Partial functional redundancy between Hoxa5 and Hoxb5 paralog genes during lung morphogenesis.

Hox genes encode transcription factors governing complex developmental processes in several organs. A subset of Hox genes are expressed in the developing lung. Except for Hoxa5, the lack of overt lung phenotype in single mutants suggests that Hox genes may not play a predominant role in lung ontogeny or that functional redundancy may mask anomalies. In the Hox5 paralog group, both Hoxa5 and Hoxb5 genes are expressed in the lung mesenchyme whereas Hoxa5 is also expressed in the tracheal mesenchyme. Herein, we generated Hoxa5;Hoxb5 compound mutant mice to evaluate the relative contribution of each gene to lung development. Hoxa5;Hoxb5 mutants carrying the four mutated alleles displayed an aggravated lung phenotype, resulting in the death of the mutant pups at birth. Characterization of the phenotype highlighted the role of Hoxb5 in lung formation, the latter being involved in branching morphogenesis, goblet cell specification, and postnatal air space structure, revealing partial functional redundancy with Hoxa5. However, the Hoxb5 lung phenotypes were less severe than those seen in Hoxa5 mutants, likely because of Hoxa5 compensation. New specific roles for Hoxa5 were also unveiled, demonstrating the extensive contribution of Hoxa5 to the developing respiratory system. The exclusive expression of Hoxa5 in the trachea and the phrenic motor column likely underlies the Hoxa5-specific trachea and diaphragm phenotypes. Altogether, our observations establish that the Hoxa5 and Hoxb5 paralog genes shared some functions during lung morphogenesis, Hoxa5 playing a predominant role.

Unsuspected effects of a lung-specific Cre deleter mouse line.

Cre-expressing mouse lines constitute an important asset to mammalian genetics, allowing the deletion of genes in a spatio-temporal specific manner. Our study on Hox gene function in lung development has led us to use a lung endoderm-specific deletion with the Sftpc-cre mouse line expressing the Cre recombinase gene under the control of human surfactant protein C regulatory sequences. In control experiments, the Cre recombinase faithfully activated the Rosa26-lacZ reporter gene in lung epithelium. However as early as e15.5, lungs from Sftp-Cre(+) embryos showed abnormal dilated cysts. This unexpected phenotype was also observed in mice carrying the conditional lung epithelial Hoxa5 deletion, indicating some bias due to Cre deleterious effects. Excessive apoptosis, likely due to Cre toxicity, could explain the abnormal cysts. Our findings illustrate the need for appropriate control experiments and careful interpretation of data to discriminate between the phenotype due to the targeted mutation and the confounding effects of the Cre recombinase.

Influence of Hoxa5 on p53 tumorigenic outcome in mice.

Hox genes encode transcription factors of crucial importance in the pattern formation of a large spectrum of species. Several studies have now proposed a role for these developmental genes in cancer biology. It has been suggested that HOXA5 possesses growth-suppressive properties through activation of p53 expression in human breast tissue. To assess the genetic cooperation that may exist between Hoxa5 and p53 in tumorigenesis, we generated Hoxa5/p53 compound mutant mice. The presence of Hoxa5 null alleles increased the susceptibility of p53(-/-) mice to develop tumors with a high prevalence for thymic lymphoma, suggesting that the loss of function of the two genes collaborate in tumor formation. To extend our analysis to mammary tumorigenesis, we performed Hoxa5/p53 whole mammary gland transplantations into wild-type hosts. In the p53(-/-) background, the presence of one Hoxa5 mutant allele had no impact on mammary tumor formation. In contrast, the complete loss of Hoxa5 function influenced the tumorigenic outcome of p53(+/-) mammary glands. However, the collaborative nature of this interaction did not depend on the transcriptional regulation of p53 by Hoxa5. Altogether, our data establish that Hoxa5 and p53 cooperate in mammary tumorigenesis in vivo.

