An essential function of the mitogen-activated protein kinase Erk2 in mouse trophoblast development

The closely related mitogen-activated protein kinase isoforms extracellular signal-regulated kinase 1 (ERK1) and ERK2 have been implicated in the control of cell proliferation, differentiation and survival. However, the specific in vivo functions of the two ERK isoforms remain to be analysed. Here, we show that disruption of the Erk2 locus leads to embryonic lethality early in mouse development after the implantation stage. Erk2 mutant embryos fail to form the ectoplacental cone and extra-embryonic ectoderm, which give rise to mature trophoblast derivatives in the fetus. Analysis of chimeric embryos showed that Erk2 functions in a cell-autonomous manner during the development of extra-embryonic cell lineages. We also found that both Erk2 and Erk1 are widely expressed throughout early-stage embryos. The inability of Erk1 to compensate for Erk2 function suggests a specific function for Erk2 in normal trophoblast development in the mouse, probably in regulating the proliferation of polar trophectoderm cells.     

Essential role of the CUL4B ubiquitin ligase in extra-embryonic tissue development during mouse embryogenesis

Mutations of the CUL4B ubiquitin ligase gene are causally linked to syndromic X-linked mental retardation (XLMR). However, the pathogenic role of CUL4B mutations in neuronal and developmental defects is not understood. We have generated mice with targeted disruption of Cul4b, and observed embryonic lethality with pronounced growth inhibition and increased apoptosis in extra-embryonic tissues. Cul4b, but not its paralog Cul4a, is expressed at high levels in extra-embryonic tissues post implantation. Silencing of CUL4B expression in an extra-embryonic cell line resulted in the robust accumulation of the CUL4 substrate p21^Cip1/WAF and G2/M cell cycle arrest, which could be partially rescued by silencing of p21^Cip1/WAF. Epiblast-specific deletion of Cul4b prevented embryonic lethality and gave rise to viable Cul4b null mice. Therefore, while dispensable in the embryo proper, Cul4b performs an essential developmental role in the extra-embryonic tissues. Our study offers a strategy to generate viable Cul4b-deficient mice to model the potential neuronal and behavioral deficiencies of human CUL4B XLMR patients.     

Extra-embryonic endoderm cells derived from ES cells induced by GATA Factors acquire the character of XEN cells

Background  

Three types of cell lines have been established from mouse blastocysts: embryonic stem (ES) cells, trophoblast stem (TS) cells, and extra-embryonic endoderm (XEN) cells, which have the potential to differentiate into their respective cognate lineages. ES cells can differentiate in vitro not only into somatic cell lineages but into extra-embryonic lineages, including trophectoderm and extra-embryonic endoderm (ExEn) as well. TS cells can be established from ES cells by the artificial repression of Oct3/4 or the upregulation of Cdx2 in the presence of FGF4 on feeder cells. The relationship between these embryo-derived XEN cells and ES cell-derived ExEn cell lines remains unclear, although we have previously reported that overexpression of Gata4 or Gata6 induces differentiation of mouse ES cells into extra-embryonic endoderm in vitro.     

A Tlx2‐Cre mouse line uncovers essential roles for hand1 in extraembryonic and lateral mesoderm

Hand1 regulates development of numerous tissues within the embryo, extraembryonic mesoderm, and trophectoderm. Systemic loss of Hand1 results in early embryonic lethality but the cause has remained unknown. To determine if Hand1 expression in extraembryonic mesoderm is essential for embryonic survival, Hand1 was conditionally deleted using the HoxB6‐Cre mouse line that expresses Cre in extraembryonic and lateral mesoderm. Deletion of Hand1 using HoxB6‐Cre resulted in embryonic lethality identical to systemic knockout. To determine if lethality is due to Hand1 function in extraembryonic mesoderm or lateral mesoderm, we generated a Tlx2‐Cre mouse line expressing Cre in lateral mesoderm but not extraembryonic tissues. Deletion of Hand1 using the Tlx2‐Cre line results in embryonic survival with embryos exhibiting herniated gut and thin enteric smooth muscle. Our results show that Hand1 regulates development of lateral mesoderm derivatives and its loss in extraembryonic mesoderm is the primary cause of lethality in Hand1‐null embryos. genesis 48:479–484, 2010. © 2010 Wiley‐Liss, Inc.     

