Concerted involvement of Cdx/Hox genes and Wnt signaling in morphogenesis of the caudal neural tube and cloacal derivatives from the posterior growth zone

Decrease in Cdx dosage in an allelic series of mouse Cdx mutants leads to progressively more severe posterior vertebral defects. These defects are corrected by posterior gain of function of the Wnt effector Lef1. Precocious expression of Hox paralogous 13 genes also induces vertebral axis truncation by antagonizing Cdx function. We report here that the phenotypic similarity also applies to patterning of the caudal neural tube and uro-rectal tracts in Cdx and Wnt3a mutants, and in embryos precociously expressing Hox13 genes. Cdx2 inactivation after placentation leads to posterior defects, including incomplete uro-rectal septation. Compound mutants carrying one active Cdx2 allele in the Cdx4-null background (Cdx2/4), transgenic embryos precociously expressing Hox13 genes and a novel Wnt3a hypomorph mutant all manifest a comparable phenotype with similar uro-rectal defects. Phenotype and transcriptome analysis in early Cdx mutants, genetic rescue experiments and gene expression studies lead us to propose that Cdx transcription factors act via Wnt signaling during the laying down of uro-rectal mesoderm, and that they are operative in an early phase of these events, at the site of tissue progenitors in the posterior growth zone of the embryo. Cdx and Wnt mutations and premature Hox13 expression also cause similar neural dysmorphology, including ectopic neural structures that sometimes lead to neural tube splitting at caudal axial levels. These findings involve the Cdx genes, canonical Wnt signaling and the temporal control of posterior Hox gene expression in posterior morphogenesis in the different embryonic germ layers. They shed a new light on the etiology of the caudal dysplasia or caudal regression range of human congenital defects.

Hoxb6 can interfere with somitogenesis in the posterior embryo through a mechanism independent of its rib-promoting activity

Formation of the vertebrate axial skeleton requires coordinated Hox gene activity. Hox group 6 genes are involved in the formation of the thoracic area owing to their unique rib-promoting properties. Here we show that the linker region (LR) connecting the homeodomain and the hexapeptide is essential for Hoxb6 rib-promoting activity in mice. The LR-defective Hoxb6 protein was still able to bind a target enhancer together with Pax3, producing a dominant-negative effect, indicating that the LR brings additional regulatory factors to target DNA elements. We also found an unexpected association between Hoxb6 and segmentation in the paraxial mesoderm. In particular, Hoxb6 can disturb somitogenesis and anterior-posterior somite patterning by dysregulation of Lfng expression. Interestingly, this interaction occurred differently in thoracic versus more caudal embryonic areas, indicating functional differences in somitogenesis before and after the trunk-to-tail transition. Our results suggest the requirement of precisely regulated Hoxb6 expression for proper segmentation at tailbud stages.

Evidence for a Myotomal Hox/Myf Cascade Governing Nonautonomous Control of Rib Specification within Global Vertebral Domains

Hox genes are essential for the patterning of the axial skeleton. Hox group 10 has been shown to specify the lumbar domain by setting a rib-inhibiting program in the presomitic mesoderm (PSM). We have now produced mice with ribs in every vertebra by ectopically expressing Hox group 6 in the PSM, indicating that Hox genes are also able to specify the thoracic domain. We show that the information provided by Hox genes to specify rib-containing and rib-less areas is first interpreted in the myotome through the regional-specific control of Myf5 and Myf6 expression. This information is then transmitted to the sclerotome by a system that includes FGF and PDGF signaling to produce vertebrae with or without ribs at different axial levels. Our findings offer a new perspective of how Hox genes produce global patterns in the axial skeleton and support a redundant nonmyogenic role of Myf5 and Myf6 in rib formation.

Switching Axial Progenitors from Producing Trunk to Tail Tissues in Vertebrate Embryos

The vertebrate body is made by progressive addition of new tissue from progenitors at the posterior embryonic end. Axial extension involves different mechanisms that produce internal organs in the trunk but not in the tail. We show that Gdf11 signaling is a major coordinator of the trunk-to-tail transition. Without Gdf11 signaling, the switch from trunk to tail is significantly delayed, and its premature activation brings the hindlimbs and cloaca next to the forelimbs, leaving extremely short trunks. Gdf11 activity includes activation of Isl1 to promote formation of the hindlimbs and cloaca-associated mesoderm as the most posterior derivatives of lateral mesoderm progenitors. Gdf11 also coordinates reallocation of bipotent neuromesodermal progenitors from the anterior primitive streak to the tail bud, in part by reducing the retinoic acid available to the progenitors. Our findings provide a perspective to understand the evolution of the vertebrate body plan.