[Hoxa5: a master gene with multifaceted roles]. / Hoxa5: un maître d'oeuvre à multiples facettes.

The Hox gene family occupies a central position in the control of body patterning by regulating the transcription of downstream effectors that, in turn, direct the morphogenetic events leading to the complex body forms along the axes. Analysis of Hox mutant mouse lines has revealed a panoply of phenotypes indicative of the broad range of Hox genes action throughout embryonic and postnatal life. Although Hox genes have been the subject of extensive research in the last two decades, the comprehension of the mechanisms involved in their regulation and function still remains elusive. Here, we present an overview of our current knowledge about one Hox gene family member, Hoxa5. The phenotypic survey of Hoxa5 mutant mice has unveiled the crucial role of this gene in regulating morphogenesis and specifying regional identity along the embryo. A majority of Hoxa5 mutant pups die at birth from defective respiratory tract. Surviving mutants present deficient alveolar septation revealing the importance of Hoxa5 during formation and maturation of the lung. Hoxa5 also participates in the morphogenesis of the digestive tract as well as that of the thyroid and mammary glands. Hoxa5 expression is restricted to the mesenchyme, and its action appears to be mediated through the control of mesenchymal-epithelial interactions during organogenesis. The implication of Hoxa5 in tumorigenesis has also been documented. In breast cancer, Hoxa5 down-regulation may impact on p53 gene expression, contributing to the oncogenic process. In contrast, the loss of Hoxa5 function limits leukaemia associated with specific chromosomal translocations. Thus, inappropriate Hoxa5 gene expression may disrupt normal growth and differentiation programs causing neoplasia. Hox gene function is intimately linked to its correct expression. Regulation of Hoxa5 expression requires multiple cis-acting regions, some encompassing coding sequences from neighboring genes. Moreover, it is complicated by the presence of several transcription units. Together these data enlighten the importance of Hox cluster organization in Hoxa5 function.

The loss of Hoxa5 function causes estrous acyclicity and ovarian epithelial inclusion cysts.

Hox genes encode transcription factors that play essential roles during embryo morphogenesis and organogenesis. Expression of several Hox members persists at the adult age, indicating a wide spectrum of action from embryonic to postnatal life. In the present study, we reported that in adult mice, the Hoxa5 gene shows a dynamic expression profile in the ovary that depends on the estrous cycle, the gestational status, and the age of the female, suggesting that Hoxa5 may have distinct physiological functions in the ovary. Consistent with a role for Hoxa5 in ovarian function, Hoxa5(-/-) nulliparous females exhibit precocious puberty and an early onset of estrous acyclicity. They show a prolonged estrous cycle with increased metestrus-diestrus length, a phenotype that worsens with age. Older mutant females also develop ovarian epithelial inclusion cysts reminiscent of human endosalpingiosis. Immunolabeling studies suggest that these cysts originate from the ovarian surface epithelium, a source of epithelial ovarian carcinomas. Staining of the Hoxa5(-/-) ovarian cysts by the ovarian cancer markers paired box gene 8 (PAX8) and Wilms' tumor 1 (WT1) further strengthens the notion that these cysts may constitute preneoplastic lesions. Moreover, the deregulation of the estrous cycle and the presence of ovarian epithelial cysts in Hoxa5(-/-) older females correlate with a reduced expression of specific epidermal growth factor receptor signaling components, namely Egfr, Areg, and Btc. Altogether, our data unveil that Hoxa5, a stroma-specific gene, plays a significant role in ovarian biology and may be involved in ovarian cancer predisposition.

Sustained Hox5 gene activity is required for respiratory motor neuron development.

