negative and -positive cells. These results suggest that our culture system partially reproduced the differentiation process of trophoblast cells invivo.

85 Elongation of trophoblastic vesicles between Days 15 and 18 in cattle

Once formed, bovine blastocysts differentiate while growing exponentially from 150-200μm (Days 7 or 8) to 200-250mm (Days 17 to 18; Sandra et al. 2017 Annu. Rev. Anim. Biosci. 5, 205-228). Thus, the length of the conceptus doubles every day between Days 9 and 16 (Berg et al. 2010 Theriogenology 73, 250-260); however, this was observed on whole conceptuses. The objective of the current study was to test whether this elongation rate is similar when the embryonic disc has been excised. Six heifers were used to produce Day-15 conceptuses, either fully developed in vivo or developed in vivo for a week after embryo transfer of Day 8in vitro-produced blastocysts. Day 15 conceptuses were recovered, measured, and cut into pieces to produce trophoblastic vesicles (TV; Heyman et al. 1984 J. Reprod. Fertil. 70, 533-540) of 4±0.07 or 4.4±0.65mm long (mean±standard error of the means) for in vivo- or in vitro-produced TV, respectively. All TV were transferred into oestrus-synchronized recipients (5 heifers and 1 cow). Each female received 8-9 TV so that a total of 24in vivo-derived and 26in vitro-produced TV were transferred in utero for a period of 3 days. The TV originating from different Day-15 conceptuses were mixed at the time of transfer, so that each recipient received the TV from different origins (conceptus and donor cow). Transcervical collection was used at Day 15 and 18 for conceptus and TV recovery (Richard et al. 2015 Theriogenology 83, 1101-1109). At Day 18, TV elongation size was analysed (mean±standard error of the means) by unpaired t-test using GraphPad Prism software (GraphPad Inc., San Diego, CA, USA). At Day 15, conceptuses from the in vivo and in vitro groups displayed different sizes and length variabilities (24-32v. 2-24mm, respectively). At Day 18, TV recovery rate was 79% for the in vivo- v. 62% for the in vitro-derived group and mean elongation rate (over 3 days) was ×5.4 (minimum=2.5, maximum=10) v. ×7.6 (minimum=0.7, maximum=20.5), respectively. There was no significant difference for TV size between groups at Day 18 (21.75±2.24 mm v. 33.38±11.63mm, respectively). Altogether, the variability in length at Day 15 was previously reported, the difference in TV recovery between in vivo and in vitro groups reached 17% and was similar to the loss of 11% that occurs in the first week after classical embryo transfer. In opposition to studies where in vitro-produced conceptuses were shorter than in vivo-developed ones, in vivo and in vitro groups of TV likely followed similar growth. Whether this reflects a normal growth awaits further studies.

The extracellular protease AdamTS-B inhibits vein formation in the Drosophila wing.

Vein patterning in the Drosophila wing provides a powerful tool to study regulation of various signaling pathways. Here we show that the ADAMTS extracellular protease AdamTS-B (CG4096) is expressed in the embryonic wing imaginal disc precursor cells and the wing imaginal disc, and functions to inhibit wing vein formation. Knock-down of AdamTS-B displayed posterior crossveins (PCVs) with either extra branches or deltas, or wider PCVs, and a wandering distal tip of the L5 longitudinal vein. Conversely, over-expression of AdamTS-B resulted in a complete absence of the PCV, an incomplete anterior crossvein, and missing distal end of the L5 longitudinal vein. We conclude that AdamTS-B inhibits wing vein formation through negative regulation of signaling pathways, possibly BMP as well as Egfr, displaying the complexity of roles for this family of extracellular proteases.

Runx2 is required for postnatal intervertebral disc tissue growth and development.

Runx2 plays an essential role in embryonic disc tissue development in mice. However, the role of runt-related transcription factor 2 (Runx2) in postnatal disc tissue growth and development has not been defined. In the present studies, we generated Runx2 conditional knockout (KO) mice (Runx2Agc1ER ), in which Runx2 was deleted in Aggrecan-expressing cells in disc tissue at postnatal 2-weeks of age. We then analyzed changes in disc tissue growth and development using histology and immunohistochemical methods in 3-month-old mice. We found that large vacuolated notochordal cells were accumulated in the nucleus pulposus (NP) in Runx2 KO mice. The growth plate cartilage tissue in the disc was thicker in Runx2 KO mice. We also found a significant upregulation of Indian hedgehog (Ihh) expression in the cells in NP cells and in annulus fibrosus cells of Runx2 KO mice. These results demonstrated that Runx2 may play an important role in postnatal disc tissue development through interacting with Ihh signaling.

