staff

Jozsef Zakany

Senior Lecturer in Developmental Genomics

  • T: +41 22 379 67 88
  • office 4003b (Sciences III)
  • Control of growth and gut maturation by HoxD genes and the associated lncRNA Haglr. Proc. Natl. Acad. Sci. U.S.A. 2017 Oct;():. 1712511114. 10.1073/pnas.1712511114.

    abstract

    During embryonic development, Hox genes participate in the building of a functional digestive system in metazoans, and genetic conditions involving these genes lead to important, sometimes lethal, growth retardation. Recently, this phenotype was obtained after deletion of Haglr, the Hoxd antisense growth-associated long noncoding RNA (lncRNA) located between Hoxd1 and Hoxd3 In this study, we have analyzed the function of Hoxd genes in delayed growth trajectories by looking at several nested targeted deficiencies of the mouse HoxD cluster. Mutant pups were severely stunted during the suckling period, but many recovered after weaning. After comparing seven distinct HoxD alleles, including CRISPR/Cas9 deletions involving Haglr, we identified Hoxd3 as the critical component for the gut to maintain milk-digestive competence. This essential function could be abrogated by the dominant-negative effect of HOXD10 as shown by a genetic rescue approach, thus further illustrating the importance of posterior prevalence in Hox gene function. A role for the lncRNA Haglr in the control of postnatal growth could not be corroborated.

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  • Control of Hoxd gene transcription in the mammary bud by hijacking a preexisting regulatory landscape. Proc. Natl. Acad. Sci. U.S.A. 2016 Nov;():. 1617141113. 10.1073/pnas.1617141113.

    abstract

    Vertebrate Hox genes encode transcription factors operating during the development of multiple organs and structures. However, the evolutionary mechanism underlying this remarkable pleiotropy remains to be fully understood. Here, we show that Hoxd8 and Hoxd9, two genes of the HoxD complex, are transcribed during mammary bud (MB) development. However, unlike in other developmental contexts, their coexpression does not rely on the same regulatory mechanism. Hoxd8 is regulated by the combined activity of closely located sequences and the most distant telomeric gene desert. On the other hand, Hoxd9 is controlled by an enhancer-rich region that is also located within the telomeric gene desert but has no impact on Hoxd8 transcription, thus constituting an exception to the global regulatory logic systematically observed at this locus. The latter DNA region is also involved in Hoxd gene regulation in other contexts and strongly interacts with Hoxd9 in all tissues analyzed thus far, indicating that its regulatory activity was already operational before the appearance of mammary glands. Within this DNA region and neighboring a strong limb enhancer, we identified a short sequence conserved in therian mammals and capable of enhancer activity in the MBs. We propose that Hoxd gene regulation in embryonic MBs evolved by hijacking a preexisting regulatory landscape that was already at work before the emergence of mammals in structures such as the limbs or the intestinal tract.

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  • Transgene- and locus-dependent imprinting reveals allele-specific chromosome conformations. Proc. Natl. Acad. Sci. U.S.A. 2013 Jul;110(29):11946-51. 1310704110. 10.1073/pnas.1310704110. PMC3718164.

    abstract

    When positioned into the integrin α-6 gene, an Hoxd9lacZ reporter transgene displayed parental imprinting in mouse embryos. While the expression from the paternal allele was comparable with patterns seen for the same transgene when present at the neighboring HoxD locus, almost no signal was scored at this integration site when the transgene was inherited from the mother, although the Itga6 locus itself is not imprinted. The transgene exhibited maternal allele-specific DNA hypermethylation acquired during oogenesis, and its expression silencing was reversible on passage through the male germ line. Histone modifications also corresponded to profiles described at known imprinted loci. Chromosome conformation analyses revealed distinct chromatin microarchitectures, with a more compact structure characterizing the maternally inherited repressed allele. Such genetic analyses of well-characterized transgene insertions associated with a de novo-induced parental imprint may help us understand the molecular determinants of imprinting.

