Current issue: Volume 55, Issue 10-11-12 2011-12-01 http://www.intjdevbiol.com http://www.intjdevbiol.com/web/covers/5510-11-12o.jpg http://www.intjdevbiol.com 2011-12-01

The mammalian heart is a complex organ composed of diverse components and various cell types. Heart organogenesis requires the contribution of distinct pools of heart progenitors positioned in separate embryonic regions and subject to particular developmental signals. Moreover, these embryonic heart lineages have different transcriptional profiles expressing specific genes which activate pathways involved in heart lineage specification. Understanding the molecular control of heart organogenesis has major implications for treating congenital and adult heart diseases since specific heart lineages have been associated with particular human cardiovascular malformations. Collagen and calcium-binding EGF-like domain 1 (Ccbe1) was identified in our laboratory using an Affymetrix GeneChip system approach to identify the transcriptome of chick heart/hemangioblast precursor cells. Here, we present a detailed and systematic analysis of the expression of Ccbe1 during early mouse development using whole-mount in situ hybridization (WISH), immunohistochemistry and histological techniques. Ccbe1 mRNA was initially detected in the early cardiac progenitors of the two bilateral cardiogenic fields (E7.0) and in the cardiogenic mesoderm (E7.5 to E8.0). Ccbe1 mRNA was then persistently detected in the pericardium and transiently expressed in the myocardial tissue of the primitive heart tube (E8.25), being later expressed in the proepicardium. By E9.5, the Ccbe1 and Prox1 proteins were found to be expressed in common regions, including the septum transversum and in the proximity of the anterior cardinal vein. Here, it is shown that Ccbe1 is expressed in the FHF, SHF and proepicardium during heart organogenesis (E7.0 to E8.75). Later in development, Ccbe1 expression is localized in the septum transversum and in the vicinity of the anterior cardinal vein, embryonic structures related to hepatic and lymphatic development, respectively.

João Facucho-OliveiraMargaret BentoJosé-António Belo http://www.intjdevbiol.com 2011-12-01

Hox collinearity is a spectacular phenomenon that has excited life scientists since its discovery in 1978. Two mechanisms have been proposed to explain the spatially sequential pattern of Hox gene expression in animal embryonic development: interactions among Hox genes, or the progressive opening of chromatin in the Hox clusters, from 3' to 5'. A review of the evidence across different species and developmental stages points to the universal involvement of trans-acting factors and cell-cell interactions. The evidence focuses attention on interactions between Hox genes and on the vertebrate somitogenesis clock. These novel conclusions open new perspectives for the field.

Antony J. DurstonHans J. JansenPaul In der RiedenMichiel H. W. Hooiveld http://www.intjdevbiol.com 2011-12-01

Rhox6 is one of the Reproductive Homeobox genes on the X chromosome (Rhox) that is expressed in the placenta and the post-migratory primordial germ cells (PGCs) in the nascent gonad. Despite its novel expression pattern, the significance of Rhox6 expression in the differentiation of these cell types remains unknown. To investigate the role that Rhox6 plays in PGCs, cDNA encoding Rhox6 and short-hairpin (sh) RNA directed against Rhox6 transcripts were introduced by unique expression vectors into a genetically engineered mouse embryonic stem cell (ESC) line. This ESC line expresses enhanced green fluorescent protein (EGFP) under the Oct3/4 promoter, thereby allowing us to monitor the presence of undifferentiated ESCs and PGCs in culture in real time. This ESC line was used to isolate clones that stably expressed Rhox6 cDNA, shRNA against Rhox6 transcripts, or controls. Quantitative RT-PCR results validated that overexpression had been achieved, as well as knockdown of Rhox6 transcripts in these ESC clones. However, these clones exhibited a normal appearance of undifferentiated ESCs and expressed EGFP. Next, these ESC clones were induced to differentiate into PGCs by generating embryoid bodies (EBs) in culture medium without leukemia inhibitory factor. Detection of EGFP expression by fluorescence microscopy and germ cell markers by RT-PCR validated the differentiation of PGCs in EBs. The Rhox6 transgene had little, if any, effect on EGFP expression in EBs, whereas Rhox6 knockdown significantly decreased EGFP expression in EBs. Thus, it is suggested with these results that Rhox6 is necessary for determination of the germ cell lineage.