Embryonic development of a collembolan,Tomocerus cuspidatus Börner, 1909: With special reference to the development and developmental potential of serosa (Hexapoda: Collembola,Tomoceridae)

The embryogenesis of a collembolan, Tomocerus cuspidatus, was examined and described, with special reference to the development of serosa and its developmental potential. As a result of cleavage, which starts with holoblastic cleavage and changes to the superficial type, the blastoderm forms. At the center of the dorsal side of the egg, the primary dorsal organ develops. The mesoderm is segregated beneath the entire blastoderm, excluding the primary dorsal organ. The mesoderm then migrates to the presumptive embryonic area, and the embryonic and extra-embryonic areas differentiate. The area lined with mesoderm is the embryo, and that devoid of it is the serosa. Owing to blastokinesis completion, the extra-embryonic area or the serosa is highly stretched, and the serosal cells are often found to undergo mitosis. The serosa possesses the ability to differentiate into the body wall. It was confirmed, in contrast to the previous understanding, that the serosal cells do not degenerate, but participate in the formation of the body wall or definitive dorsal closure. Integrating this newly obtained information and other embryological evidence, the basal splitting of Hexapoda was phylogenetically discussed and reconstructed, and a phylogeny formulated as “Ellipura (= Protura + Collembola) + Cercophora (= Diplura and Ectognatha)” was proposed.     

CUL4B ubiquitin ligase in mouse development: A model for human X-linked mental retardation syndrome?

