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Local knowledge has been proposed as a place-based tool to ground-truth climate models and to narrow their geographic sensitivity. To assess the potential role of local knowledge in our quest to understand better climate change and its impacts, we first need to critically review the strengths and weaknesses of local knowledge of climate change and the potential complementarity with scientific knowledge. With this aim, we conducted a systematic, quantitative meta-analysis of published peer-reviewed documents reporting local indicators of climate change (including both local observations of climate change and observed impacts on the biophysical and the social systems). Overall, primary data on the topic are not abundant, the methodological development is incipient, and the geographical extent is unbalanced. On the 98 case studies documented, we recorded the mention of 746 local indicators of climate change, mostly corresponding to local observations of climate change (40%), but also to observed impacts on the physical (23%), the biological (19%), and the socioeconomic (18%) systems. Our results suggest that, even if local observations of climate change are the most frequently reported type of change, the rich and fine-grained knowledge in relation to impacts on biophysical systems could provide more original contributions to our understanding of climate change at local scale., Competing Interests: The authors declare they do not have any conflict of interest.

P53 is a key regulator of many cellular processes and is negatively regulated by the human homolog of murine double minute-2 (MDM2) E3 ubiquitin ligase. Single nucleotide polymorphisms (SNPs) of either gene alone, and in combination, are linked to cancer susceptibility, disease progression, and therapy response. We analyzed the interaction of TP53 R72P and MDM2 SNP309 SNPs in relationship to outcome in patients with myelodysplastic syndromes (MDS). Sanger sequencing was performed on DNA isolated from 208 MDS cases. Utilizing a novel functional SNP scoring system ranging from +2 to -2 based on predicted p53 activity, we found statistically significant differences in overall survival (OS) (p = 0.02) and progression-free survival (PFS) (p = 0.02) in non-del(5q) MDS patients with low functional scores. In univariate analysis, only IPSS and the functional SNP score predicted OS and PFS in non-del(5q) patients. In multivariate analysis, the functional SNP score was independent of IPSS for OS and PFS. These data underscore the importance of TP53 R72P and MDM2 SNP309 SNPs in MDS, and provide a novel scoring system independent of IPSS that is predictive for disease outcome.

International Prognostic Scoring Systems are used to determine the individual risk profile of myelodysplastic syndrome patients. For the assessment of International Prognostic Scoring Systems, an adequate chromosome banding analysis of the bone marrow is essential. Cytogenetic information is not available for a substantial number of patients (5%-20%) with dry marrow or an insufficient number of metaphase cells. For these patients, a valid risk classification is impossible. In the study presented here, the International Prognostic Scoring Systems were validated based on fluorescence in situ hybridization analyses using extended probe panels applied to cluster of differentiation 34 positive (CD34(+)) peripheral blood cells of 328 MDS patients of our prospective multicenter German diagnostic study and compared to chromosome banding results of 2902 previously published patients with myelodysplastic syndromes. For cytogenetic risk classification by fluorescence in situ hybridization analyses of CD34(+) peripheral blood cells, the groups differed significantly for overall and leukemia-free survival by uni- and multivariate analyses without discrepancies between treated and untreated patients. Including cytogenetic data of fluorescence in situ hybridization analyses of peripheral CD34(+) blood cells (instead of bone marrow banding analysis) into the complete International Prognostic Scoring System assessment, the prognostic risk groups separated significantly for overall and leukemia-free survival. Our data show that a reliable stratification to the risk groups of the International Prognostic Scoring Systems is possible from peripheral blood in patients with missing chromosome banding analysis by using a comprehensive probe panel (clinicaltrials.gov identifier:01355913)., (Copyright© Ferrata Storti Foundation.)

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., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The casein kinase 1A1 gene (CSNK1A1) is a putative tumor suppressor gene located in the common deleted region for del(5q) myelodysplastic syndrome (MDS). We generated a murine model with conditional inactivation of Csnk1a1 and found that Csnk1a1 haploinsufficiency induces hematopoietic stem cell expansion and a competitive repopulation advantage, whereas homozygous deletion induces hematopoietic stem cell failure. Based on this finding, we found that heterozygous inactivation of Csnk1a1 sensitizes cells to a CSNK1 inhibitor relative to cells with two intact alleles. In addition, we identified recurrent somatic mutations in CSNK1A1 on the nondeleted allele of patients with del(5q) MDS. These studies demonstrate that CSNK1A1 plays a central role in the biology of del(5q) MDS and is a promising therapeutic target., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The Hoxd(Del(11-13)) mutant is one of the animal models for human synpolydactyly, characterized by short and syndactylous digits. Here we have characterized in detail the cartilage and bone defects in these mutants. We report two distinct phenotypes: (i) a delay and change in pattern of chondrocyte maturation of metacarpals/metatarsals and (ii) formation of a poor and not centrally positioned primary ossification center in the proximal-intermediate phalanx. In the metacarpals of Hoxd(Del(11-13)) mutants, ossification occurs postnataly, in the absence of significant Ihh expression and without the establishment of growth plates, following patterns similar to those of short bones. The strong downregulation in Ihh expression is associated with a corresponding increase of the repressor form of Gli3. To evaluate the contribution of this alteration to the phenotype, we generated double Hoxd(Del(11-13));Gli3 homozygous mutants. Intriguingly, these double mutants showed a complete rescue of the phenotype in metatarsals but only partial phenotypic rescue in metacarpals. Our results support Hox genes being required in a dose-dependent manner for long bone cartilage maturation and suggest that and excess of Gli3R mediates a significant part of the Hoxd(Del(11-13)) chondrogenic phenotype., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Limb innervation is established by opposing Hox-dependent activities. In this issue of Developmental Cell, Jung et al. (2014) show that Hoxc9 restriction of Foxp1, high levels of which specify limb-innervating motor neurons, first appeared in vertebrates concomitantly with paired appendages. Spatial control of this activity shapes neural networks controlling locomotion patterns., (Copyright © 2014 Elsevier Inc. All rights reserved.)