Pitx2 and nodal as conserved early markers of the anterior-posterior axis in the rabbit embryo

Attaining molecular and morphological axial polarity during gastrulation is a fundamental early requirement for normal development of the embryo. In mammals, the first morphological sign of the anterior–posterior axis appears anteriorly in the form of the anterior marginal crescent (or anterior visceral endoderm) while in the avian the first such sign is the Koller’s sickle at the posterior pole of the embryonic disc. Despite this inverse mode of axis formation many genes and molecular pathways involved in various steps of this process seem to be evolutionarily conserved amongst amniotes, the nodal gene being a well-known example with its functional involvement prior and during gastrulation. The pitx2 gene, however, is a new candidate described in the chick as an early marker for anterior–posterior polarity and as a regulator of axis formation including twinning. To find out whether pitx2 has retained its inductive and early marker function during the evolution of mammals this study analyses pitx2 and nodal expression at parallel stages during formation of the anterior–posterior polarity in the early rabbit embryo using whole-mount in situ hybridization and serial light-microscopical sections. At a late pre-gastrulation stage a localized reduction of nodal expression presages the position of the anterior pole of the embryonic disc and thus serves as the earliest molecular marker of anterior–posterior polarity known so far. Pitx2 is expressed in a polarized manner in the anterior marginal crescent and in the posterior half of the embryonic disc during further development. In the anterior segment of the posterior pitx2 expression domain, the anterior streak domain (ASD) is defined by nodal expression as a hypothetical progenitor region of the anterior half of the primitive streak. The expression patterns of both genes thus serve as signs of a conserved involvement in early axis formation in amniotes and, possibly, in twinning in mammals as well.

A mathematical model of the interaction between bovine blastocyst developmental stage and progesterone-stimulated uterine factors on differential embryonic development observed on Day 15 of gestation

A complex interaction between the developing bovine embryo and the growth potential of the uterine milieu it inhabits results in an embryo capable of developing past the maternal recognition stage and on to a successful pregnancy. Previously, we observed variation in the lengths of embryos recovered 8 d after bulk transfer of Day 7 in vitro-produced (IVP) blastocysts into the same uterus. Potential causes of the differential embryonic growth were examined and modeled using 2 rounds of bulk (n = 4-6) IVP transfers and recovery of these embryos 8 d later. Morphological and gene expression measurements of the embryos were determined and the progesterone concentration of the cows was measured throughout the reproductive cycle as a reflection of the status of the uterine environment. These data were used to develop and evaluate a model that describes the interaction between the uterine environment and the growth rate of the developing embryo. Expression of 6 trophectoderm genes (IFNT, TKDP1, PAG11, PTGS2, DKK1, and PDPN) was correlated with conceptus length. The model determined that if the embryo develops to blastocyst stage, the uterine environment, driven by progesterone, is a more important component than blastocyst size in the stimulation of embryonic growth rate to ensure adequate interferon tau (IFNT) for pregnancy recognition. We detected an effect of Day 7 progesterone on the expression of all 6 genes, embryonic disc size, and trophectoderm length on Day 15. We also found effects of embryo transfer size on trophectoderm length and expression of IFNT and PAG11 on Day 15. Lower energy balance over the period from transfer to recovery was associated with reduced embryo growth to Day 15, and this effect was independent of progesterone. Energy balance also affected expression of PDPN and TKDP1 on Day 15. We observed an effect of energy balance from transfer to recovery on embryo survival in cows with partial embryo losses, where embryo factors dominate embryo survival, with cows with greater energy balance having lower embryo losses. This effect was independent of energy balance 40 d before transfer and suggests that energy balance has direct, immediate effects on the embryo and maternal environment during this period. Furthermore, energy balance effects on embryo survival in cows with partial embryo losses were largely mediated by expression of TKDP1, PAG11, and PDPN. These results provide candidate signaling pathways for the effect of progesterone and energy balance on embryo growth and survival.