CUL4B, a member of the cullin-RING ubiquitin ligase family, is frequently mutated in X-linked mental retardation (XLMR) patients. The study by Liu et al. showed that Cul4b plays an essential developmental role in the extra-embryonic tissues, while it is dispensable in the embryo proper during mouse embryogenesis. Viable Cul4b-null mice provide the first animal model to study neuronal and behavioral deficiencies seen in human CUL4B XLMR patients.CUL4 is a member of the cullin-RING ubiquitin ligase family, the largest E3 ligase family, which appears to account for ∼20% of total protein degradation by the ubiquitin-proteasome system^1,2,3. CUL4 is conserved during evolution from yeast to human. In yeast, CUL4 encodes a single gene, but mammalian cells express two closely related paralogs, CUL4A and CUL4B with about 82% sequence identity. CUL4A and CUL4B assemble structurally similar E3 complexes through binding to an adaptor protein (DDB1) and a substrate receptor protein (DCAF) at the N-terminus, and a RING protein RBX1 at the C-terminus (Figure 1), and share functional redundancy in targeting substrates such as p21 and Cdt1 for ubiquitination and degradation^1,2. The Cul4a-null mice are viable and display no abnormal development and growth phenotypes, likely due to functional compensation from Cul4b^4,5. The only phenotype associated with Cul4a abrogation is the reproductive defects seen with male but not female mice, resulting from differential non-overlapping expression patterns of the two Cul4 genes during male meiosis⁶. On the other hand, germline deletion of Cul4b resulted in embryonic lethality around E9.5⁷, indicating a unique function of Cul4b that cannot be compensated by Cul4a during embryogenesis.Open in a separate windowFigure 1Differential expression of Cul4a and Cul4b in the embryo proper and extra embryonic tissues determines their fate. Before implantation, both Cul4a and Cul4b are expressed in the blastocyst. Following implantation, Cul4a is expressed in the embryo proper, but not in extra-embryonic tissues. Upon Cul4b deletion, p21 accumulates in extra-embryonic tissues to induce G2/M arrest and eventually embryonic death due to degeneration of extra-embryonic tissues. Expression of Cul4a in embryo prevents p21 accumulation and subsequent embryonic death.Mental retardation (MR) affects approximately 1%-3% of the population and is about 30% more common in males than in females⁸, suggesting a causal relationship with gene mutations on the X chromosome. To date, mutations in about 100 genes have been identified in X-linked MR (XLMR), much more than those found on autosomes⁹. In 2007, two independent groups reported that mutations of CUL4B (Xq24) ubiquitin ligase gene are associated with XLMR^10,11. CUL4B-deficient patients display a syndrome of delayed puberty, moderate short stature, hypogonadism, relative macrocephaly, central obesity, fine intention tremor, brachydactyly, and large tongue^10,11. Similarly, the neuronal and developmental deficiencies found in XLMR patients with CUL4B mutations are not compensated by CUL4A. The studies of the molecular pathogenesis of human XLMR are lagging partly due to the lack of an animal model for the disease.In the most recent study published in Cell Research, Zhou and coworkers¹² attempted to generate conditional Cul4b knockout mice with targeted deletion of Cul4b at exons 4 and 5, giving rise to a non-functional Cul4b fragment lacking both the DDB1-binding domain and the cullin homology domain for RBX1 recruitment. The chicken-actin (CAG)-Cre was used, which drives Cre-mediated recombination at the early zygote stage, leading to Cul4b deletion in both the embryo proper and extra-embryonic tissues. Like human CUL4B, the mouse Cul4b is also located on the X-chromosome. Intercrossing of male CAG-Cre with female Cul4b^fl/+ revealed that hemizygous deletion of Cul4b causes embryonic lethality. No embryos with the genotype of Cul4b^−/y survived beyond E9.5. Interestingly, the heterozygous Cul4b^+/− embryos also die in the uterus before E13.5, suggesting that the paternal X chromosome undergoes imprinted inactivation with only trace amount, if any, of Cul4b expression remaining in extra-embryonic tissues. Detailed analysis of dissected embryos revealed that dying Cul4b^+/− embryos (E12.5) lack blood supply from the yolk sacs, whereas the Cul4b^−/y embryos (E8.5) showed remarkable reduction in proliferation with growth arrest at G2/M and enhanced apoptosis. The authors went on and investigated why Cul4a failed to compensate the loss of Cul4b, and found a dynamic expression pattern, differing between two forms, during early embryonic development. Prior to implantation, both Cul4 proteins are detectable in the blastocysts. Shortly after implantation, while both forms are expressed in the embryo proper, only Cul4b is expressed in the extra-embryonic tissues. Thus, upon Cul4b deletion, extra-embryonic tissues without Cul4a compensation degenerate, eventually leading to embryonic death. Consistently, when the authors deleted Cul4b in the epiblast using the Sox2-Cre (targeted Cul4b deletion in embryos proper only), viable Cul4b-null mice are produced likely due to Cul4a compensation. Thus, Cul4b is essential for the development of extra-embryonic tissues, but is dispensable for embryogenesis itself.To study the potential underlying mechanism(s) of embryonic lethality upon Cul4b deletion in extra-embryonic tissues, the authors used an extra-embryonic cell line (XEN). Cul4b knockdown induced a remarkable cell cycle arrest at the G2/M phase, consistent with observation made in Cul4b-null embryos, and robust accumulation of p21, a universal inhibitor of cyclin dependent kinase and a known substrate of Cul4¹. To determine whether accumulated p21 is responsible for the G2/M arrest, the authors simultaneously knocked down both Cul4b and p21 in XEN cells and observed a partial abrogation of growth arrest, suggesting that p21 plays a causal role, at least in part. Unfortunately, due to unavailability of anti-mouse p21 antibody specific for immunohistochemical staining, the authors were not able to show if p21 is indeed accumulated in extra-embryonic tissues upon Cul4b deletion. However, whether p21 indeed plays a causal role in embryonic death upon Cul4b deletion can be unequivocally determined by a rescuing experiment in which simultaneous deletion of p21 should abrogate or at least delay embryonic lethality, if it is causal. Nevertheless, the study by Zhou''s group can be summarized as follows. Before implantation, both Cul4a and Cul4b ubiquitin ligases are expressed in the blastocyst (inner cell mass and trophoblast cells). Following embryo implantation, while Cul4b is expressed in both the embryo proper and extra embryonic tissues, Cul4a is only expressed in the embryo proper. The CAG-Cre-driven Cul4b deletion (in both the embryo proper and extra-embryonic tissues) causes significant p21 accumulation in Cul4a non-expressing extra-embryonic tissues, resulting in G2/M arrest, followed by embryonic death due to degeneration of extra-embryonic tissues. On the embryo side, Cul4b deletion has no detrimental consequence, benefiting from the compensatory effect of Cul4a for p21 targeting. The same holds true when Cul4b is deleted driven by embryonic specific Sox2-Cre (Figure 1).It is noteworthy that the studies by Zhou''s group revealed two distinct differences between Cul4b KO mice and CUL4B-associated XLMR patients. First, Cul4b deletion at the zygote stage causes embryonic lethality, whereas XLMR patients with CUL4B mutations live to adulthood. Second, the Cul4b-null allele cannot be transmitted from the mother to the offspring, whereas human XLMR patients inherit X-linked CUL4B mutations from their mothers. Nevertheless, viable Cul4b-null mice (upon epiblast ablation by Sox2-Cre) provide the first mouse model for mechanistic study of human XLMR diseases associated with CUL4B mutations in the following three aspects:First, as noted earlier, human CUL4B XLMR patients have multiple neuronal and developmental defects. An obvious follow-up study will be to use this mouse model for neurological and behavioral analyses to determine whether Cul4b-null mice indeed present some of human XLMR symptoms.Second, this model can also be used to validate whether accumulation of Cul4b substrates during various stages of brain development indeed plays a pathogenic role and contributes to the clinical symptoms of XLMR patients. For instance, WDR5, a recently identified gene affecting general cognitive ability¹³, was found to be a novel nuclear substrate of CUL4B, but not CUL4A¹⁴. Investigation into whether WDR5 is abnormally accumulated upon Cul4b deletion in vivo would rule in or rule out its potential association with human XLMR, although it was not the case in this study using an extra-embryonic cell line in vitro.Third, the viability of Cul4b-null mice upon epiblast-specific deletion provides opportunities to study neuronal specific ablation of Cul4b in association with the pathogenesis of CUL4B-associated XLMR. For example, Cul4b is expressed at high levels in the hippocampus and cerebrum of mouse brains; both regions are affected in MR patients¹⁵. Thus, the use of Cre mouse lines that target the deletion of Cul4b in the entire brain, selected brain areas, or specific neuronal cells in both spatial and temporal manners¹⁶ would reveal potential contributions of particular regions and cell types to the development and symptoms of CUL4B-associated XLMR.A number of questions that warrant future investigation remain unanswered. First, in addition to p21, what are the other Cul4B substrates, which also contribute to degeneration of extra-embryonic tissues upon Cul4b deletion, since simultaneous deletion of p21 only partially rescues the growth defects? Second, besides the difference in tissue/cell specific expression seen in this study, are Cul4a and Cul4b targeting a unique set of substrates non-redundantly, thus differentiating their physiological functions? A related question will be why CUL4A cannot compensate for the loss of CUL4B in CUL4B-associated XLMR patients? Third, what is the pathogenic mechanism for CUL4B-associated XLMR? Is it mainly due to pathological accumulation of many CUL4B substrates? Answers to these questions may offer insights into potential therapeutic strategies for the treatment of CUL4B-associated XLMR patients.In summary, the findings reported by Zhou''s group provide the first convincing evidence that demonstrates an essential role of Cul4b in the development of extra-embryonic tissues during mouse embryogenesis. The viable Cul4b conditional knockout mice, generated in this study, may serve as the first mouse model for future mechanistic studies of neuronal and behavioral deficiencies of human XLMR associated with CUL4B mutations. We look forward to more exciting discoveries of how Cul4b deficiency leads to the development of XLMR in years to come.     