Respiration in mammals relies on the rhythmic firing of neurons in the phrenic motor column (PMC), a motor neuron group that provides the sole source of diaphragm innervation. Despite their essential role in breathing, the specific determinants of PMC identity and patterns of connectivity are largely unknown. We show that two Hox genes, Hoxa5 and Hoxc5, control diverse aspects of PMC development including their clustering, intramuscular branching, and survival. In mice lacking Hox5 genes in motor neurons, axons extend to the diaphragm, but fail to arborize, leading to respiratory failure. Genetic rescue of cell death fails to restore columnar organization and branching patterns, indicating these defects are independent of neuronal loss. Unexpectedly, late Hox5 removal preserves columnar organization but depletes PMC number and branches, demonstrating a continuous requirement for Hox function in motor neurons. These findings indicate that Hox5 genes orchestrate PMC development through deployment of temporally distinct wiring programs.

Multiple promoters and alternative splicing: Hoxa5 transcriptional complexity in the mouse embryo.

BACKGROUND: The genomic organization of Hox clusters is fundamental for the precise spatio-temporal regulation and the function of each Hox gene, and hence for correct embryo patterning. Multiple overlapping transcriptional units exist at the Hoxa5 locus reflecting the complexity of Hox clustering: a major form of 1.8 kb corresponding to the two characterized exons of the gene and polyadenylated RNA species of 5.0, 9.5 and 11.0 kb. This transcriptional intricacy raises the question of the involvement of the larger transcripts in Hox function and regulation. METHODOLOGY/PRINCIPAL FINDINGS: We have undertaken the molecular characterization of the Hoxa5 larger transcripts. They initiate from two highly conserved distal promoters, one corresponding to the putative Hoxa6 promoter, and a second located nearby Hoxa7. Alternative splicing is also involved in the generation of the different transcripts. No functional polyadenylation sequence was found at the Hoxa6 locus and all larger transcripts use the polyadenylation site of the Hoxa5 gene. Some larger transcripts are potential Hoxa6/Hoxa5 bicistronic units. However, even though all transcripts could produce the genuine 270 a.a. HOXA5 protein, only the 1.8 kb form is translated into the protein, indicative of its essential role in Hoxa5 gene function. The Hoxa6 mutation disrupts the larger transcripts without major phenotypic impact on axial specification in their expression domain. However, Hoxa5-like skeletal anomalies are observed in Hoxa6 mutants and these defects can be explained by the loss of expression of the 1.8 kb transcript. Our data raise the possibility that the larger transcripts may be involved in Hoxa5 gene regulation. SIGNIFICANCE: Our observation that the Hoxa5 larger transcripts possess a developmentally-regulated expression combined to the increasing sum of data on the role of long noncoding RNAs in transcriptional regulation suggest that the Hoxa5 larger transcripts may participate in the control of Hox gene expression.

Functional resolution of duplicated hoxb5 genes in teleosts.

The duplication-degeneration-complementation (DDC) model predicts that subfunctionalization of duplicated genes is a common mechanism for their preservation. The additional Hox complexes of teleost fish constitute a good system in which to test this hypothesis. Zebrafish have two hoxb complexes, with two hoxb5 genes, hoxb5a and hoxb5b, the expression patterns of which suggest subfunctionalization of an ancestral hoxb5 gene. We characterized conserved non-coding elements (CNEs) near the zebrafish hoxb5 genes. One CNE, J3, is only retained in the hoxb5a locus, whereas the others, J1 and J2, are present in both hoxb5 loci. When tested individually, the enhancer activity of individual CNEs, including J3, extensively overlapped and did not support a role in subfunctionalization. By contrast, reporter transgene constructs encompassing multiple CNEs were able to target reporter gene expression to unique domains of hoxb5a and hoxb5b expression. The deletion of J3 from the hoxb5a locus resulted in expression that approached that of hoxb5b, whereas its insertion in the hoxb5b locus increased reporter expression and rendered it more similar to that of hoxb5a. Our results highlight the importance of interactions between CNEs in the execution of complementary subfunctions of duplicated genes.

Comparative analysis of Hoxa5 allelic series.