It has long been known that Hox genes are central players in patterning the vertebrate axial skeleton. Extensive genetic studies in the mouse have revealed that the combinatorial activity of Hox genes along the anterior-posterior body axis specifies different vertebral identities. In addition, Hox genes were instrumental for the evolutionary diversification of the vertebrate body plan. In this review, we focus on fundamental questions regarding the intricate mechanisms controlling Hox gene activity. In particular, we discuss the functional relevance of the precise timing of Hox gene activation in the embryo. Moreover, we provide insight into the epigenetic regulatory mechanisms that are likely to control this process and are responsible for the maintenance of spatially restricted Hox expression domains throughout embryonic development. We also analyze how specific features of each Hox protein may contribute to the functional diversity of Hox family. Altogether, the work reviewed here further supports the notion that the Hox program is far more complex than initially assumed. Exciting new findings will surely emerge in the years ahead., (Copyright © 2013 Wiley Periodicals, Inc.)

Cytogenetic aberrations identified by metaphase cytogenetics (MC) have diagnostic, prognostic, and therapeutic implications in myelodysplastic syndromes (MDS). However, in some MDS patients MC study is unsuccesful. Single nucleotide polymorphism array (SNP-A) based karyotyping could be helpful in these cases. We performed SNP-A in 62 samples from bone marrow or peripheral blood of primary MDS with an unsuccessful MC study. SNP-A analysis enabled the detection of aberrations in 31 (50%) patients. We used the copy number alteration information to apply the International Prognostic Scoring System (IPSS) and we observed differences in survival between the low/intermediate-1 and intermediate-2/high risk patients. We also saw differences in survival between very low/low/intermediate and the high/very high patients when we applied the revised IPSS (IPSS-R). In conclusion, SNP-A can be used successfully in PB samples and the identification of CNA by SNP-A improve the diagnostic and prognostic evaluation of this group of MDS patients., (Copyright © 2013 Wiley Periodicals, Inc.)

A 72-year-old woman was diagnosed with 5q- myelodysplastic syndrome in the course of an indolent multiple myeloma (MM). Bone marrow (BM) cytogenetics disclosed two unrelated clones: 46,XX,del(5)(q13q33), and [47,X,-X,der(1;21)(q10;q10),-4,-4,+5,del(5)(q13q31),+7,der(7)t(1;7)(p34.2;p22),add(8)(p23),-13,+15,der(16) t(1;16)(q23;q12.2),+19,-21,+mar1,+mar2]. The last complex karyotype belonged to malignant plasma cells. FISH and SKY techniques demonstrated different 5q deletions. EGR1 gene (on 5q31) lost in 5q- syndrome remained in 5q- plasma cells. Biclonal evolution was noted: myeloid 5q- cells added a deletion 13q and plasma cells showed monosomy 13. Patient achieved complete cytogenetic response of 5q- syndrome with low-dose of lenalidomide, and a partial remission of MM with high-dose of lenalidomide/dexamethasone combination., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Hox genes encode a family of transcriptional regulators that elicit distinct developmental programmes along the head-to-tail axis of animals. The specific regional functions of individual Hox genes largely reflect their restricted expression patterns, the disruption of which can lead to developmental defects and disease. Here, we examine the spectrum of molecular mechanisms controlling Hox gene expression in model vertebrates and invertebrates and find that a diverse range of mechanisms, including nuclear dynamics, RNA processing, microRNA and translational regulation, all concur to control Hox gene outputs. We propose that this complex multi-tiered regulation might contribute to the robustness of Hox expression during development.