Signaling dynamics of feather tract formation from the chick somatopleure

In the chick, most feathers are restricted to specific areas of the skin, the feather tracts or pterylae, while other areas, such as the apteria, remain bare. In the embryo, the expansion and closure of the somatopleure leads to the juxtaposition of the ventral pteryla, midventral apterium and amnion. The embryonic proximal somatopleural mesoderm is determined to form a feather-forming dermis at 2 days of incubation (E2), while the embryonic distal and the extra-embryonic somatopleure remain open to determination. We found a progressive, lateral expression of Noggin in the embryonic area, and downregulation of Msx1, a BMP4 target gene, with Msx1 expression being ultimately restricted to the most distal embryonic and extra-embryonic somatopleural mesoderm. Msx1 downregulation thus correlates with the formation of the pterylae, and its maintenance to that of the apterium. Suspecting that the inhibition of BMP4 signaling might be linked to the determination of a feather-forming dermis, we grafted Noggin-expressing cells in the distal somatopleure at E2. This elicited the formation of a supplementary pteryla in the midventral apterium. Endogenous Noggin, which is secreted by the intermediate mesoderm at E2, then by the proximal somatopleure at E4, could be sufficient to suppress BMP4 signaling in the proximal somatopleural mesoderm and then in part of the distal somatopleure, thus in turn allowing the formation of the dense dermis of the future pterylae. The same result was obtained with the graft of Shh-producing cells, but Noggin and Shh are both required in order to change the future amnion into a feather-bearing skin. A possible synergistic role of endogenous Shh from the embryonic endoderm remains to be confirmed.     