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  • A genetic basis for altered sexual behavior in mutant female mice. Curr. Biol. 2012 Sep;22(18):1676-80. S0960-9822(12)00743-9. 10.1016/j.cub.2012.06.067.

    abstract

    Although neural substrates of mammalian female mating behavior have been described, the association between complex courtship activity and specific underlying mechanisms remains elusive. We have isolated a mouse line that unexpectedly shows altered female social behavior with increased investigation of males and increased genital biting. We investigated adult individuals by behavioral observation and genetic and molecular neuroanatomy methods. We report exacerbated inverse pursuits and incapacitating bites directed at the genitals of stud males. This extreme deviation from wild-type female courtship segregates with a deletion of the Hoxd1 to Hoxd9 genomic region. This dominant Atypical female courtship allele (HoxD(Afc)) induces ectopic Hoxd10 gene expression in several regions in newborn forebrain transitorily and stably in a sparse subpopulation of cells in the cornu ammonis fields of adult hippocampus, which may thus lead to an abnormal modulation in the sexual behavior of mutant females. The resulting compulsive sexual solicitation behavior displayed by the most affected individuals suggests new avenues to study the genetic and molecular bases of normal and pathological mammalian affect and raises the potential involvement of the hippocampus in the control of female courtship behavior. The potential relevance to human 2q.31.1 microdeletion syndrome is discussed.

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  • A function for all posterior Hoxd genes during digit development? Dev. Dyn. 2012 Apr;241(4):792-802. 10.1002/dvdy.23756.

    abstract

    Four posterior Hoxd genes, from Hoxd13 to Hoxd10, are collectively regulated during the development of tetrapod digits. Besides the well-documented role of Hoxd13, the function of the neighboring genes has been difficult to evaluate due to the close genetic linkage and potential regulatory interferences. We used a combination of five small nested deletions in cis, involving from two to four consecutive genes of the Hoxd13 to Hoxd9 loci, in mice, to evaluate their combined functional importance.

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  • A systematic enhancer screen using lentivector transgenesis identifies conserved and non-conserved functional elements at the Olig1 and Olig2 locus. PLoS ONE 2010 ;5(12):e15741. 10.1371/journal.pone.0015741. PMC3012086.

    abstract

    Finding sequences that control expression of genes is central to understanding genome function. Previous studies have used evolutionary conservation as an indicator of regulatory potential. Here, we present a method for the unbiased in vivo screen of putative enhancers in large DNA regions, using the mouse as a model. We cloned a library of 142 overlapping fragments from a 200 kb-long murine BAC in a lentiviral vector expressing LacZ from a minimal promoter, and used the resulting vectors to infect fertilized murine oocytes. LacZ staining of E11 embryos obtained by first using the vectors in pools and then testing individual candidates led to the identification of 3 enhancers, only one of which shows significant evolutionary conservation. In situ hybridization and 3C/4C experiments suggest that this enhancer, which is active in the neural tube and posterior diencephalon, influences the expression of the Olig1 and/or Olig2 genes. This work provides a new approach for the large-scale in vivo screening of transcriptional regulatory sequences, and further demonstrates that evolutionary conservation alone seems too limiting a criterion for the identification of enhancers.

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  • Uncoupling time and space in the collinear regulation of Hox genes. PLoS Genet. 2009 Mar;5(3):e1000398. 10.1371/journal.pgen.1000398. PMC2642670.

    abstract

    During development of the vertebrate body axis, Hox genes are transcribed sequentially, in both time and space, following their relative positions within their genomic clusters. Analyses of animal genomes support the idea that Hox gene clustering is essential for coordinating the various times of gene activations. However, the eventual collinear ordering of the gene specific transcript domains in space does not always require genomic clustering. We analyzed these complex regulatory relationships by using mutant alleles at the mouse HoxD locus, including one that splits the cluster into two pieces. We show that both positive and negative regulatory influences, located on either side of the cluster, control an early phase of collinear expression in the trunk. Interestingly, this early phase does not systematically impact upon the subsequent expression patterns along the main body axis, indicating that the mechanism underlying temporal collinearity is distinct from those acting during the second phase. We discuss the potential functions and evolutionary origins of these mechanisms, as well as their relationship with similar processes at work during limb development.