We hypothesized that the dynamic acquisition of cytogenetic abnormalities (ACA) during the follow up of myelodysplastic syndromes (MDS) could be associated with poor prognosis. We conducted a retrospective analysis of 365 patients with IPSS low or intermediate-1 risk MDS who had at least two consecutive cytogenetic analyses during the follow up. Acquisition of cytogenetic abnormalities was detected in 107 patients (29%). The most frequent alteration involved chromosome 7 in 21% of ACA cases. Median transformation-free and overall survival for patients with and without ACA were 13 vs. 52 months (P = 0.01) and 17 vs. 62 months (P = 0.01), respectively. By fitting ACA as a time-dependent covariate, multivariate Cox regression analysis showed that patients with ACA had increased risk of transformation (HR = 1.40; P = 0.03) or death (HR = 1.45; P = 0.02). Notably, female patients with therapy-related MDS (t-MDS) had an increased risk of developing ACA (OR = 5.26; P < 0.0001), although subgroup analysis showed that prognostic impact of ACA was not evident in t-MDS. In conclusion, ACA occurs in close to one third of patients with IPSS defined lower risk MDS, more common among patients with t-MDS, but has a significant prognostic impact on de novo MDS., (Copyright © 2013 Wiley Periodicals, Inc.)

Lenalidomide is an effective drug in low-risk myelodysplastic syndromes (MDS) with isolated del(5q), although not all patients respond. Studies have suggested a role for TP53 mutations and karyotype complexity in disease progression and outcome. In order to assess the impact of complex karyotypes on treatment response and disease progression in 52 lenalidomide-treated patients with del(5q) MDS, conventional G-banding cytogenetics (CC), single nucleotide polymorphism array (SNP-A), and genomic sequencing methods were used. SNP-A analysis (with control sample, lymphocytes CD3+, in 30 cases) revealed 5q losses in all cases. Other recurrent abnormalities were infrequent and were not associated with lenalidomide responsiveness. Low karyotype complexity (by CC) and a high baseline platelet count (>280 × 10(9) /l) were associated with the achievement of haematological response (P = 0·020, P = 0·013 respectively). Unmutated TP53 status showed a tendency for haematological response (P = 0·061). Complete cytogenetic response was not observed in any of the mutated TP53 cases. By multivariate analysis, the most important predictor for lenalidomide treatment failure was a platelet count <280 × 10(9) /l (Odds Ratio = 6·17, P = 0·040). This study reveals the importance of a low baseline platelet count, karyotypic complexity and TP53 mutational status for response to lenalidomide treatment. It supports the molecular study of TP53 in MDS patients treated with lenalidomide., (© 2013 John Wiley & Sons Ltd.)

Patterning of the vertebrate skeleton requires the coordinated activity of Hox genes. In particular, Hox10 proteins are essential to set the transition from thoracic to lumbar vertebrae because of their rib-repressing activity. In snakes, however, the thoracic region extends well into Hox10-expressing areas of the embryo, suggesting that these proteins are unable to block rib formation. Here, we show that this is not a result of the loss of rib-repressing properties by the snake proteins, but rather to a single base pair change in a Hox/Paired box (Pax)-responsive enhancer, which prevents the binding of Hox proteins. This polymorphism is also found in Paenungulata, such as elephants and manatees, which have extended rib cages. In vivo, this modified enhancer failed to respond to Hox10 activity, supporting its role in the extension of rib cages. In contrast, the enhancer could still interact with Hoxb6 and Pax3 to promote rib formation. These results suggest that a polymorphism in the Hox/Pax-responsive enhancer may have played a role in the evolution of the vertebrate spine by differently modulating its response to rib-suppressing and rib-promoting Hox proteins.

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., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Chromosomal abnormalities are detected in 40-60% of patients with de novo myelodysplastic syndromes (MDS). This study used the FISH technique in 773 patients with de novo MDS without evidence of monosomy 7 (-7) or 7q deletion (7q-) by conventional G-banding cytogenetics (CC) to analyze their prognostic impact by FISH alone. FISH detected -7/7q- in 5.2% of patients. Presence of -7/7q- was associated with shorter overall survival than absence of such aberrations. Our results suggest that FISH 7q could be beneficial in patients with intermediate WHO morphologic risk stratification and no evidence of -7/7q- by CC., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Development of the vertebrate axial skeleton requires the concerted activity of several Hox genes. Among them, Hox genes belonging to the paralog group 10 are essential for the formation of the lumbar region of the vertebral column, owing to their capacity to block rib formation. In this work, we explored the basis for the rib-repressing activity of Hox10 proteins. Because genetic experiments in mice demonstrated that Hox10 proteins are strongly redundant in this function, we first searched for common motifs among the group members. We identified the presence of two small sequences flanking the homeodomain that are phylogenetically conserved among Hox10 proteins and that seem to be specific for this group. We show here that one of these motifs is required but not sufficient for the rib-repressing activity of Hox10 proteins. This motif includes two potential phosphorylation sites, which are essential for protein activity as their mutation to alanines resulted in a total loss of rib-repressing properties. Our data indicates that this motif has a significant regulatory function, modulating interactions with more N-terminal parts of the Hox protein, eventually triggering the rib-repressing program. In addition, this motif might also regulate protein activity by alteration of the protein's DNA-binding affinity through changes in the phosphorylation state of two conserved tyrosine residues within the homeodomain.