Reprogramming of rabbit induced pluripotent stem cells toward epiblast and chimeric competency using Krüppel-like factors

Rabbit induced pluripotent stem cells (rbiPSCs) possess the characteristic features of primed pluripotency as defined in rodents and primates. In the present study, we reprogrammed rbiPSCs using human Krüppel-like factors (KLFs) 2 and 4 and cultured them in a medium supplemented with fetal calf serum and leukemia inhibitory factor. These cells (designated rbEKA) were propagated by enzymatic dissociation for at least 30 passages, during which they maintained a normal karyotype. This new culturing protocol resulted in transcriptional and epigenetic reconfiguration, as substantiated by the expression of transcription factors and the presence of histone modifications associated with naïve pluripotency. Furthermore, microarray analysis of rbiPSCs, rbEKA cells, rabbit ICM cells, and rabbit epiblast showed that the global gene expression profile of the reprogrammed rbiPSCs was more similar to that of rabbit ICM and epiblast cells. Injection of rbEKA cells into 8-cell stage rabbit embryos resulted in extensive colonization of ICM in 9% early-blastocysts (E3.5), epiblast in 10% mid-blastocysts (E4.5), and embryonic disk in 1.4% pre-gastrulae (E6). Thus, these results indicate that KLF2 and KLF4 triggered the conversion of rbiPSCs into epiblast-like, embryo colonization-competent PSCs. Our results highlight some of the requirements to achieve bona fide chimeric competency.

Whole Transcriptome Analysis of Notochord-Derived Cells during Embryonic Formation of the Nucleus Pulposus

Recapitulation of developmental signals represents a promising strategy for treating intervertebral disc degeneration. During development, embryonic notochord-derived cells (NDCs) are the direct progenitors of cells that populate the adult nucleus pulposus (NP) and are an important source of secreted signaling molecules. The objective of this study was to define global gene expression profiles of NDCs at key stages of embryonic disc formation. NDCs were isolated from Shh-cre;ROSA:YFP mice at embryonic day 12.5 and postnatal day 0, representing opposite ends of the notochord to NP transformation. Differences in global mRNA abundance across this developmental window were established using RNA-Seq. Protein expression of selected molecules was confirmed using immunohistochemistry. Principal component analysis revealed clustering of gene expression at each developmental stage with more than 5000 genes significantly differentially expressed between E12.5 and P0. There was significantly lower mRNA abundance of sonic hedgehog pathway elements at P0 vs E12.5, while abundance of elements of the transforming growth factor-beta and insulin-like growth factors pathways, and extracellular matrix components including collagen 6 and aggrecan, were significantly higher at P0. This study represents the first transcriptome-wide analysis of embryonic NDCs. Results suggest signaling and biosynthesis of NDCs change dramatically as a function of developmental stage.

A pluripotent stem cell-based model for post-implantation human amniotic sac development

Development of the asymmetric amniotic sac—with the embryonic disc and amniotic ectoderm occupying opposite poles—is a vital milestone during human embryo implantation. Although essential to embryogenesis and pregnancy, amniotic sac development in humans remains poorly understood. Here, we report a human pluripotent stem cell (hPSC)-based model, termed the post-implantation amniotic sac embryoid (PASE), that recapitulates multiple post-implantation embryogenic events centered around amniotic sac development. Without maternal or extraembryonic tissues, the PASE self-organizes into an epithelial cyst with an asymmetric amniotic ectoderm-epiblast pattern that resembles the human amniotic sac. Upon further development, the PASE initiates a process that resembles posterior primitive streak development in a SNAI1-dependent manner. Furthermore, we observe asymmetric BMP-SMAD signaling concurrent with PASE development, and establish that BMP-SMAD activation/inhibition modulates stable PASE development. This study reveals a previously unrecognized fate potential of human pluripotent stem cells and provides a platform for advancing human embryology.

Negative uterine asynchrony retards early equine conceptus development and upregulation of placental imprinted genes

IntroductionPlacental imprinted genes appear to be sensitive indicators of an inappropriate pre-implantation environment. This study examined the effects of negative uterine asynchrony after embryo transfer (ET) on early horse embryo development, and yolk-sac membrane expression of DNA methyltransferases (DNMTs) and equine specific placental imprinted genes. MethodsDay 8 embryos were transferred to recipient mares on day 8 (synchronous) or day 3 (asynchronous) after ovulation, and conceptuses were recovered 6 or 11 days later (day 14 or 19 of development). ResultsDay 14 conceptuses recovered from an asynchronous uterus had a smaller embryonic disc, in which primitive streak development was visibly retarded compared to conceptuses from a synchronous uterus. Similarly, length, somite number and organogenesis were retarded in day 19 embryos after asynchronous ET. Maternal (GRB10, H19, IGF2R, PHLDA2) and paternal (IGF2, INSR, PEG3, PEG10, DIO3, NDN, SNRPN) imprinted genes and DNMTs (DNMT1, 3A and 3B) were all up-regulated between day 14 and 19 of pregnancy and, for most, mRNA expression was higher in synchronous than asynchronous day 19 yolk-sac membrane. Expression of the paternally imprinted gene HAT1 increased between day 14 and 19 of pregnancy, but was not affected by the asynchrony. DiscussionConceptus development and upregulation of DNMTs and imprinted genes were delayed rather than dysregulated after transfer into a negatively asynchronous uterus. We propose that this ability to ‘reset’ conceptus development to uterine stage is an adaptation that explains why horse embryos are unusually tolerant of asynchrony after ET.