Chang LiuPaichi TsaiAna-Marie GarcíaBrandon LogemanTetsuya S. Tanaka http://www.intjdevbiol.com 2011-12-01

Immediate early response 2 (Ier2) is a downstream target of fibroblast growth factor (FGF) signaling. In zebrafish, Ier2 is involved in left-right asymmetry establishment and in convergent extension movements. We isolated the Xenopus ier2 gene based on sequence similarity searches using multiple vertebrate species. Xenopus Ier2 has high homology in the N-terminal region to other vertebrate Ier2 proteins, and Xier2 transcripts were observed from oocytes through larval stages. Except for the maternal expression of xier2, the expression of this gene in the marginal region at gastrulation and in somites and the notochord at later stages is similar to the expression pattern of zebrafish ier2. XIer2 knockdown using antisense morpholinos resulted in defects of convergent extension leading to severe neural tube defects; overexpression of Ier2 showed similar, albeit milder phenotypes. Assays in animal cap explants likewise showed inhibition of elongation after blocking XIer2 expression. These results indicate that Xenopus Ier2 is essential for the execution of convergent extension movements during early Xenopus development.

Sung-Kook HongKosuke TanegashimaIgor B. Dawid http://www.intjdevbiol.com 2011-12-01

In amphibian embryos, calcium (Ca2+) signalling is a necessary and sufficient event to induce neural fate. Transient elevations of [Ca2+]i are recorded in neural tissue precursor cells in whole embryos during gastrulation. Using a subtractive cDNA library between control ectoderm (animal caps) and ectoderm induced toward a neural fate by Ca2+ release, we have isolated several Ca2+-induced target genes. Among the isolated genes, Xp54nrb encodes a protein which exhibits the RRM domains characteristic of RNA binding proteins, and is implicated in pre-mRNA splicing steps. Here we show that the Xp54nrb transcripts are expressed throughout early developmental stages, specifically in the neural and sensorial territories and that Xp54nrb could be involved in anterior neural patterning.

Isabelle NeantNina DeisigPierluigi ScerboCatherine LeclercMarc Moreau http://www.intjdevbiol.com 2011-12-01

We previously demonstrated that retinoic acid (RA) induces epidermis to transdifferentiate to mucosal epithelium with goblet cells in chick embryonic cultured skin. To characterize the molecular mechanism of this transdifferentiation process, we used rat embryonic cultured skin and immunohistochemistry to confirm that RA-induced epidermal transdifferentiation accompanies the expression of markers of esophagus epithelium. Because Gbx1, TG2/Gh (transglutaminase2) and TGF-beta2 are reported individually to be induced by RA in cultures of chick embryonic skin, mouse epidermal cells and human hair follicles respectively, here, we investigated whether cooperative interplay of Gbx1, TG2/Gh and TGF-beta2 is required for the transdifferentiation of epidermal cells to mucosal cells. We have shown that expression of Gbx1, TG2/Gh and TGF-beta proteins were all upregulated in RA-induced transdifferentiated skin and that the former two were expressed in the epidermis, while TGF-beta was expressed in the dermis. Inhibitors of the TGF-beta signal pathway partially inhibited transdifferentiation. Overexpression of both hTG2/Gh and mGbx1 together in the epidermis by electroporation resulted in cuboidal cells in the upper cell layers of the epidermis without keratinized layers, although epidermal keratinization was observed in skin by overexpression of either of them. Labeling DNA with BrdU indicated that RA directly transdifferentiated transient amplifying epidermal cells, not stem cells, to mucosal cells. This study showed that coexpression of TG/2 and Gbx1 in the epidermis was required for esophagus-like mucosal transdifferentiation, and that increase in TGF-beta2 expression by RA in the dermis was essential to induce transdifferentiation through epithelial-mesenchymal interaction.

Akiko ObinataKeitarou OsakabeMari YamaguchiIyo MorimotoYoshihiro Akimoto http://www.intjdevbiol.com 2011-12-01

An odd family gene drumstick (drm) encodes a zinc finger protein, and is necessary for the development of the small intestine, an anterior domain of the ectodermal hindgut of Drosophila melanogaster. However, mechanisms that specify the small intestine, as well as gene regulatory pathways leading to transcriptional activation of drm, are still unclear. We found that drm is expressed in two different tissues abutting the anterior end of the hindgut primordium, that is, the posterior-most region of the midgut (endoderm) and basal portion of the Malpighian tubules. A small intestine marker gene, unpaired (upd), begins to be expressed at the anterior-most region of the hindgut primordium that abuts the basal portion of Malpighian tubules, and the upd-positive region expands, resulting in a short tube during stages 11-13. The small intestine develops in both of the mutant embryos, serpent (srp) and Krüppel (Kr), that lack the drm-positive midgut or Malpighian tubules, respectively, while it fails to develop in the Kr srp double-mutant embryos that lack both of the drm-positive tissues. These results demonstrate that drm expressed in the abutting tissues cell-non-autonomously induces development of the small intestine in the hindgut primordium, probably by deploying some extracellular signaling factor. drm expression in the posterior gut region disappears and the small intestine fails to form in tailless (tll) mutant embryos, whereas over-expression of tll causes expansion of drm expression throughout the midgut, inducing a longer small intestine. These results indicate that drm is activated under the control of tll and triggers development of the small intestine cell-non-autonomously through some extracellular signaling.