Purpose: The karyotype is a strong independent prognostic factor in myelodysplastic syndromes (MDS). Since the implementation of the International Prognostic Scoring System (IPSS) in 1997, knowledge concerning the prognostic impact of abnormalities has increased substantially. The present study proposes a new and comprehensive cytogenetic scoring system based on an international data collection of 2,902 patients., Patients and Methods: Patients were included from the German-Austrian MDS Study Group (n = 1,193), the International MDS Risk Analysis Workshop (n = 816), the Spanish Hematological Cytogenetics Working Group (n = 849), and the International Working Group on MDS Cytogenetics (n = 44) databases. Patients with primary MDS and oligoblastic acute myeloid leukemia (AML) after MDS treated with supportive care only were evaluated for overall survival (OS) and AML evolution. Internal validation by bootstrap analysis and external validation in an independent patient cohort were performed to confirm the results., Results: In total, 19 cytogenetic categories were defined, providing clear prognostic classification in 91% of all patients. The abnormalities were classified into five prognostic subgroups (P < .001): very good (median OS, 61 months; hazard ratio [HR], 0.5; n = 81); good (49 months; HR, 1.0 [reference category]; n = 1,809); intermediate (26 months; HR, 1.6; n = 529); poor (16 months; HR, 2.6; n = 148); and very poor (6 months; HR, 4.2; n = 187). The internal and external validations confirmed the results of the score., Conclusion: In conclusion, these data should contribute to the ongoing efforts to update the IPSS by refining the cytogenetic risk categories.

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.

The clone size has been postulated as a prognostic factor in myelodysplastic syndromes (MDS), though it has not been studied systematically. We tested its impact (<100% vs. 100%) in a population of 216 MDS with chromosome 7 abnormalities (-7/7q-) (n=84), trisomy 8 (n=99), 20q deletion (n=28) and loss of Y chromosome (n=26). Focusing on the survival the bad prognosis of -7/7q- was independent of the clone size (9.3 vs. 5.0 months, P=0.188, not significant) but trisomy 8 cases with 100% aberrant metaphases did reveal a worse prognosis (13.9 vs. 5.9 months, P=0.003)., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Background: The prognostic value of cytogenetic findings in chronic myelomonocytic leukemia is unclear. Our purpose was to evaluate the independent prognostic impact of cytogenetic abnormalities in a large series of patients with chronic myelomonocytic leukemia included in the database of the Spanish Registry of Myelodysplastic Syndromes., Design and Methods: We studied 414 patients with chronic myelomonocytic leukemia according to WHO criteria and with a successful conventional cytogenetic analysis at diagnosis. Different patient and disease characteristics were examined by univariate and multivariate methods to establish their relationship with overall survival and evolution to acute myeloid leukemia., Results: Patients with abnormal karyotype (110 patients, 27%) had poorer overall survival (P=0.001) and higher risk of acute myeloid leukemia evolution (P=0.010). Based on outcome analysis, three cytogenetic risk categories were identified: low risk (normal karyotype or loss of Y chromosome as a single anomaly), high risk (presence of trisomy 8 or abnormalities of chromosome 7, or complex karyotype), and intermediate risk (all other abnormalities). Overall survival at five years for patients in the low, intermediate, and high risk cytogenetic categories was 35%, 26%, and 4%, respectively (P<0.001). Multivariate analysis confirmed that this new CMML-specific cytogenetic risk stratification was an independent prognostic variable for overall survival (P=0.001). Additionally, patients belonging to the high-risk cytogenetic category also had a higher risk of acute myeloid leukemia evolution on univariate (P=0.001) but not multivariate analysis., Conclusions: Cytogenetic findings have a strong prognostic impact in patients with chronic myelomonocytic leukemia.

Hox genes encode transcription factors that regulate morphogenesis in all animals with bilateral symmetry. Although Hox genes have been extensively studied, their molecular function is not clear in vertebrates, and only a limited number of genes regulated by Hox transcription factors have been identified. Hoxa2 is required for correct development of the second branchial arch, its major domain of expression. We now show that Meox1 is genetically downstream from Hoxa2 and is a direct target. Meox1 expression is downregulated in the second arch of Hoxa2 mouse mutant embryos. In chromatin immunoprecipitation (ChIP), Hoxa2 binds to the Meox1 proximal promoter. Two highly conserved binding sites contained in this sequence are required for Hoxa2-dependent activation of the Meox1 promoter. Remarkably, in the absence of Meox1 and its close homolog Meox2, the second branchial arch develops abnormally and two of the three skeletal elements patterned by Hoxa2 are malformed. Finally, we show that Meox1 can specifically bind the DNA sequences recognized by Hoxa2 on its functional target genes. These results provide new insight into the Hoxa2 regulatory network that controls branchial arch identity.