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  • Hox gene function in vertebrate gut morphogenesis: the case of the caecum. Development 2007 Nov;134(22):3967-73. dev.010991. 10.1242/dev.010991.

    abstract

    The digestive tract is made of different subdivisions with various functions. During embryonic development, the developing intestine expresses combinations of Hox genes along its anterior to posterior axis, suggesting a role for these genes in this regionalization process. In particular, the transition from small to large intestine is labelled by the transcription of all Hoxd genes except Hoxd12 and Hoxd13, the latter two genes being transcribed only near the anus. Here, we describe two lines of mice that express Hoxd12 ectopically within this morphological transition. As a consequence, budding of the caecum is impeded, leading to complete agenesis in homozygous individuals. This effect is concurrent with a dramatic reduction of both Fgf10 and Pitx1 expression. Furthermore, the interactions between ;anterior' Hox genes and ectopic Hoxd12 suggest a model whereby anterior and posterior Hox products compete in controlling Fgf10 signalling, which is required for the growth of this organ in mice. These results illuminate components of the genetic cascade necessary for the emergence of this gut segment, crucial for many vertebrates.

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  • The role of Hox genes during vertebrate limb development. Curr. Opin. Genet. Dev. 2007 Aug;17(4):359-66. S0959-437X(07)00116-5. 10.1016/j.gde.2007.05.011.

    abstract

    The potential role of Hox genes during vertebrate limb development was brought into focus by gene expression analyses in mice (P Dolle, JC Izpisua-Belmonte, H Falkenstein, A Renucci, D Duboule, Nature 1989, 342:767-772), at a time when limb growth and patterning were thought to depend upon two distinct and rather independent systems of coordinates; one for the anterior-to-posterior axis and the other for the proximal-to-distal axis (see D Duboule, P Dolle, EMBO J 1989, 8:1497-1505). Over the past years, the function and regulation of these genes have been addressed using both gain-of-function and loss-of-function approaches in chick and mice. The use of multiple mutations either in cis-configuration in trans-configuration or in cis/trans configurations, has confirmed that Hox genes are essential for proper limb development, where they participate in both the growth and organization of the structures. Even though their molecular mechanisms of action remain somewhat elusive, the results of these extensive genetic analyses confirm that, during the development of the limbs, the various axes cannot be considered in isolation from each other and that a more holistic view of limb development should prevail over a simple cartesian, chess grid-like approach of these complex structures. With this in mind, the functional input of Hox genes during limb growth and development can now be re-assessed.

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  • Interactions between HOXD and Gli3 genes control the limb apical ectodermal ridge via Fgf10. Dev. Biol. 2007 Jun;306(2):883-93. S0012-1606(07)00242-4. 10.1016/j.ydbio.2007.03.517.

    abstract

    The development of the vertebrate limb is dependent upon two signaling centers, the apical ectodermal ridge (AER), which provides the underlying mesenchyme with essential growth factors, and the zone of polarizing activity (ZPA), the source of the Sonic hedgehog (SHH) product. Recent work involving gain and loss of function of Hox genes has emphasized their impact both on AER maintenance and Shh transcriptional activation. Here, we describe antagonistic interactions between posterior Hoxd genes and Gli3, suggesting that the latter product protects the AER from the deleterious effect of the formers, and we present evidence that Fgf10 is the mediator of HOX-dependent AER expansion. Furthermore, the striking similarity between some of the hereby observed Hox/Gli3-dependent morphogenetic defects and those displayed by fetuses with severely altered retinoic acid metabolism suggests a tight connection between these various pathways. The nature of these potential interactions is discussed in the context of proximal-distal growth and patterning.

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  • A dual role for Hox genes in limb anterior-posterior asymmetry. Science 2004 Jun;304(5677):1669-72. 10.1126/science.1096049. 304/5677/1669.

    abstract

    Anterior-to-posterior patterning, the process whereby our digits are differently shaped, is a key aspect of limb development. It depends on the localized expression in posterior limb bud of Sonic hedgehog (Shh) and the morphogenetic potential of its diffusing product. By using an inversion of and a large deficiency in the mouse HoxD cluster, we found that a perturbation in the early collinear expression of Hoxd11, Hoxd12, and Hoxd13 in limb buds led to a loss of asymmetry. Ectopic Hox gene expression triggered abnormal Shh transcription, which in turn induced symmetrical expression of Hox genes in digits, thereby generating double posterior limbs. We conclude that early posterior restriction of Hox gene products sets up an anterior-posterior prepattern, which determines the localized activation of Shh. This signal is subsequently translated into digit morphological asymmetry by promoting the late expression of Hoxd genes, two collinear processes relying on opposite genomic topographies, upstream and downstream Shh signaling.