Analysis of the Hoxa5(-/-) mutants has revealed the critical role of Hoxa5 in survival, specification of axial identity, and ontogeny of organs, including the respiratory tract. The presence of the selection cassette in the original Hoxa5(-/-) mutation may interfere with the interpretation of the phenotypes. To circumvent this aspect and to bypass the lethality of the Hoxa5 mutation, we have designed a conditional approach and generated Hoxa5 allelic variants. The conditional allele (Hoxa5(floxed)) behaves as a wild-type allele. In contrast, both the Hoxa5(Delta) and the Hoxa5(floxneo) alleles are characterized by the loss of the functional transcript and protein, the lethality due to lung defects and the skeletal homeotic transformations similar to those of the Hoxa5(-/-) mutants. Analysis of neighboring Hox gene expression patterns in the Hoxa5 mutants produced further confirmed that the Hoxa5 allelic variants are true null alleles.

Impact of the loss of Hoxa5 function on lung alveogenesis.

The involvement of genes controlling embryonic processes in the etiology of diseases often escapes attention because of the focus given to their inherent developmental role. Hoxa5 belongs to the Hox gene family encoding transcription factors known for their role in skeletal patterning. Hoxa5 is required for embryonic respiratory tract morphogenesis. We now show that the loss of Hoxa5 function has severe repercussions on postnatal lung development. Hoxa5-/- lungs present an emphysema-like morphology because of impaired alveogenesis. Chronic inflammation characteristics, including goblet cell hyperplasia, mucus hypersecretion, and recruitment of inflammatory cells, were also observed. Altered cell specification during lung morphogenesis triggered goblet cell anomalies. In addition, the defective motility of alveolar myofibroblast precursors in the embryonic lung led to the mispositioning of the alveolar myofibroblasts and to abnormal elastin deposition postnatally. Both goblet cell hyperplasia and elastic fiber abnormalities contributed to the chronic physiopathological features of Hoxa5-/- lungs. They constituted an attractive stimulus to recruit activated macrophages that in turn generated a positive feedback loop that perpetuated macrophage accumulation in the lung. The present work corroborates the notion that altered Hox gene expression may predispose to lung pathologies.

Ephrin-B1 forward and reverse signaling are required during mouse development.

Eph receptors and ephrin ligands are key players in many developmental processes including embryo patterning, angiogenesis, and axon guidance. Eph/ephrin interactions lead to the generation of a bidirectional signal, in which both the Eph receptors and the ephrins activate downstream signaling cascades simultaneously. To understand the role of ephrin-B1 and the importance of ephrin-B1-induced reverse signaling during embryonic development, we have generated mouse lines carrying mutations in the efnb1 gene. Complete ablation of ephrin-B1 resulted in perinatal lethality associated with a range of phenotypes, including defects in neural crest cell (NCC)-derived tissues, incomplete body wall closure, and abnormal skeletal patterning. Conditional deletion of ephrin-B1 demonstrated that ephrin-B1 acts autonomously in NCCs, and controls their migration. Last, a mutation in the PDZ binding domain indicated that ephrin-B1-induced reverse signaling is required in NCCs. Our results demonstrate that ephrin-B1 acts both as a ligand and as a receptor in a tissue-specific manner during embryogenesis.

In vivo convergence of BMP and MAPK signaling pathways: impact of differential Smad1 phosphorylation on development and homeostasis.