Abnormalities of chromosome 5 are common aberrations in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), being del(5q) the most frequent. In contrast, monosomy 5 is not so frequent and, usually, is associated with complex karyotypes, conferring poor prognosis. The aim of this study was to analyze, by FISH for 5q31 (EGR1), a series of 28 MDS and AML cases with monosomy 5 detected by G-banding. FISH results revealed deletion of 5q31 in 24 of them (85.7%) and monosomy 5 just in three. FISH for 5q31 can complement conventional cytogenetics and improve the karyotype definition, leading to a better diagnostic and prognostic stratification., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Several decades have passed since the discovery of Hox genes in the fruit fly Drosophila melanogaster. Their unique ability to regulate morphologies along the anteroposterior (AP) axis (Lewis, 1978) earned them well-deserved attention as important regulators of embryonic development. Phenotypes due to loss- and gain-of-function mutations in mouse Hox genes have revealed that the spatio-temporally controlled expression of these genes is critical for the correct morphogenesis of embryonic axial structures. Here, we review recent novel insight into the modalities of Hox protein function in imparting specific identity to anatomical regions of the vertebral column, and in controlling the emergence of these tissues concomitantly with providing them with axial identity. The control of these functions must have been intimately linked to the shaping of the body plan during evolution., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

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., (Copyright 2010 Elsevier Inc. All rights reserved.)

Hox and Cdx transcription factors regulate embryonic positional identities. Cdx mutant mice display posterior body truncations of the axial skeleton, neuraxis, and caudal urorectal structures. We show that trunk Hox genes stimulate axial extension, as they can largely rescue these Cdx mutant phenotypes. Conversely, posterior (paralog group 13) Hox genes can prematurely arrest posterior axial growth when precociously expressed. Our data suggest that the transition from trunk to tail Hox gene expression successively regulates the construction and termination of axial structures in the mouse embryo. Thus, Hox genes seem to differentially orchestrate posterior expansion of embryonic tissues during axial morphogenesis as an integral part of their function in specifying head-to-tail identity. In addition, we present evidence that Cdx and Hox transcription factors exert these effects by controlling Wnt signaling. Concomitant regulation of Cyp26a1 expression, restraining retinoic acid signaling away from the posterior growth zone, may likewise play a role in timing the trunk-tail transition.

In vertebrates, the paraxial mesoderm differentiates into several structures, including the axial skeleton. The genetic mechanisms that control positional information in the paraxial mesoderm along the anterior-posterior axis are responsible for the development of a skeleton with the appropriate vertebral formula, i.e. a specific number of cervical, thoracic, lumbar, sacral and caudal vertebrae. These control mechanisms are complex and involve molecules of different kinds, including transcription factors, like those encoded by the Hox genes, and signalling molecules, like those involved in Gdf11, FGF, retinoic acid or WNT signalling. Recent experiments indicate that most of the positional information for the paraxial mesoderm is encoded during the initial steps of its development in the presomitic mesoderm, although it is only decoded later during differentiation of the somites. The genesis of positional identity may be linked to the process of somitogenesis, which also occurs in the presomitic mesoderm as a result of complex interactions involving oscillatory activity of components of the Notch and WNT signalling pathways and antagonistic gradients of FGF/WNT and retinoic acid. The possible connections between Hox genes and all these signalling processes to generate a properly patterned axial skeleton are discussed in this review.

Granulocytic sarcomas (GS) are tumor masses of immature myeloid cells presenting at an extramedullary site, mainly the skin, bone, and lymph node. They are often associated with acute myeloid leukemia (AML) with monoblastic or myelomonocytic differentiation, including either AML M2 with t(8;21)(q22;q22) or AML M4Eo with inv(16)(p13q22). We present a case diagnosed with GS associated with AML M4 that presented a normal karyotype with conventional cytogenetic analysis. Although the myeloblasts did not show the inv(16)(p13q22) (CBFB/MYH11), a gain of multiple copies of the CBFB gene was detected with fluorescence in situ hybridization analysis. To our knowledge, no cases with this rare genetic anomaly have been previously described.

Homeodomain-containing transcription factors are important regulators of stem cell behavior. HOXB4 mediates expansion of adult and embryo-derived hematopoietic stem cells (HSCs) when expressed ectopically. To define the underlying molecular mechanisms, we performed gene expression profiling in combination with subsequent functional analysis with enriched adult HSCs and embryonic derivatives expressing inducible HOXB4. Thereby, we identified a set of overlapping genes that likely represent "universal" targets of HOXB4. A substantial number of loci are involved in signaling pathways important for controlling self-renewal, maintenance, and differentiation of stem cells. Functional assays performed on selected pathways confirmed the biological coherence of the array results. HOXB4 activity protected adult HSCs from the detrimental effects mediated by the proinflammatory cytokine TNF-alpha. This protection likely contributes to the competitive repopulation advantage of HOXB4-expressing HSCs observed in vivo. The concept of TNF-alpha inhibition may also prove beneficial for patients undergoing bone marrow transplantation. Furthermore, we demonstrate that HOXB4 activity and FGF signaling are intertwined. HOXB4-mediated expansion of adult and ES cell-derived HSCs was enhanced by specific and complete inhibition of FGF receptors. In contrast, the expanding activity of HOXB4 on hematopoietic progenitors in day 4-6 embryoid bodies was blunted in the presence of basic FGF (FGF2), indicating a dominant negative effect of FGF signaling on the earliest hematopoietic cells. In summary, our results strongly suggest that HOXB4 modulates the response of HSCs to multiple extrinsic signals in a concerted manner, thereby shifting the balance toward stem cell self-renewal.