Confined chorionic mosaicism in prenatal diagnosis

Summary Confined chorionic mosaicism, detected commonly on chorionic villus sampling (CVS) and occasionally in cultured amniotic fluid cells, is described in five pregnancies that showed confined chorionic mosaicism for trisomies 12, 13, 14, 17 and a marker chromosome. Cytogenetic findings in these pregnancies support the conclusion that within chorion some chromosomal mosaicism are confined to the trophectoderm derivatives while others to the extra-embryonic mesoderm. The etiology of confined chorionic mosaicism is discussed in relation to a significant role of multiple cell lineages contributing to the early development of placenta. The need is indicated for the use of both direct and long-term cultures in CVS prenatal diagnosis, and for the confirmatory testing of fetal blood or amniotic fluid in cases where mosaicism is detected in chorionic villi.     

Erk2 signaling and early embryo stem cell self-renewal

Differentiation of the mammalian blastocyst generates two distinct cell lineages: the trophectoderm, which contributes to the trophoblast layers of the placenta, and the inner cell mass, which forms the embryo. We and others recently demonstrated that the MAP kinase ERK2 is essential for trophoblast development. Erk2 mutant embryos fail to form extra-embryonic ectoderm and the ectoplacental cone, suggesting a role for ERK2 activation in the proliferation of trophoblast stem (TS) cells. Previous studies have documented that ERK1/2 activity is dispensable for proliferation of embryonic stem (ES) cells and rather interferes with self-renewal. Thus, signaling by the ERK1/2 MAP kinase pathway appears to be critical for the regulation of self-renewal and propagation of early embryo stem cell populations.     

In Vivo Function and Evolution of the Eutherian-Specific Pluripotency Marker UTF1

Embryogenesis in placental mammals is sustained by exquisite interplay between the embryo proper and placenta. UTF1 is a developmentally regulated gene expressed in both cell lineages. Here, we analyzed the consequence of loss of the UTF1 gene during mouse development. We found that homozygous UTF1 mutant newborn mice were significantly smaller than wild-type or heterozygous mutant mice, suggesting that placental insufficiency caused by the loss of UTF1 expression in extra-embryonic ectodermal cells at least in part contributed to this phenotype. We also found that the effects of loss of UTF1 expression in embryonic stem cells on their pluripotency were very subtle. Genome structure and sequence comparisons revealed that the UTF1 gene exists only in placental mammals. Our analyses of a family of genes with homology to UTF1 revealed a possible mechanism by which placental mammals have evolved the UTF1 genes.     

Requirement for Map2k1 (Mek1) in extra-embryonic ectoderm during placentogenesis

Map2k1(-/-) embryos die at mid-gestation from abnormal development and hypovascularization of the placenta. We now show that this phenotype is associated with a decreased labyrinth cell proliferation and an augmented cell apoptosis. Although the activation of MAP2K1 and MAP2K2 is widespread in the labyrinthine region, MAPK1 and MAPK3 activation is restricted to the cells lining the maternal sinuses, suggesting an important role for the ERK/MAPK cascade in these cells. In Map2k1(-/-) placenta, ERK/MAPK cascade activation is perturbed. Abnormal localization of the syncytiotrophoblasts is also observed in Map2k1(-/-) placenta, even though this cell lineage is specified at the correct time during placentogenesis. The placental phenotype can be rescued in tetraploid experiments. In addition, Map2k1-specific deletion in the embryo leads to normal embryo development and to the birth of viable Map2k1(-/-) mice. Altogether, these data enlighten the essential role of Map2k1 in extra-embryonic ectoderm during placentogenesis. In the embryo, the Map2k1 gene function appears dispensable.     

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.