Integration of diverse signaling pathways is essential in development and homeostasis for cells to interpret context-dependent cues. BMP and MAPK signaling converge on Smads, resulting in differential phosphorylation. To understand the physiological significance of this observation, we have generated Smad1 mutant mice carrying mutations that prevent phosphorylation of either the C-terminal motif required for BMP downstream transcriptional activation (Smad1(C) mutation) or of the MAPK motifs in the linker region (Smad1(L) mutation). Smad1(C/C) mutants recapitulate many Smad1(-/-) phenotypes, including defective allantois formation and the lack of primordial germ cells (PGC), but also show phenotypes that are both more severe (head and branchial arches) and less severe (allantois growth) than the null. Smad1(L/L) mutants survive embryogenesis but exhibit defects in gastric epithelial homeostasis correlated with changes in cell contacts, actin cytoskeleton remodeling, and nuclear beta-catenin accumulation. In addition, formation of PGCs is impaired in Smad1(L/L) mutants, but restored by allelic complementation in Smad1(C/L) compound mutants. These results underscore the need to tightly balance BMP and MAPK signaling pathways through Smad1.

Perturbed thyroid morphology and transient hypothyroidism symptoms in Hoxa5 mutant mice.

The Hox family of transcriptional regulators has been extensively studied for their role in axial and appendicular patterning. Genetic analyses have also unveiled Hox gene function in organogenesis and postnatal development. A phenotypical survey of the Hoxa5(-/-) mutant mice shows that the surviving mutants display symptoms of hypothyroidism, including transient growth retardation, and delayed eye opening and ear elevation. Thyroid gland morphogenesis initiates normally, but follicle formation and thyroglobulin processing are abnormal at late gestation. The expression of several molecular markers essential for thyroid gland formation and function, namely Nkx2.1, Pax8, and Titf2, is affected in the developing thyroid gland of Hoxa5(-/-) mutants. As a consequence, the expression of thyroid effector genes, including the thyroglobulin and thyroperoxidase genes, is perturbed. Our characterization reveals that the loss of Hoxa5 function transiently affects thyroid development in a non-cell autonomous manner.

Stomach regional specification requires Hoxa5-driven mesenchymal-epithelial signaling.

The genetic control of gut regionalization relies on a hierarchy of molecular events in which the Hox gene family of transcription factors is suspected to be key participant. We have examined the role of Hox genes in gut patterning using the Hoxa5(-/-) mice as a model. Hoxa5 is expressed in a dynamic fashion in the mesenchymal component of the developing gut. Its loss of function results in gastric enzymatic anomalies in Hoxa5(-/-) surviving mutants that are due to perturbed cell specification during stomach development. Histological, biochemical and molecular characterization of the mutant stomach phenotype may be compatible with a homeotic transformation of the gastric mucosa. As the loss of mesenchymal Hoxa5 function leads to gastric epithelial defects, Hoxa5 should exert its action by controlling molecules involved in mesenchymal-epithelial signaling. Indeed, in the absence of Hoxa5 function, the expression of genes encoding for signaling molecules such as sonic hedgehog, Indian hedgehog, transforming growth factor beta family members and fibroblast growth factor 10, is altered. These findings provide insight into the molecular controls of patterning events of the stomach, supporting the notion that Hoxa5 acts in regionalization and specification of the stomach by setting up the proper domains of expression of signaling molecules.

Cooperation of Hoxa5 and Pax1 genes during formation of the pectoral girdle.

Hox and Pax transcription factors are master regulators of skeletal and organ morphogenesis. Some skeletal malformations encountered in Hoxa5 mutants are shared by the undulated (un) mice, which bear a point mutation in the Pax1 gene. To investigate whether Hoxa5 and Pax1 act in common pathways during skeletal development, we analyzed Hoxa5;un compound mutants. Our genetic studies show that Hoxa5 and Pax1 cooperate in the vertebral patterning of the cervicothoracic transition region and in acromion morphogenesis. The dynamics of expression of Hoxa5 and Pax1 in the pectoral girdle region suggest that both genes function in a complementary fashion during acromion formation. Whereas Pax1 is required for the recruitment of acromion precursor cells, Hoxa5 may provide regional cues essential for the correct formation of the acromion by ensuring Pax1 expression at the proper time and position during morphogenesis of the pectoral girdle. Hoxa5 also has a distinctive role in specifying the fate of perichondrial and chondrogenic cell lineages in a Sox9-dependent way.