Sarder N. UddinMasahiro YanoRyutaro Murakami http://www.intjdevbiol.com 2011-12-01

The role of the prion protein (PrP) in transmissible spongiform encephalopathies has been the focus of intense investigation. However, less is known about the physiological function of normal cellular PrP (PrPC). In adult human teeth, PrPC has been identified in odontoblasts, cementoblasts and epithelial remnants of Malassez. In this study, we have localized PrPC in developing human and mouse teeth, and investigated the function of PrP using a PrP-knockout (Prnp0/0 ) mouse model. PrPC was detected in developing human and mouse ameloblasts and odontoblasts. In vitro, undifferentiated dental mesenchymal cells from embryonic day 18 (E18) Prnp0/0 mouse molars proliferated much more rapidly compared to age-matched, wild-type (wt) mouse molar dental mesenchymal cells. Histochemistry and immunohistochemical analyses showed a subtle but measurable phenotype, with the absence of PrP resulting in earlier initiation of both dentin and enamel formation. Consistent with this finding, laser microdissected odontoblasts from newborn Prnp0/0 mouse incisors had a reduced proliferation rate, as measured by the expression of proliferating cell nuclear antigen (PCNA), and increased type 1 collagen mRNA expression. Dentin microhardness of the fully erupted molars was reduced and incisal enamel mineralization was delayed in Prnp0/0 compared to age-matched wt mouse teeth. Taken together, these results suggest that PrPC affects multiple processes involved in tooth formation, through regulating the differentiation of ameloblasts and odontoblasts.

Yan ZhangSeong-Oh KimSibylle Opsahl-VitalSunita P. HoJean-Baptiste SouronCharles KimKurt GilesPamela K. Den Besten http://www.intjdevbiol.com 2011-12-01

The angiogenic process is precisely regulated by different molecular mechanisms, with a balance between stimulatory and inhibitory factors in embryonic development. Transmembrane proteins of the ADAM (a disintegrin and metalloprotease) family play a critical role in embryogenesis and are involved in protein ectodomain shedding, as well as cell-cell and cell-matrix interactions. In the present study, we found that ADAM17 is expressed spatiotemporally in the tectal layers during chicken embryonic development. To investigate the effect of ADAM17 overexpression on angiogenesis, chicken ADAM17 plasmids were transfected into the developing tectum in vivo by electroporation. Results showed that overexpression of ADAM17 induces morphological changes of brain microvessels, such as an increase in diameter, of capillary sprouting from radial microvessels and an increase in the number of pericytes, but not of endothelial cells. Our data suggest that overexpression of ADAM17 in the developing tectum promotes angiogenesis by increasing the number of pericytes and capillary sprouting in the radial vessels.

Juntang LinCornelius LemkeChristoph RediesXin YanEilhard MixArndt RolfsJiankai Luo http://www.intjdevbiol.com 2011-12-01

With the increased use of gene expression profiling to identify molecular regulators of cellular and developmental mechanisms, developmental biologists face a new challenge in dissecting tissues without cross-contamination or change in RNA profile, and with intact RNA integrity. We have developed a technique that overcomes these problems. We took the dissection of rudimentary mouse embryonic mammary glands as an example, as these structures are particularly difficult to separate from their contiguous ectoderm and strongly adhering mesenchyme. Contrary to conventional enzymatic tissue-separation methods, we blocked transcriptional activity prior to dissection and protected RNA from degradation during dissection, by the use of RNAlater. While RNAlater dehydrates specimens so severely that it interferes with visibility and clean dissection of organs or tissues, we established rehydration conditions that in fact facilitated tissue separation and shortened dissection time to about 10 minutes. The extracted RNA had an excellent quality, rendering it perfectly suitable for transcriptional profiling. Visual inspection of separated tissues and tissue specific gene expression analysis by microarray and RT-PCR confirmed that the tissues were separated with minimal or no cross-contamination. We show that this dissection method can be applied to a broad variety of organs, and that the tissue is still amenable to protein detection. In conclusion, this is a rapid, cheap and effective non-enzymatic tissue separation method which greatly facilitates the exploration of molecular mechanisms in organ formation.

Li SunMay-Yin LeeJacqueline M. Veltmaat http://www.intjdevbiol.com 2011-12-01

The transformer-2 gene is involved in sex determination in tephritid flies (Tephritidae). It is required for the auto-regulation of the transformer gene (the memory device for sex determination in these insects) and for the female-specific splicing of doublesex pre-mRNA, the last gene in the sex determination gene cascade. The present manuscript addressed the question of the functional conservation of the tephritid Anastrepha Tra2 protein to direct sexual development in Drosophila (Drosophilidae). To express this protein in Drosophila, the GAL4-UAS system was used. The Anastrepha Tra2 protein supplies tra-2 function in Drosophila: this protein would form a complex with the endogenous Drosophila Tra protein to promote the female-specific splicing of the Drosophila doublesex pre-mRNA. The feminisation produced by the Anastrepha Tra2 protein was, however, partial.