Bone morphogenetic protein (BMP) signaling is essential for neural crest development in several vertebrates. Genetic experiments in the mouse have shown that Bmp2 is essential for the genesis of migratory neural crest cells. Using several markers and a transgenic reporter approach, we now show that neural crest cells are induced in Bmp2 null mutant embryos, but that these cells fail to migrate out of the neural tube. The absence of migratory neural crest cells in these mutants is not due to their elimination by cell death. The neuroectoderm of Bmp2-/- embryos fail to close and create abnormal folds both along the anterior-posterior and medio-lateral axes, which are associated with an apparent medio-lateral expansion of the neural tube. Finally, our data suggest that the molecular cascade downstream of BMP signaling in early neural crest development may be different in mouse and avian embryos., ((c) 2007 Wiley-Liss, Inc.)

The vertebrate body is organized in segments, easily visible in the consecutive vertebrae of the skeleton. These are first defined in the embryo by the formation of somites. Somites are generated at regular intervals from the presomitic mesoderm by a combination of oscillating signals, known as the segmentation clock, which establish the pace at which new somites are formed, and signaling gradients that set the location of new intersomitic borders. Using a microarray approach, Dequéant et al. have now shown that the segmentation clock is more complex than previously thought and includes oscillating expression of genes from at least three signaling pathways organized in coordinated networks., ((c) 2007 Wiley Periodicals, Inc.)

SOX9 is an evolutionary conserved transcription factor that is expressed in a variety of tissues, with essential functions in cartilage, testis, heart, glial cell, inner ear and neural crest development. By comparing human and pufferfish genomic sequences, we previously identified eight highly conserved sequence elements between 290 kb 5' and 450 kb 3' to human SOX9. In this study, we assayed the regulatory potential of elements E1 to E7 in transgenic mice using a lacZ reporter gene driven by a 529 bp minimal mouse Sox9 promoter. We found that three of these elements and the Sox9 promoter control distinct subsets of the tissue-specific expression pattern of Sox9. E3, located 251 kb 5' to SOX9, directs lacZ expression to cranial neural crest cells and to the inner ear. E1 is located 28 kb 5' to SOX9 and controls expression in the node, notochord, gut, bronchial epithelium and pancreas. Transgene expression in the neuroectoderm is mediated by E7, located 95 kb 3' to SOX9, which regulates expression in the telencephalon and midbrain, and by the Sox9 minimal promoter which controls expression in the ventral spinal cord and hindbrain. We show that E3-directed reporter gene expression in neural crest cells of the first but not of the second and third pharyngeal arch is dependent on beta-catenin, revealing a complex regulation of Sox9 in cranial neural crest cells. Moreover, we identify and discuss highly conserved transcription factor binding sites within enhancer E3 that are in good agreement with current models for neural crest and inner ear development. Finally, we identify enhancer E1 as a cis-regulatory element conserved between vertebrates and invertebrates, indicating that some cis-regulatory sequences that control developmental genes in vertebrates might be phylogenetically ancient.

The emergence of techniques that allow fine manipulation of gene expression in the mouse have changed the way biomedically relevant processes are studied, as they allow their analysis in the living animal. In addition, this has opened the possibility to generate animal models for several human diseases, which are useful both for understanding the disease' s physiopathological mechanisms and for the eventual evaluation of novel therapeutic approaches. Many of the gene manipulation systems currently employed in the mouse are based on regulatory mechanisms normally operating in yeast and prokaryotic organisms. This has allowed specific experimental control with very limited unspecific interference with the normal physiology of the cell. Some of these systems use elements that permit transcriptional regulation of a particular gene or genes. Among them, I will discuss in detail the tet, lac and Gal4/UAS systems, which are among the most popular of these transcriptional systems because they can be used to achieve spatial and temporal control on the expression of a specific gene in a reversible fashion. The other major group of systems currently employed to manipulate gene expression in the mouse is based on site-specific recombination reactions. The Cre/lox and FLP/FRT systems are the most popular of these. I will discuss how these recombination systems are used in the mouse with special focus on their use to achieve specific gene activation.