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  • The loss of circadian PAR bZip transcription factors results in epilepsy. Genes Dev. 2004 Jun;18(12):1397-412. 10.1101/gad.301404. 301404. PMC423191.

    abstract

    DBP (albumin D-site-binding protein), HLF (hepatic leukemia factor), and TEF (thyrotroph embryonic factor) are the three members of the PAR bZip (proline and acidic amino acid-rich basic leucine zipper) transcription factor family. All three of these transcriptional regulatory proteins accumulate with robust circadian rhythms in tissues with high amplitudes of clock gene expression, such as the suprachiasmatic nucleus (SCN) and the liver. However, they are expressed at nearly invariable levels in most brain regions, in which clock gene expression only cycles with low amplitude. Here we show that mice deficient for all three PAR bZip proteins are highly susceptible to generalized spontaneous and audiogenic epilepsies that frequently are lethal. Transcriptome profiling revealed pyridoxal kinase (Pdxk) as a target gene of PAR bZip proteins in both liver and brain. Pyridoxal kinase converts vitamin B6 derivatives into pyridoxal phosphate (PLP), the coenzyme of many enzymes involved in amino acid and neurotransmitter metabolism. PAR bZip-deficient mice show decreased brain levels of PLP, serotonin, and dopamine, and such changes have previously been reported to cause epilepsies in other systems. Hence, the expression of some clock-controlled genes, such as Pdxk, may have to remain within narrow limits in the brain. This could explain why the circadian oscillator has evolved to generate only low-amplitude cycles in most brain regions.

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  • Localized and transient transcription of Hox genes suggests a link between patterning and the segmentation clock. Cell 2001 Jul;106(2):207-17. S0092-8674(01)00436-6.

    abstract

    During development, Hox gene transcription is activated in presomitic mesoderm with a time sequence that follows the order of the genes along the chromosome. Here, we show that Hoxd1 and other Hox genes display dynamic stripes of expression within presomitic mesoderm. The underlying transcriptional bursts may reflect the mechanism that coordinates Hox gene activation with somitogenesis. This mechanism appears to depend upon Notch signaling, as mice deficient for RBPJk, the effector of the Notch pathway, showed severely reduced Hoxd gene expression in presomitic mesoderm. These results suggest a molecular link between Hox gene activation and the segmentation clock. Such a linkage would efficiently keep in phase the production of novel segments with their morphological specification.

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  • Hox genes and the making of sphincters. Nature 1999 Oct;401(6755):761-2. 10.1038/44511.

    abstract

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  • Hox genes in digit development and evolution. Cell Tissue Res. 1999 Apr;296(1):19-25.

    abstract

    Homeobox genes located in the 5' part of the HoxA and HoxD complexes are required for proliferation of skeletal progenitor cells of the vertebrate limb. Specific combinations of gene products determine the length of the upper arm (genes belonging to groups 9 and 10), the lower arm (groups 10, 11 and 12) and the digits (groups 11, 12 and 13). In these different domains, individual gene products quantitatively contribute to an overall protein dose, with predominant roles for groups 11 and 13. Quantitative reduction in the gene dose in each set results in truncations of the corresponding anatomical regions. The physical order of the genes in the HoxA and HoxD complexes, as well as a unidirectional sequence in gene activation, allow for completion of the process in a precise order, which in turn makes possible the sequential outgrowth of the respective primordia. While the skeletal patterns of upper and lower limb are relatively stable throughout the tetrapods, more variation is seen in the digits. Molecular analysis of the underlying regulatory processes promises further exciting insights into the genetic control of development, pathology and the course of evolution.

