Isabel Palmeirim

Avian hairy Gene Expression Identifies a Molecular Clock Linked to Vertebrate Segmentation and Somitogenesis

We have identified and characterized c-hairy1, an avian homolog of the Drosophila segmentation gene, hairy. c-hairy1 is strongly expressed in the presomitic mesoderm, where its mRNA exhibits cyclic waves of expression whose temporal periodicity corresponds to the formation time of one somite (90 min). The apparent movement of these waves is due to coordinated pulses of c-hairy1 expression, not to cell displacement along the anteroposterior axis, nor to propagation of an activating signal. Rather, the rhythmic c-hairy mRNA expression is an autonomous property of the paraxial mesoderm. These results provide molecular evidence for a developmental clock linked to segmentation and somitogenesis of the paraxial mesoderm, and support the possibility that segmentation mechanisms used by invertebrates and vertebrates have been conserved.

Monocarboxylate transporters (MCTs) in gliomas: Expression and exploitation as therapeutic targets

BACKGROUND Gliomas exhibit high glycolytic rates, and monocarboxylate transporters (MCTs) play a major role in the maintenance of the glycolytic metabolism through the proton-linked transmembrane transport of lactate. However, their role in gliomas is poorly studied. Thus, we aimed to characterize the expression of MCT1, MCT4, and their chaperone CD147 and to assess the therapeutic impact of MCT inhibition in gliomas. METHODS MCTs and CD147 expressions were characterized by immunohistochemistry in nonneoplastic brain and glioma samples. The effect of CHC (MCT inhibitor) and MCT1 silencing was assessed in in vitro and in vivo glioblastoma models. RESULTS MCT1, MCT4, and CD147 were overexpressed in the plasma membrane of glioblastomas, compared with diffuse astrocytomas and nonneoplastic brain. CHC decreased glycolytic metabolism, migration, and invasion and induced cell death in U251 cells (more glycolytic) but only affected proliferation in SW1088 (more oxidative). The effectiveness of CHC in glioma cells appears to be dependent on MCT membrane expression. MCT1 downregulation showed similar effects on different glioma cells, supporting CHC as an MCT1 inhibitor. There was a synergistic effect when combining CHC with temozolomide treatment in U251 cells. In the CAM in vivo model, CHC decreased the size of tumors and the number of blood vessels formed. CONCLUSIONS This is the most comprehensive study reporting the expression of MCTs and CD147 in gliomas. The MCT1 inhibitor CHC exhibited anti-tumoral and anti-angiogenic activity in gliomas and, of importance, enhanced the effect of temozolomide. Thus, our results suggest that development of therapeutic approaches targeting MCT1 may be a promising strategy in glioblastoma treatment.

Uncoupling segmentation and somitogenesis in the chick presomitic mesoderm.

Little is known about the tissue interactions and the molecular signals implicated in the sequence of events leading to the subdivision of the somite into its rostral and caudal compartments. It has been demonstrated that rostrocaudal identity of the sclerotome is acquired at the presomitic (PSM) level. However, it is not known whether this compartment specification is fully determined in the PSM or whether it is dependent upon maintenance cues from the surrounding environment, as is the case for somite epithelialization. In this report, we address this issue by examining the expression profiles of C-Delta-1 and C-Notch-1, the avian homologues of mouse Delta-like1 (Delta1) and Notch1 which have been implicated in the specification of the somite rostrocaudal polarity in mouse. In chick, these genes are expressed in distinct but partially overlapping domains in the PSM and subsequently in the caudal regions of the somites. We have used an in vitro assay that consists of culturina PSM explants to examine the regulation of these genes in this tissue. We find that PSM explants cultured without overlying ectoderm continue to lay down stripes of C-Delta-1 expression, although epithelialization is blocked. These results suggest that somite rostrocaudal patterning is an autonomous property of the PSM. In addition, they demonstrate that segmentation is not necessarily coupled with the formation of somites.

terra is a left–right asymmetry gene required for left–right synchronization of the segmentation clock

To establish the vertebrate body plan, it is fundamental to create left–right asymmetry in the lateral-plate mesoderm to correctly position the organs. However, it is also crucial to maintain symmetry between the left and the right sides of the presomitic mesoderm, ensuring the allocation of symmetrical body structures, such as the axial skeleton and skeletal muscles. Here, we show that terra is an early left-sided expressed gene that links left–right patterning with bilateral synchronization of the segmentation clock.