We show here that expression of Hoxa10 in the presomitic mesoderm is sufficient to confer a Hox group 10 patterning program to the somite, producing vertebrae without ribs, an effect not achieved when Hoxa10 is expressed in the somites. In addition, Hox group 11-dependent vertebral sacralization requires Hoxa11 expression in the presomitic mesoderm, while their caudal differentiation requires that Hoxa11 is expressed in the somites. Therefore, Hox gene patterning activity is different in the somites and presomitic mesoderm, the latter being very prominent for Hox gene-mediated patterning of the axial skeleton. This is further supported by our finding that inactivation of Gbx2, a homeobox-containing gene expressed in the presomitic mesoderm but not in the somites, produced Hox-like phenotypes in the axial skeleton without affecting Hox gene expression.

Duplication of genomic regions is an important biological process associated with the appearance of gene families, the origin of alternative splicing, and the etiopathogenesis of genetic diseases. Different mechanisms for the genesis of duplications have been suggested, based mainly on structural analyses. However, experimental confirmation of those mechanisms is scarce, mostly because of a lack of information about the circumstances that triggered the rearrangements. Here, I characterize a duplication of about 300 kbp (kilobase pairs) that occurred in the course of a gene targeting experiment. Considering the structure of the locus and the triggering event, I suggest a likely mechanism for the genesis of this duplication which involves anomalous processing of contiguous Okazaki fragments during lagging strand replication. Most importantly, I provide experimental evidence to substantiate that the proposed mechanism can indeed lead to duplication of genomic segments. The model presented represents a novel mechanistic pathway that can explain a variety of rearrangements, including genomic tandem duplications and deletions.

Tbx1 belongs to the family of T-box containing transcription factors. In humans, TBX1 is implicated in the etiology of the DiGeorge syndrome. Inactivation of the Tbx1 gene in mice produces a variety of malformations including abnormal branching of the heart outflow tract, deficiencies in the branchial arch derivatives, agenesis of pharyngeal glands and abnormal development of the auditory system. We analyze here the middle and inner ear phenotypes of the Tbx1 null mice. The middle ear is strongly affected. Its skeletal components are malformed to varying degrees, some being slightly hypoplastic and others completely absent. However, a seemingly normal-looking tympanic membrane can still be recognized. Middle ear anomalies are associated with other skeletal deficiencies in the branchial arch-derived skeleton. These phenotypes derive from a combination of the failure of the posterior branchial arches to develop and the misrouting of neural crest cells. The inner ears of Tbx1(-/-) animals are hypoplastic. No vestibular or cochlear structures are detectable, but the endolymphatic duct, the cochleovestibular ganglia and residual sensory patches are still identifiable. Molecular analyses revealed a seemingly normal spatial distribution of a variety of patterning markers in the otic vesicles of Tbx1 null mutants at E9.0. However, 1 day later, several of these markers presented altered domains of expression in the otocysts of these mutant embryos, suggesting that Tbx1 is not required for the establishment of spatial patterns in the otocyst, but rather for their maintenance. The inability of the Tbx1(-/-) embryos to keep properly segregated functional domains in the otocyst is likely the cause of the strong inner ear phenotypes observed in these mutants.

The Hoxa2 transcription factor acts during development of the second branchial arch. As for most of the developmental processes controlled by Hox proteins, the mechanism by which Hoxa2 regulates the morphology of second branchial arch derivatives is unclear. We show that Six2, another transcription factor, is genetically downstream of Hoxa2. High levels of Six2 are observed in the Hoxa2 loss-of-function mutant. By using a transgenic approach to overexpress Six2 in the embryonic area controlled by Hoxa2, we observed a phenotype that is reminiscent of the Hoxa2 mutant phenotype. Furthermore, we demonstrate that Hoxa2 regulation of Six2 is confined to a 0.9 kb fragment of the Six2 promoter and that Hoxa2 binds to this promoter region. These results strongly suggest that Six2 is a direct target of Hoxa2.

Hox genes are known key regulators of embryonic segmental identity, but little is known about the mechanisms of their action. To address this issue, we have analyzed how Hoxa2 specifies segmental identity in the second branchial arch. Using a subtraction approach, we found that Ptx1 was upregulated in the second arch mesenchyme of Hoxa2 mutants. This upregulation has functional significance because, in Hoxa2(-/-);Ptx1(-/-) embryos, the Hoxa2(-/-) phenotype is partially reversed. Hoxa2 interferes with the Ptx1 activating process, which is dependent on Fgf signals from the epithelium. Consistently, Lhx6, another target of Fgf8 signaling, is also upregulated in the Hoxa2(-/-) second arch mesenchyme. Our findings have important implications for the understanding of developmental processes in the branchial area and suggest a novel mechanism for mesenchymal patterning by Hox genes that acts to define the competence of mesenchymal cells to respond to skeletogenic signals.

Gene-inactivation techniques in the mouse have become an essential tool for modern biomedical research. Both ubiquitous and tissue-specific inactivation are possible with current approaches, and recent developments facilitate a temporal control of the inactivation process. However, one of the limitations of current procedures is that inactivation is irreversible. We have produced complete and reversible inactivation of the Hoxa2 gene in the mouse using the control elements of the tetracycline-resistance operon. We show that a Hoxa2 allele containing tetracycline operator (tetO) sequences is susceptible to controlled regulation by tTS, a chimeric molecule containing the tetracycline repressor and a transcriptional repressing domain. This inhibition was specific to the tetO-modified allele, did not affect neighboring genes, and was reversible by administration of doxycycline to the pregnant female. This procedure allows the production of gene inactivation that is complete, is reversible, and can be controlled at the spatial and temporal levels.