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  • Regulation of number and size of digits by posterior Hox genes: a dose-dependent mechanism with potential evolutionary implications. Proc. Natl. Acad. Sci. U.S.A. 1997 Dec;94(25):13695-700. PMC28368.

    abstract

    The proper development of digits, in tetrapods, requires the activity of several genes of the HoxA and HoxD homeobox gene complexes. By using a variety of loss-of-function alleles involving the five Hox genes that have been described to affect digit patterning, we report here that the group 11, 12, and 13 genes control both the size and number of murine digits in a dose-dependent fashion, rather than through a Hox code involving differential qualitative functions. A similar dose-response is observed in the morphogenesis of the penian bone, the baculum, which further suggests that digits and external genitalia share this genetic control mechanism. A progressive reduction in the dose of Hox gene products led first to ectrodactyly, then to olygodactyly and adactyly. Interestingly, this transition between the pentadactyl to the adactyl formula went through a step of polydactyly. We propose that in the distal appendage of polydactylous short-digited ancestral tetrapods, such as Acanthostega, the HoxA complex was predominantly active. Subsequent recruitment of the HoxD complex contributed to both reductions in digit number and increase in digit length. Thus, transition through a polydactylous limb before reaching and stabilizing the pentadactyl pattern may have relied, at least in part, on asynchronous and independent changes in the regulation of HoxA and HoxD gene complexes.

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  • Deletion of a HoxD enhancer induces transcriptional heterochrony leading to transposition of the sacrum. EMBO J. 1997 Jul;16(14):4393-402. 10.1093/emboj/16.14.4393. PMC1170065.

    abstract

    A phylogenetically conserved transcriptional enhancer necessary for the activation of Hoxd-11 was deleted from the HoxD complex of mice by targeted mutagenesis. While genetic and expression analyses demonstrated the role of this regulatory element in the activation of Hoxd-11 during early somitogenesis, the function of this gene in developing limbs and the urogenital system was not affected, suggesting that Hox transcriptional controls are different in different axial structures. In the trunk of mutant embryos, transcriptional activation of Hoxd-11 and Hoxd-10 was severely delayed, but subsequently resumed with appropriate spatial distributions. The resulting caudal transposition of the sacrum indicates that proper vertebral specification requires a precise temporal control of Hox gene expression, in addition to spatial regulation. A slight time delay in expression (transcriptional heterochrony) cannot be compensated for at a later developmental stage, eventually leading to morphological alterations.

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  • Synpolydactyly in mice with a targeted deficiency in the HoxD complex. Nature 1996 Nov;384(6604):69-71. 10.1038/384069a0.

    abstract

    The morphogenesis of mammalian digits requires the function of several genes of the HoxD complex during development of limb buds. Using embryonic stem (ES) cells and a site-specific recombination system (loxP/Cre), we have induced a deficiency that eliminates the products of the Hoxd-13, Hoxd-12 and Hoxd-11 genes simultaneously. A Hoxd-11/lacz reporter gene replaced the deleted region in order to monitor the effect of this triple inactivation at the cellular level. Mice homozygous for this deficiency showed small digit primordia, a disorganized cartilage pattern and impaired skeletal mass. These alterations are similar to the defects seen in a human synpolydactyly, suggesting that this syndrome, which is associated with a subtle mutation in HOXD13 (ref. 8), may involve the loss of function of several Hoxd genes. These results indicate the existence of a functional hierarchy among these genes and provide us with an animal model to study human digit malformations.

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  • Functional equivalence and rescue among group 11 Hox gene products in vertebral patterning. Dev. Biol. 1996 Jun;176(2):325-8. S0012-1606(96)90137-2. 10.1006/dbio.1996.0137.

    abstract

    Hoxa-11 and Hoxd-11 are paralogous genes required for proper development of the vertebral column, the limbs, and the urogenital system. To further explore the functional relationship between these genes, as well as the potential rescue of one function by the other, we have introduced a Hoxd-11-expressing transgene into Hoxa-11/Hoxd-11 mutant genetic backgrounds. A range of phenotypes was observed, with transgenic mice displaying as few as four lumbar vertebrae while double mutant mice had as many as eight. When transgenic, double homozygote mutant animals showed six lumbar vertebrae, instead of the eight usually observed. The phenotypic rescue of these genotypes shows that the Hoxa-11 and Hoxd-11 products are functionally equivalent and that extra doses of Hoxd-11 can rescue Hoxa-11 loss of function.

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