Gene expression analysis of bovine embryonic disc, trophoblast and parietal hypoblast at the start of gastrulation.

In cattle early gastrulation-stage embryos (Stage 5), four tissues can be discerned: (i) the top layer of the embryonic disc consisting of embryonic ectoderm (EmE); (ii) the bottom layer of the disc consisting of mesoderm, endoderm and visceral hypoblast (MEH); (iii) the trophoblast (TB); and (iv) the parietal hypoblast. We performed microsurgery followed by RNA-seq to analyse the transcriptome of these four tissues as well as a developmentally earlier pre-gastrulation embryonic disc. The cattle EmE transcriptome was similar at Stages 4 and 5, characterised by the OCT4/SOX2/NANOG pluripotency network. Expression of genes associated with primordial germ cells suggest their presence in the EmE tissue at these stages. Anterior visceral hypoblast genes were transcribed in the Stage 4 disc, but no longer by Stage 5. The Stage 5 MEH layer was equally similar to mouse embryonic and extraembryonic visceral endoderm. Our data suggest that the first mesoderm to invaginate in cattle embryos is fated to become extraembryonic. TGFβ, FGF, VEGF, PDGFA, IGF2, IHH and WNT signals and receptors were expressed, however the representative members of the FGF families differed from that seen in equivalent tissues of mouse embryos. The TB transcriptome was unique and differed significantly from that of mice. FGF signalling in the TB may be autocrine with both FGFR2 and FGF2 expressed. Our data revealed a range of potential inter-tissue interactions, highlighted significant differences in early development between mice and cattle and yielded insight into the developmental events occurring at the start of gastrulation.

English

The aim of this study is to demonstrate the teratogenicity of Riparin III in embryos of Gallus gallus (domestic) at an early stage of development. This study is unprecedented in the literature. Chicken eggs were used to investigate the morphometry. All the groups were incubated at 37.2 ± 0.1°C and 60 ± 5% relative humidity for 24 h. The embryonic disc was identified and the eggs of the control group were administered 0.1 ml of saline tamponade (PBS, pH 7.0) and those of the other group were administered Riparin III in 25, 50 and 100 μg/ml doses. The eggs were closed with sterile adhesive strips and incubation was continued for another 24 h. All the eggs were then reopened and the embryos dissected from embryonic membranes and evaluated histopathologically with haematoxylin eosin dye for 15 s and washed with distilled water. An embryological development within twelve stages as classified by Hamburger and Hamilton (1951) was obtained. When compared with the control group, the width of the neural tube of the embryos was modified with all Riparin III concentrations. Longer somites resulted from the 50 and 100 μg/ml Riparina III concentrations. The function of the structures was preserved. This drug can potentially be used in pregnant women as an anxiolytic and for treating depression. Key words: Aniba Riparia, depression, Riparin III, chicken embryo, neural tube defect.

Prosurvival effect of cumulus prostaglandin G/H synthase 2/prostaglandin2 signaling on bovine blastocyst: impact on in vivo posthatching development.

