RESEARCH INTERESTS

INTRODUCTION and BACKGROUND

Our group is studying the functions of retinoic acid, the main active derivative of vitamin A (retinol), in development, morphogenesis and cell differentiation. Development of a vertebrate embryo is a complex process, involving numerours cell-cell communication events and inductive processes. These require the action of various classes of signaling molecules, which in turn lead to alterations in gene expression programs in target cells (see Box 1). Many of these signaling molecules and/or their target genes are involved, when mutated, in the etiology of human congenital defects or pathologies, including cancer.

Retinoic acid (RA) was identified almost 20 years ago as the active ligand of a subfamily of nuclear receptors, the RARs and RXRs (see Box 2). This molecule, which is a small lipophilic compound, can act both as an intracrine and a paracrine (diffusible) signal to regulate gene expression, and it is estimated that several hundred genes could be regulated by this signal. RA and/or other retinoid compounds also have several indications in therapy, especially for the treatment of skin disorders such as acne and psoriasis, and are used in standard treatment or clinical trials for several types of cancers.

Our work aims at characterizing the functions of endogenous RA signaling in the context of mammalian development. We have developed several lines of genetically engineered mice that are used as models to investigate how perturbations of endogenous RA levels affect developmental processes, and to correlate the resulting morphological and cellular abnormalities to changes in gene regulatory pathways. Our current work focuses on forebrain, retinal, spinal cord and mesodermal patterning. We are using in particular the early enhancer of the proneural gene Neurogenin2 as a model system to study the interactions between the retinoid signal and other major signaling pathways, such as the FGF and Sonic hedgehog pathways, during early spinal neurogenesis.

RECENT RESULTS and ONGOING PROJECTS

Retinaldehyde dehydrogenase 2 (RALDH2) knockout mice: a model for early embryonic retinoic acid deficiency

Our work has contributed to establish that precise a regulation of the production and availability of RA within embryonic tissues is essential for development to proceed normally.
A first level of control is at the level of its intracellular synthesis (see Box 1). A small number of aldehyde dehydrogenases (the retinaldehyde dehydrogenases, RALDH) efficiently act as RA-synthesizing enzymes, and each of these enzymes exhibits highly regulated patterns of expression during mouse development (7). Raldh2 is expressed in specific embryonic regions, from gastrulation onwards. These include the somitic mesoderm, the posterior hindbrain mesoderm, the flank region or the spinal cord lateral motor columns (see Fig. 1). A null mutation of Raldh2 was generated via homologous recombination in mouse embryonic stem (ES) cells (1). Raldh2_^-/- null mutants are early embryonic lethal and display complex morphogenetic abnormalities (see Fig. 1). In-depth phenotypic analyses led to a characterization of the consequences of a lack of embryonic RA synthesis for specific developmental processes, such as hindbrain patterning (3) or early heart development (9).

To circumvent the early lethality of the Raldh2^-/- null mutants, we have established a rescue system in which maternal RA supplementation at early gestational stages allows to correct the abnormal heart phenotype and to extend the viability of mutants until fetal stages. This system has allowed to reveal and molecularly characterize additional tissue-specific functions of embryonic RA in forelimb (13) or branchial arch (18) patterning (see Fig. 2). Current work aims at understanding the molecular basis of the alterations in somite formation in the Raldh2^-/- embryos.We are assessing how RA signaling interferes with other embryonic signals to regulate the activity and/or the output of the ‘molecular oscillator’ mechanism that allows sequential formation of somites (20). Additional functions of RALDH2-mediated RA synthesis in patterning or differentiation of specific organ systems are also being studied in the context of several international collaborations.

Genetic redundancy between RA-synthesizing enzymes

Despite their essentially distinct expression profiles, RALDH enzymes may act in a combinatorial manner to produce RA in certain embryonic areas. For instance, there is a temporal overlap between RALDH2 and RALDH3 expression within the early embryonic forebrain and optic area. At later stages, all three RALDH enzymes are expressed in distinct regions and/or cell types of the developing eye and retina. To fully apprehend the role of RA for development of these structures, we are analyzing mice with compound mutations of Raldh2 and Raldh3 (or Raldh1) (see Ghyselinck/Mark laboratory for a description of the Raldh1 and Raldh3 mutants). According to the phenotypic defects, compound mutants will be used, along with the corresponding single mutants, for functional genomic analyses (using DNA microarrays) to unravel the genetic pathways involved in these defects.