Compartment-dependent activities of Wnt3a/β-catenin signaling during vertebrate axial extension

Extension of the vertebrate body results from the concerted activity of many signals in the posterior embryonic end. Among them, Wnt3a has been shown to play relevant roles in the regulation of axial progenitor activity, mesoderm formation and somitogenesis. However, its impact on axial growth remains to be fully understood. Using a transgenic approach in the mouse, we found that the effect of Wnt3a signaling varies depending on the target tissue. High levels of Wnt3a in the epiblast prevented formation of neural tissues, but did not impair axial progenitors from producing different mesodermal lineages. These mesodermal tissues maintained a remarkable degree of organization, even within a severely malformed embryo. However, from the cells that failed to take a neural fate, only those that left the epithelial layer of the epiblast activated a mesodermal program. The remaining tissue accumulated as a folded epithelium that kept some epiblast-like characteristics. Together with previously published observations, our results suggest a dose-dependent role for Wnt3a in regulating the balance between renewal and selection of differentiation fates of axial progenitors in the epiblast. In the paraxial mesoderm, appropriate regulation of Wnt/β-catenin signaling was required not only for somitogenesis, but also for providing proper anterior-posterior polarity to the somites. Both processes seem to rely on mechanisms with different requirements for feedback modulation of Wnt/β-catenin signaling, once segmentation occurred in the presence of high levels of Wnt3a in the presomitic mesoderm, but not after permanent expression of a constitutively active form of β-catenin. Together, our findings suggest that Wnt3a/β-catenin signaling plays sequential roles during posterior extension, which are strongly dependent on the target tissue. This provides an additional example of how much the functional output of signaling systems depends on the competence of the responding cells.

Revisiting the involvement of signaling gradients in somitogenesis

During embryonic development, formation of individual vertebrae requires that the paraxial mesoderm becomes divided into regular segmental units known as somites. Somites are sequentially formed at the anterior end of the presomitic mesoderm (PSM) resulting from functional interactions between the oscillatory activity of signals promoting segmentation and a moving wavefront of tissue competence to those signals, eventually generating a constant flow of new somites at regular intervals. According to the current model for somitogenesis, the wavefront results from the combined activity of two opposing functional gradients in the PSM involving the Fgf, Wnt and retinoic acid (RA) signaling pathways. Here, I use published data to evaluate the wavefront model. A critical analysis of those studies seems to support a role for Wnt signaling, but raise doubts regarding the extent to which Fgf and RA signaling contribute to this process.

Prdm1 acts downstream of a sequential RA, Wnt and Fgf signaling cascade during zebrafish forelimb induction

Vertebrate limb induction is triggered in the lateral plate mesoderm (LPM) by a cascade of signaling events originating in the axial mesoderm. While it is known that Fgf, Wnt and retinoic acid (RA) signals are involved in this cascade, their precise regulatory hierarchy has not been determined in any species. tbx5 is the earliest gene expressed in the limb bud mesenchyme. Recently, another transcription factor, Prdm1, has been shown to be crucial for zebrafish forelimb development. Here, we show that Prdm1 is downstream of RA, Wnt2b and Tbx5 activity. We find that RA activity, but not Fgf signaling, is necessary for wnt2b expression. Fgf signaling is required for prdm1 expression in the fin bud, but is not necessary for the initiation of tbx5 expression. We propose a model in which RA signaling from the somitic mesoderm leads to activation of wnt2b expression in the intermediate mesoderm, which then signals to the LPM to trigger tbx5 expression. tbx5 is required for Fgf signaling in the limb bud leading to activation of prdm1 expression, which in turn is required for downstream activation of fgf10 expression.

Opposing RA and FGF signals control proximodistal vertebrate limb development through regulation of Meis genes.

Vertebrate limbs develop in a temporal proximodistal sequence, with proximal regions specified and generated earlier than distal ones. Whereas considerable information is available on the mechanisms promoting limb growth, those involved in determining the proximodistal identity of limb parts remain largely unknown. We show here that retinoic acid (RA) is an upstream activator of the proximal determinant genes Meis1 and Meis2. RA promotes proximalization of limb cells and endogenous RA signaling is required to maintain the proximal Meis domain in the limb. RA synthesis and signaling range, which initially span the entire lateral plate mesoderm, become restricted to proximal limb domains by the apical ectodermal ridge (AER) activity following limb initiation. We identify fibroblast growth factor (FGF) as the main molecule responsible for this AER activity and propose a model integrating the role of FGF in limb cell proliferation, with a specific function in promoting distalization through inhibition of RA production and signaling.

Memoirs. no. 1-7 ...

Continued as the American anatomical memoirs.

Identifying the molecular phenotype of renal progenitor cells

Although many of the molecular interactions in kidney development are now well understood, the molecules involved in the specification of the metanephric mesenchyme from surrounding intermediate mesoderm and, hence, the formation of the renal progenitor population are poorly characterized. In this study, cDNA microarrays were used to identify genes enriched in the murine embryonic day 10.5 (E10.5) uninduced metanephric mesenchyme, the renal progenitor population, in comparison with more rostral derivatives of the intermediate mesoderm. Microarray data were analyzed using R statistical software to determine accurately genes differentially expressed between these populations. Microarray outliers were biologically verified, and the spatial expression pattern of these genes at E10.5 and subsequent stages of early kidney development was determined by RNA in situ hybridization. This approach identified 21 genes preferentially expressed by the E10.5 metanephric mesenchyme, including Ewing sarcoma homolog, 14-3-3 theta, retinoic acid receptor-alpha, stearoyl-CoA desaturase 2, CD24, and cadherin-11, that may be important in formation of renal progenitor cells. Cell surface proteins such as CD24 and cadherin-11 that were strongly and specifically expressed in the uninduced metanephric mesenchyme and mark the renal progenitor population may prove useful in the purification of renal progenitor cells by FACS. These findings may assist in the isolation and characterization of potential renal stem cells for use in cellular therapies for kidney disease.