Apoptotic activity is a common physiological process which culminates at the blastocyst stage in the preimplantation embryo of many mammals. The degree of embryonic cell death can be influenced by the oocyte microenvironment. However, the prognostic significance of the incidence of apoptosis remains undefined. Prostaglandin E2 (PGE2) derived from prostaglandin G/H synthase-2 (PTGS2) activity is a well-known prosurvival factor that is mainly studied in oncology. PGE2 is the predominant PTGS2-derived prostaglandin present in the oocyte microenvironment during the periconceptional period. Using an in vitro model of bovine embryo production followed by transfer and collection procedures, we investigated the impact of periconceptional PGE2 on the occurrence of spontaneous apoptosis in embryos and on subsequent in vivo posthatching development. Different periconceptional PGE2 environments were obtained using NS-398, a specific inhibitor of PTGS2 activity, and exogenous PGE2. We assessed the level of embryonic cell death in blastocysts at day 8 postfertilization by counting total cell numbers, by the immunohistochemical staining of active caspase-3, and by quantifying terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling signals and apoptosis regulator (BCL-2/BAX) mRNA expression. Morphometric parameters were used to estimate the developmental stage of the embryonic disk and the extent of trophoblast elongation on day 15 conceptuses. Our findings indicate that periconceptional PGE2 signaling durably impacts oocytes, conferring increased resistance to spontaneous apoptosis in blastocysts and promoting embryonic disk development and the elongation process during preimplantation development.

TGF-β Family Signaling in Drosophila.

The transforming growth factor β (TGF-β) family signaling pathway is conserved and ubiquitous in animals. In Drosophila, fewer representatives of each signaling component are present compared with vertebrates, simplifying mechanistic study of the pathway. Although there are fewer family members, the TGF-β family pathway still regulates multiple and diverse functions in Drosophila. In this review, we focus our attention on several of the classic and best-studied functions for TGF-β family signaling in regulating Drosophila developmental processes such as embryonic and imaginal disc patterning, but we also describe several recently discovered roles in regulating hormonal, physiological, neuronal, innate immunity, and tissue homeostatic processes.

Integrated and Quantitative Proteomics of Human Tumors.

Quantitative proteomics represents a powerful approach for the comprehensive analysis of proteins expressed under defined conditions. These properties have been used to investigate the proteome of disease states, including cancer. It has become a major subject of studies to apply proteomics for biomarker and therapeutic target identification. In the last decades, technical advances in mass spectrometry have increased the capacity of protein identification and quantification. Moreover, the analysis of posttranslational modification (PTM), especially phosphorylation, has allowed large-scale identification of biological mechanisms. Even so, increasing evidence indicates that global protein quantification is often insufficient for the explanation of biology and has shown to pose challenges in identifying new and robust biomarkers. As a consequence, to improve the accuracy of the discoveries made using proteomics in human tumors, it is necessary to combine (i) robust and reproducible methods for sample preparation allowing statistical comparison, (ii) PTM analyses in addition to global proteomics for additional levels of knowledge, and (iii) use of bioinformatics for decrypting protein list. Herein, we present technical specificities for samples preparation involving isobaric tag labeling, TiO2-based phosphopeptides enrichment and hydrazyde-based glycopeptides purification as well as the key points for the quantitative analysis and interpretation of the protein lists. The method is based on our experience with tumors analysis derived from hepatocellular carcinoma, chondrosarcoma, human embryonic intervertebral disk, and chordoma experiments.

Conserved and divergent expression patterns of markers of axial development in the laboratory opossum, Monodelphis domestica.

Previous comparative studies suggest that the requirement for Nodal in epiblast and hypoblast development is unique to mammalians. Expression of anterior visceral endoderm (AVE) genes in the visceral endoderm and of their orthologs in the hypoblast may be unique to mammalians and avians, and is absent in the reptilian hypoblast. Axis formation in reptiles is signaled by the formation of the posterior marginal epiblast (PME), which expresses a series of primitive streak genes. To assess the phylogenetic origin of Nodal and AVE gene expression and axis formation in amniotes, we examined marker gene expression in gray short-tailed opossum, a metatherian. Nodal was expressed in neither epiblast nor hypoblast of opossum embryos. No AVE genes were expressed in the opossum hypoblast. Attainment of polarity in the embryonic disk was signaled by Nodal, Wnt3a, Fgf8, and Bra expression in the PME at 8.5 days post-coitus. Nodal expression in epiblast or hypoblast may be unique to eutherians. AVE gene expression in visceral endoderm and hypoblast may have been independently acquired in eutherian and avian lineages. PME formation appears to be the event that signals axis formation in reptilian and metatherian embryos, and thus may be an ancestral characteristic of basal amniotes. Developmental Dynamics 245:1176-1188, 2016. © 2016 Wiley Periodicals, Inc.