Francesca SarnoMaría-Fernanda RuizLucas Sánchez http://www.intjdevbiol.com 2011-12-01

Uncoordinated-5 homologs 1-4 (UNC5H1-4) transmembrane netrin receptors are reported to control a number of cellular processes, including axonal guidance, angiogenesis and cell proliferation. These receptors are known as "dependence receptors" because they are able to induce apoptosis in the absence of their ligand, netrin. We have recently reported the localization of netrin-1 and its uncoordinated-5-B (UNC5B) receptor in both villous and extravillous cytotrophoblasts in the human placenta. However, the roles that netrin-1 and UNC5B play in the development of the placenta, as well as the regulation of their expression during the early stages of placental development, remain unexplored. Placental explants were used to demonstrate a proliferative effect of netrin-1 on cytotrophoblasts, as assessed by Ki67 staining. Primary cytotrophoblasts collected at different gestational ages during the first trimester of pregnancy indicated that netrin-1 mRNA expression decreased after 6 weeks of gestation (wg), whereas UNC5B expression increased gradually up to 13-14 wg. The BeWo cell line was used to evaluate the effect of hypoxia on the expression of netrin-1 and UNC5B. Primary cytotrophoblast and BeWo cells cultured under hypoxic conditions exhibited a decrease in the expression of UNC5B both at the mRNA and protein levels; in contrast, hypoxia induced no change in the levels of netrin-1. When hypoxia-inducible factor 1α (HIF-1α) was knocked down by siRNA, we found a significant increase in UNC5B expression, indicating that the HIF-1 pathway is involved in hypoxia-induced UNC5B transcriptional down-regulation. Altogether, these results demonstrate the role of netrin-1 as a new mitogenic factor for cytotrophoblastic cells, report the pattern of expression of netrin-1 and its receptor, UNC5B, in the human placenta during the first trimester of pregnancy, and bring insights into the direct control of the expression of UNC5B by HIF-1.

Mbarka Dakouane-GiudicelliNadia AlfaidyPerrine BayleAlexandre Tassin de NonnevilleVivien StuderPatrick RozenbergPhilippe de Mazancourt http://www.intjdevbiol.com 2011-12-01

Pdzrn3, a member of the PDZRN/SEMCAP/LNX protein family containing a RING finger and two PDZ domains, has been implicated in myoblast and osteoblast differentiation. However, its spatio-temporal expression pattern during embryonic development has not been defined. Here, we describe the cloning and expression pattern of pdzrn3 during zebrafish development. We found that in addition to being expressed in several mesodermal structures, this gene displays specific expression in the central nervous system including rhombomere 1, ventral retina, thalamus and motor neurons, indicating a novel function during neural development. In particular, the absence of expression of pdzrn3 in the ventral retina of noi mutant fish suggests a possible role for this gene in regulating fasciculation and/or navigation of retinal ganglion cell axons.

Luciana DenteGaia GestriMichael TsangTetsuhiro KudohStephen W. WilsonIgor B. DawidMassimiliano Andreazzoli http://www.intjdevbiol.com 2011-12-01

Embryonic stem cell studies have generated great interest, due to their ability to form a wide variety of matured cells. However, there remains a poor understanding of mechanisms regulating the cell state of embryonic stem cells (ESCs) and of the genes they express during early differentiation. Gene expression analysis may be a valuable tool to elucidate either the molecular pathways involved in self-renewal and pluripotency, or early differentiation and to identify potential molecular therapy targets. The aim of this study was to characterize at the molecular level the undifferentiated mouse ESC state and the early development towards embryoid bodies. To attempt this issue, we performed CodeLink Mouse Uniset I 20K bioarrays in a well-characterized mouse ESC line, MES3, 3- and 7 day-old embryoid bodies and we compared our findings with those in adult tissue cells. Gene expression results were subsequently validated in a commercial stem cell line, CGR8 (ATCC). Significance Analysis of Microarrays (SAM) was used to identify statistically significant changes in microarray data. We identified 3664 genes expressed at significantly greater levels in MES3 stem cells than in adult tissue cells, which included 611 with 3-fold higher gene expression levels versus the adult cells. We also investigated the gene expression profile during early embryoid body formation, identifying 2040 and 2243 genes that were up-regulated in 3- and 7- day-old embryoid bodies, respectively. Our gene expression results in MES3 cells were partially confirmed in CGR8 cells, showing numerous genes that are expressed in both mouse stem cells. In conclusion, our results suggest that commonly expressed genes may be strong candidates for involvement in the maintenance of a pluripotent and undifferentiated phenotype and in early development.

Juan SainzFernando García-AlcaldeArmando BlancoÁngel Concha