Analysis of specific RA functions through conditional mutagenesis

Although the RA-rescue of Raldh2^-/- null mutants (see above) allowed to phenotypically characterize specific functions of this enzyme, this system is not suitable for in depth functional genomic studies, as the exogenously administered RA may interfere with embryonic gene regulation. Another drawback of this system is that the rescued Raldh2^-/- mutants do not survive after term, due to the presence of specific cardiovascular abnormalities (9). To analyze additional functions of this enzyme and possibly generate viable mouse models, we have engineered a novel allele allowing conditional somatic mutagenesis through Cre-mediated recombination. This allele contains a ‘floxed’ intronic PGK-neo selectable marker in an intronic location. Interestingly, the PGK-neo floxed allele behaves as a hypomorph, i.e. it leads to a decrease, rather that a complete absence, of the Raldh2 transcripts and protein (see Fig. 3). This allowed us to phenotypically analyze whether certain developmental processes are particularly sensitive to a partial decrease in RA levels. Interestingly, the resulting mutant mice display abnormalities restricted to the derivatives of the posterior branchial arches (see Fig. 3) which phenocopy the defects seen in the human DiGeorge syndrome (16). Conditional Raldh2 mutants devoid of the ‘floxed’ PGK-neo sequence have also been generated, and crosses have been undertaken with transgenic mice expressing the Cre recombinase in specific tissues (e.g. within the spinal cord). We are also generating novel transgenic lines that will be useful for generating or analyzing conditional mutant mice.

Developmental functions of RA-metabolizing enzymes

A novel class of P450 cytochromes, that includes the CYP26A1, B1 and C1 enzymes, has recently been shown to metabolize RA into more polar derivatives, thus triggering its catabolism. Expression analysis of the corresponding genes has revealed complex expression patterns in several embryonic regions, such as the hindbrain rhombomeres, the branchial apparatus or the spinal cord (8,11). The Cyp26A1 gene is mainly expressed during early embryogenesis, and its expression sites are often complementary to those expressing Raldh2 (e.g. in the rostral embryonic epiblast versus the posterior mesoderm during gastrulation, and in the tail bud versus the somitic region at later stages). In collaboration with M. Petkovich (Queen’s University, Kingston, Canada), we have analyzed the phenotype of Cyp26A1^-/- knockout mice (6) and have shown that its function is required for proper development of tail bud-derived structures, as well as for regional patterning of the rostral hindbrain and axial skeleton (see Fig. 4). Furthermore, the observation that Raldh2 haploinsufficiency has a suppressive effect on the Cyp26A1^-/- phenotype established that both enzymatic activities act antagonistically to control RA levels in specific embryonic areas (12). Additional functional aspects of the CYP26A1 enzyme are being studied.

Early spinal cord neurogenesis: study of the function and the regulatory sequences of the proneural gene Neurogenin2

During spinal cord neurogenesis, neural progenitors acquire positional identity along the rostro-caudal and dorso-ventral axis in response to the spatially restricted actions of extrinsic signaling factors. The proneural gene Neurogenin2 (Ngn2) encodes a basic helix-loop-helix (bHLH) transcription factor expressed in subsets of spinal progenitors and functional studies showed that Ngn2 promotes cell cycle arrest, neuronal differentiation and is involved in the specification of motor neuron progenitors in the mouse. We have shown that the first Ngn2 positive cells are located within the caudal neural plate at the 5 somite stage (E 8.5), anterior to the node. These data strongly suggest that Ngn2 is a key factor during spinal cord neurogenesis and therefore the study of its early function and its regulatory sequences constitute a model to better understand the onset of spinal cord differentiation and the specification of motor neuron progenitors. Our ongoing experiments have three aims: (i) The characterisation of Ngn2 function at E8.5 and the establishment of the fate of Ngn2-positive cells. (ii) The identification of transcription factors directly binding to the regulatory region responsible for Ngn2 early expression. Our present data suggest that RA, FGF and Sonic hedgehog signals could be integrated at the level of the Ngn2 regulatory enhancer sequence. (iii) The characterisation of the temporal and spatial restriction of Ngn2 expression within the motor neuron progenitor domain. We showed that the same regulatory sequence controls Ngn2 expression within this domain and in the caudal neural plate at E8.5. These experiments will be carried out mostly in the mouse embryo using Ngn2 mutants, an inducible Cre recombinase mouse line, mouse embryo culture and classic mouse transgenesis complemented with chicken DNA electroporation.