Bovine conceptus of Bos indicus produced by somatic cell nuclear transfer and parthenogenesis present morphological variations since the blastocyst stage

In cattle, embryo development is characterized by the appearance of two distinct cell layers, the trophectoderm and the inner cell mass. The latter will undergo differentiation to form the embryonic disc consisting of the epiblast and hypoblast. The aim of this study was to ultrastructurally characterize the bovine embryo from different in vitro production techniques, with emphasis on trophectoderm and inner cell mass cells. Bovine embryos on day 7 (conception = D1) of pregnancy, derived via in vitro production techniques, were fixed for light and transmission electron microscopy processing. Results suggested that embryos produced by nuclear transfer of somatic cells and parthenogenesis showed significant changes in macroscopic and microscopic structure. Size was reduced, and the inner cell mass had no defined shape. Furthermore, organelles responsible for the absorption processes, communication, growth, and cellular metabolism were fewer and had changes in shape, when compared to results in embryos produced by in vitrofertilization. We concluded that embryos produced by parthenogenesis and SCNT exhibit morphological differences when compared with IVF embryos, such as undeveloped blastocoel, poorly defined distribution of ICM, and morphological differences in organelles.

Overexpression of Indian hedgehog partially rescues short stature homeobox 2-overexpression-associated congenital dysplasia of the temporomandibular joint in mice.

The role of short stature homeobox 2 (shox2) in the development and homeostasis of the temporomandibular joint (TMJ) has been well documented. Shox2 is known to be expressed in the progenitor cells and perichondrium of the developing condyle. A previous study by our group reported that overexpression of shox2 leads to congenital dysplasia of the TMJ via downregulation of the Indian hedgehog (Ihh) signaling pathway, which is essential for embryonic disc primordium formation and mandibular condylar growth. To determine whether overexpression of Ihh may rescue the overexpression of shox2 leading to congenital dysplasia of the TMJ, a mouse model in which Ihh and shox2 were overexpressed (Wnt1-Cre; pMes-stop shox2; pMes-stop Ihh mice) was utilized to assess the consequences of this overexpression on TMJ development during post-natal life. The results showed that the developmental process and expression levels of runt-related transcription factor 2 and sex determining region Y-box 9 in the TMJ of the Wnt1-Cre; pMes-stop shox2; pMes-stop Ihh mice were similar to those in wild-type mice. Overexpression of Ihh rescued shox2 overexpression-associated reduction of extracellular matrix components. However, overexpression of Ihh did not inhibit the shox2 overexpression-associated increase of matrix metalloproteinases (MMPs) MMP9, MMP13 and apoptosis in the TMJ. These combinatory cellular and molecular defects appeared to account for the observed congenital dysplasia of TMJ, suggesting that overexpression of Ihh partially rescued shox2 overexpression-associated congenital dysplasia of the TMJ in mice.

Primary Bovine Extra-Embryonic Cultured Cells: A New Resource for the Study of In Vivo Peri-Implanting Phenotypes and Mesoderm Formation.

In addition to nourishing the embryo, extra-embryonic tissues (EETs) contribute to early embryonic patterning, primitive hematopoiesis, and fetal health. These tissues are of major importance for human medicine, as well as for efforts to improve livestock efficiency, but they remain incompletely understood. In bovines, EETs are accessible easily, in large amounts, and prior to implantation. We took advantage of this system to describe, in vitro and in vivo, the cell types present in bovine EETs at Day 18 of development. Specifically, we characterized the gene expression patterns and phenotypes of bovine extra-embryonic ectoderm (or trophoblast; bTC), endoderm (bXEC), and mesoderm (bXMC) cells in culture and compared them to their respective in vivo micro-dissected cells. After a week of culture, certain characteristics (e.g., gene expression) of the in vitro cells were altered with respect to the in vivo cells, but we were able to identify “cores” of cell-type-specific (and substrate-independent) genes that were shared between in vitro and in vivo samples. In addition, many cellular phenotypes were cell-type-specific with regard to extracellular adhesion. We evaluated the ability of individual bXMCs to migrate and spread on micro-patterns, and observed that they easily adapted to diverse environments, similar to in vivo EE mesoderm cells, which encounter different EE epithelia to form chorion, yolk sac, and allantois. With these tissue interactions, different functions arose that were detected in silico and corroborated in vivo at D21–D25. Moreover, analysis of bXMCs allowed us to identify the EE cell ring surrounding the embryonic disc (ED) at D14-15 as mesoderm cells, which had been hypothesized but not shown prior to this study. We envision these data will serve as a major resource for the future in the analysis of peri-implanting phenotypes in response to the maternal metabolism and contribute to subsequent studies of placental/fetal development in eutherians.