Redefining the role of ectoderm in somitogenesis: a player in the formation of the fibronectin matrix of presomitic mesoderm.

The absence of ectoderm impairs somite formation in cultured presomitic mesoderm (PSM) explants, suggesting that an ectoderm-derived signal is essential for somitogenesis. Here we show in chick that the standard enzymatic treatments used for explant isolation destroy the fibronectin matrix surrounding the anterior PSM, which fails to form somites when cultured for 6 hours. By contrast, explants isolated with collagenase retain their fibronectin matrix and form somites under identical culture conditions. The additional presence of ectoderm enhances somite formation, whereas endoderm has no effect. Furthermore, we show that pancreatin-isolated PSM explants cultured in fibronectin-supplemented medium, form significantly more somites than control explants. Interestingly, ectoderm is the major producer of fibronectin (Fn1) transcripts, whereas all but the anterior-most region of the PSM expresses the fibronectin assembly receptor, integrinα 5 (Itga5). We thus propose that the ectoderm-derived fibronectin is assembled by mesodermal α5β1 integrin on the surface of the PSM. Finally, we demonstrate that inhibition of fibronectin fibrillogenesis in explants with ectoderm abrogates somitogenesis. We conclude that a fibronectin matrix is essential for morphological somite formation and that a major, previously unrecognised role of ectoderm in somitogenesis is the synthesis of fibronectin.

Sonic hedgehog in temporal control of somite formation

Vertebrate embryo somite formation is temporally controlled by the cyclic expression of somitogenesis clock genes in the presomitic mesoderm (PSM). The somitogenesis clock is believed to be an intrinsic property of this tissue, operating independently of embryonic midline structures and the signaling molecules produced therein, namely Sonic hedgehog (Shh). This work revisits the notochord signaling contribution to temporal control of PSM segmentation by assessing the rate and number of somites formed and somitogenesis molecular clock gene expression oscillations upon notochord ablation. The absence of the notochord causes a delay in somite formation, accompanied by an increase in the period of molecular clock oscillations. Shh is the notochord-derived signal responsible for this effect, as these alterations are recapitulated by Shh signaling inhibitors and rescued by an external Shh supply. We have characterized chick smoothened expression pattern and have found that the PSM expresses both patched1 and smoothened Shh signal transducers. Upon notochord ablation, patched1, gli1, and fgf8 are down-regulated, whereas gli2 and gli3 are overexpressed. Strikingly, notochord-deprived PSM segmentation rate recovers over time, concomitant with raldh2 overexpression. Accordingly, exogenous RA supplement rescues notochord ablation effects on somite formation. A model is presented in which Shh and RA pathways converge to inhibit PSM Gli activity, ensuring timely somite formation. Altogether, our data provide evidence that a balance between different pathways ensures the robustness of timely somite formation and that notochord-derived Shh is a component of the molecular network regulating the pace of the somitogenesis clock.

Dynamic 3D Cell Rearrangements Guided by a Fibronectin Matrix Underlie Somitogenesis

Somites are transient segments formed in a rostro-caudal progression during vertebrate development. In chick embryos, segmentation of a new pair of somites occurs every 90 minutes and involves a mesenchyme-to-epithelium transition of cells from the presomitic mesoderm. Little is known about the cellular rearrangements involved, and, although it is known that the fibronectin extracellular matrix is required, its actual role remains elusive. Using 3D and 4D imaging of somite formation we discovered that somitogenesis consists of a complex choreography of individual cell movements. Epithelialization starts medially with the formation of a transient epithelium of cuboidal cells, followed by cell elongation and reorganization into a pseudostratified epithelium of spindle-shaped epitheloid cells. Mesenchymal cells are then recruited to this medial epithelium through accretion, a phenomenon that spreads to all sides, except the lateral side of the forming somite, which epithelializes by cell elongation and intercalation. Surprisingly, an important contribution to the somite epithelium also comes from the continuous egression of mesenchymal cells from the core into the epithelium via its apical side. Inhibition of fibronectin matrix assembly first slows down the rate, and then halts somite formation, without affecting pseudopodial activity or cell body movements. Rather, cell elongation, centripetal alignment, N-cadherin polarization and egression are impaired, showing that the fibronectin matrix plays a role in polarizing and guiding the exploratory behavior of somitic cells. To our knowledge, this is the first 4D in vivo recording of a full mesenchyme-to-epithelium transition. This approach brought new insights into this event and highlighted the importance of the extracellular matrix as a guiding cue during morphogenesis.