Expression analysis of the Epha1 receptor tyrosine kinase and its high-affinity ligands Efna1 and Efna3 during early mouse development

Interaction of Eph receptor tyrosine kinases with their membrane bound ephrin ligands initiates bidirectional signaling events that regulate cell migratory and adhesive behavior. Whole-mount in situ hybridization revealed overlapping expression of the Epha1 receptor and its high-affinity ligands ephrin A1 (Efna1) and ephrin A3 (Efna3) in the primitive streak and the posterior paraxial mesoderm during early mouse development. These results show complex and dynamic expression for all three genes with expression domains that are successively complementary, overlapping, and divergent. (c) 2006 Elsevier B.V. All rights reserved.

Knockdown of zebrafish crim1 results in a bent tail phenotype with defects in somite and vascular development

The Crim1 gene encodes a transmembrane protein containing six cysteine-rich repeats similar to those found in the BMP antagonist, chordin (chd). To investigate its physiological role, zebrafish crim1 was cloned and shown to be both maternally and zygotically expressed during zebrafish development in sites including the vasculature, intermediate cell mass. notochord, and otic vesicle. Bent or hooked tails with U-shaped somites were observed in 85% of morphants from 12 hpf. This was accompanied by a loss of muscle pioneer cells. While morpholino knockdown of crim1 showed some evidence of ventralisation, including expansion of the intermediate cell mass (ICM), reduction in head size bent tails and disruption to the somites and notochord, this did not mimic the classically ventralised phenotype, as assessed by the pattern of expression of the dorsal markers chordin, otx2 and the ventral markers eve1, pax2.1, tall and gata1 between 75% epiboly and six-somites. From 24 hpf, morphants displayed an expansion of the ventral mesoderm-derived ICM, as evidenced by expansion of tall. Imo2 and crim1 itself. Analysis of the crim1 morphant phenotype in Tg(fli:EGFP) fish showed a clear reduction in the endothelial cells forming the intersegmental vessels and a loss of the dorsal longitudinal anastomotic vessel (DLAV). Hence, the primary role of zebrafish crim1 is likely to be the regulation of somitic and vascular development. (c) 2006 Elsevier Ireland Ltd. All rights reserved.

BRACHYURY confers cancer stem cell characteristics on colorectal cancer cells

Cancer stem cells (CSCs) are initiating cells in colorectal cancer (CRC). Colorectal tumours undergo epithelial to mesenchymal transition (EMT)-like processes at the invasive front, enabling invasion and metastasis, and recent studies have linked this process to the acquisition of stem cell-like properties. It is of fundamental importance to understand the molecular events leading to the establishment of cancer initiating cells and how these mechanisms relate to cellular transitions during tumourigenesis. We use an in vitro system to recapitulate changes in CRC cells at the invasive front (mesenchymal-like cells) and central mass (epithelial-like cells) of tumours. We show that the mesoderm inducer BRACHYURY is expressed in a subpopulation of CRC cells that resemble invasive front mesenchymal-like cells, where it acts to impose characteristics of CSCs in a fully reversible manner, suggesting reversible formation and modulation of such cells. BRACHYURY, itself regulated by the oncogene β-catenin, influences NANOG and other 'stemness' markers including a panel of markers defining CRC-CSC whose presence has been linked to poor patient prognosis. Similar regulation of NANOG through BRACHYURY was observed in other cells lines, suggesting this might be a pathway common to cancer cells undergoing mesenchymal transition. We suggest that BRACHYURY may regulate NANOG in mesenchymal-like CRC cells to impose a 'plastic-state', allowing competence of cells to respond to signals prompting invasion or metastasis. Copyright © 2011 UICC.

Reorganisation ofHoxdregulatory landscapes during the evolution of a snake-like body plan

Within land vertebrate species, snakes display extreme variations in their body plan, characterized by the absence of limbs and an elongated morphology. Such a particular interpretation of the basic vertebrate body architecture has often been associated with changes in the function or regulation of Hox genes. Here, we use an interspecies comparative approach to investigate different regulatory aspects at the snake HoxD locus. We report that, unlike in other vertebrates, snake mesoderm-specific enhancers are mostly located within the HoxD cluster itself rather than outside. In addition, despite both the absence of limbs and an altered Hoxd gene regulation in external genitalia, the limb-associated bimodal HoxD chromatin structure is maintained at the snake locus. Finally, we show that snake and mouse orthologous enhancer sequences can display distinct expression specificities. These results show that vertebrate morphological evolution likely involved extensive reorganisation at Hox loci, yet within a generally conserved regulatory framework.

Role of Ccbe1 during cardiac differentiation of mouse ESCs

Tese de Doutoramento, Ciências Biomédicas, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2016