Neural crest cells are essential for proper development of a variety of tissues and structures, including peripheral and autonomic nervous systems, facial skeleton, aortic arches and pharyngeal glands like the thymus and parathyroids. Previous work has shown that bone morphogenic protein (BMP) signalling is required for the production of migratory neural crest cells that contribute to the neurogenic and skeletogenic lineages. We show here that BMP-dependent neural crest cells are also required for development of the embryonic aortic arches and pharynx-derived glands. Blocking formation or migration of this crest cell population from the caudal hindbrain resulted in strong phenotypes in the cardiac outflow tract and the thymus. Thymic aplasia or hypoplasia occurs despite uncompromised gene induction in the pharyngeal endoderm. In addition, when hypoplastic thymic tissue is found, it is ectopically located, but functional in thymopoiesis. Our data indicate that thymic phenotypes produced by neural crest deficits result from aberrant formation of pharyngeal pouches and impaired migration of thymic primordia because the mesenchymal content in the branchial arches is below a threshold level.

The middle ear allows animals to hear while moving in an aerial medium. It is composed of a cavity harbouring a chain of three ossicles that transmit vibrations produced by airborne sound in the tympanic membrane into the inner ear, where they are converted into neural impulses. The middle ear develops in the branchial arches, and this requires sequential interactions between the epithelia and the underlying mesenchyme. Gene-inactivation experiments have identified genes required for the formation of different middle ear components. Some encode for signalling molecules, including Endothelin1 and Fgf8, probable mediators of epithelial-mesenchymal interactions. Other genes, including Eya1, Prx1, Hoxa1, Hoxa2, Dlx1, Dlx2, Dlx5, and Gsc, are most likely involved in patterning and morphogenetic processes in the neural crest-derived mesenchyme. Mechanisms controlling formation of a functional tympanic membrane are also discussed. Basically, the tympanic ring, which serves as support for the tympanic membrane, directs invagination of the first pharyngeal cleft ectoderm to form the external acoustic meatus (EAM), which provides the outer layer of the membrane. Gsc and Prx1 are essential for tympanic ring development. While invaginating, the EAM controls skeletogenesis in the underlying mesenchyme to form the manubrium of the malleus, the link between the membrane and the middle ear ossicles., (Copyright 2001 Academic Press.)

In this report, we present the characterization of a humanized monoclonal antibody specific for the human epidermal growth factor receptor (hEGFR). Direct analysis by matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) of peptide mixtures and chromatographically isolated fractions allowed identification of 94.0% and 85.4% of the amino acid sequence of light and heavy chains, respectively. Microheterogeneity sources were identified in light and heavy chains and a previously unreported posttranslational modification for immunoglobulins was found. One N-glycosylation site was identified in the heavy chain with non-sialylated bianntenary fucosylated structures. This study is one of the first to assess the potential of MALDI-MS in combination with more conventional protein chemistry techniques for the characterization of monoclonal antibodies.

Interferon (IFN)-gamma, a key immunoregulatory cytokine, has been thought to be produced solely by activated T cells and natural killer cells. In this study, we show that murine bone marrow- derived macrophages (BMMPhi) secrete large amounts of IFN-gamma upon appropriate stimulation. Although interleukin (IL)-12 and IL-18 alone induce low levels of IFN-gamma mRNA transcripts, the combined stimulation of BMMPhi with both cytokines leads to the efficient production of IFN-gamma protein. The macrophage-derived IFN-gamma is biologically active as shown by induction of inducible nitric oxide synthase as well as upregulation of CD40 in macrophages. Our findings uncover a novel pathway of autocrine macrophage activation by demonstrating that the macrophage is not only a key cell type responding to IFN-gamma but also a potent IFN-gamma-producing cell.

In this study we tested whether the segmental identities of the hindbrain and its derived neural crest are necessarily linked or, instead, if they can be altered independently. Using morphological criteria, we show that the hindbrains of Hoxa-2 mutant mice, in which the second arch skeletal derivatives assume first arch characteristics (Gendron-Maguire et al. [1993] Cell 75:1317-1331; Rijli et al. [1993] Cell 75:1333-1349), retain normal segmental identities. Also, by phenotypic analysis, we show that, with retinoic acid, changes can be induced in the identity of the preotic hindbrain without effects in its derived neural crest. Our data thus indicate that identity changes in the hindbrain and branchial arch neural crest can occur independently. Moreover, if Hoxa-2 is concomitantly induced by retinoic acid in the first branchial arch, the proximal derivatives of this arch are also affected. We propose a model for the patterning of the branchial region, according to which the segmental identity in this area is provided mainly by the branchial arches.