Neural crest ontogeny during secondary neurulation: a gene expression pattern study in the chick embryo

In the prospective lumbo-sacral region of the chick embryo, neurulation is achieved by cavitation of the medullary cord, a process called secondary neurulation. Neural crest cells (NCC) are generated in this region and they give rise to the same types of derivatives as in more rostral parts of the trunk where neurulation occurs by dorsal fusion of the neural plate borders (primary neurulation). However, no molecular data were available concerning the different steps of their ontogeny. We thus performed a detailed expression study of molecular players likely to participate in the generation of secondary NCC in chick embryos between Hamburger and Hamilton stages 18-20 (HH18-20) at the level of somites 30 to 43. We found that specification of secondary NCC involves, as in primary neurulation, the activity of several transcription factors such as Pax3, Pax7, Snail2, FoxD3 and Sox9, which are all expressed in the dorsal secondary neural tube as soon as full cavitation is achieved. Moreover, once specification has occurred, emigration of NCC from the dorsal neuroepithelium starts facing early dissociating somites and involves a series of changes in cell shape and adhesion, as well as interactions with the extracellular matrix. Furthermore, Bmp4 and Wnt1 expression precedes the detection of migratory secondary NCC and is coincident with maturation of adjacent somites. Altogether, this first study of molecular aspects of secondary NCC ontogeny has revealed that the mechanisms of neural crest generation occurring along the trunk region of the chick embryo are generally conserved and independent of the type of neurulation involved.

Getting a handle on embryo limb development: molecular interactions driving limb outgrowth and patterning

Development of the vertebrate embryo involves multiple segmentation processes to generate a functional, articulated organism. Cell proliferation, differentiation and patterning involve spatially and temporally regulated gene expression and signal transduction mechanisms. The developing vertebrate limb is an excellent model to study such fine-tuned regulations, whereby cells proliferate and are differentially sculptured along the proximal-distal, anterior-posterior and dorsal-ventral axes to form a functional limb. Complementary experimental approaches in different organisms have enhanced our knowledge on the molecular events underlying limb development. Herein, we summarize the current knowledge of the main signaling mechanisms governing vertebrate limb initiation, outgrowth, specification of limb segments and termination.

Comprehensive analysis of fibroblast growth factor receptor expression patterns during chick forelimb development

Specific interactions between fibroblast growth factors (Fgf1-22) and their tyrosine kinase receptors (FgfR1-4) activate different signalling pathways that are responsible for the biological processes in which Fgf signalling is implicated during embryonic development. In the chick, several Fgf ligands (Fgf2, 4, 8, 9, 10, 12, 13 and 18) and the four FgfRs (FgfR 1, 2, 3 and 4) have been reported to be expressed in the developing limb. The precise spatial and temporal expression of these transcripts is important to guide the limb bud to develop into a wing/leg. In this paper, we present a detailed and systematic analysis of the expression patterns of FgfR1, 2, 3 and 4 throughout chick wing development, by in situ hybridisation on whole mounts and sections. Moreover, we characterize for the first time the different isoforms of FGFR1-3 by analysing their differential expression in limb ectoderm and mesodermal tissues, using RT-PCR and in situ hybridisation on sections. Finally, isoform-specific sequences for FgfR1IIIb, FgfR1IIIc, FgfR3IIIb and FgfR3IIIc were determined and deposited in GenBank with the following accession numbers: GU053725, GU065444, GU053726, GU065445, respectively.