References

1. Niederreither, K., V. Subbarayan, P. Dollé and P. Chambon.
Embryonic retinoic acid synthesis is essential for early mouse post-implantation development.
Nature Genet. 21, 444-448, 1999.

2. Chazaud, C., P. Chambon and P. Dollé.
Retinoic acid is required in the mouse embryo for left-right asymmetry determination and heart morphogenesis.
Development 126, 2589-2596, 1999.

3. Niederreither, K., J. Vermot, B. Schuhbaur, P. Chambon and P. Dollé.
Retinoic acid synthesis and hindbrain patterning in the mouse embryo.
Development 127, 75-85, 2000.

4. Vermot, J., V. Fraulob, P. Dollé and K. Niederreither
Expression of enzymes synthetizing (Aldh1 and Raldh2) and metabolizing (CYP26) retinoic acid in the mouse female reproductive system.
Endocrinology 141, 3638-3645, 2000.

5. Mollard, R., S. Viville, S. J. Ward, D. Décimo, P. Chambon and P. Dollé.
Tissue-specific expression of retinoic acid receptor isoform transcripts in the mouse embryo.
Mech. Dev. 94, 223-232, 2000.

6. Abu-Abed, S *., P. Dollé *, D. Metzger, B. Beckett, P. Chambon and M. Petkovich. (* equal authors)
The retinoic acid-metabolizing enzyme, CYP26A1, is essential for normal hindbrain patterning, vertebral identity, and development of posterior structures
Genes Dev. 15, 226-240, 2001.

7. Romand, R., E. Albuisson, K. Niederreither, V. Fraulob, P. Chambon and P. Dollé.
Specific expression of the retinoic acid-synthesizing enzyme RALDH2 during mouse inner ear development.
Mech. Dev. 106, 185-189, 2001.

8. MacLean, G., S. Abu-Abed, P. Dollé, A. Tahayato, P.Chambon and M. Petkovich.
Cloning of a novel retinoic-acid metabolizing cytochrome P450, Cyp26B1, and comparative expression analysis with Cyp26A1 during early murine development.
Mech Dev. 107, 195-201, 2001.

9. Niederreither, K., J. Vermot, N. Messaddeq, B. Schuhbaur, P. Chambon and P. Dollé.
Embryonic retinoic acid synthesis is essential for heart patterning in the mouse.
Development 128, 1019-1031, 2001.

10. Niederreither, K., V. Fraulob, J.M. Garnier, P. Chambon and P. Dollé.
Differential expression of retinoic acid-synthesizing (RALDH) enzymes during fetal development and organ differentiation in the mouse.
Mech Dev. 110, 165-171, 2002.

11. Abu-Abed, S., G. MacLean, V. Fraulob , P. Chambon, M. Petkovich and P. Dollé.
Differential expression of the retinoic acid-metabolizing enzymes CYP26A1 and CYP26B1 during murine organogenesis.
Mech Dev. 110, 173-177, 2002.

12. Niederreither, K., S. Abu-Abed, B. Schuhbaur, M. Petkovich, P. Chambon and P. Dollé.
Genetic evidence that oxidative derivatives of retinoic acid are not involved in retinoid signaling during mouse development.
Nature Genet. 31, 84-88, 2002.

13. Niederreither, K., J. Vermot, B. Schuhbaur, P. Chambon and P. Dollé.
Embryonic retinoic acid synthesis is required for forelimb growth and anteroposterior patterning in the mouse.
Development 129, 3563-3574, 2002.

14. Niederreither, K, J. Vermot, V. Fraulob, P. Chambon and P. Dollé.
Retinaldehyde dehydrogenase 2 (RALDH2)-independent patterns of retinoic acid synthesis in the mouse embryo.
Proc. Natl. Acad. Sci. U.S.A. 99, 16111-16116, 2002.

15. Oosterveen, T., K. Niederreither, P. Dollé, P. Chambon, F. Meijlink and J. Deschamps.
Retinoids regulate the anterior expression boundaries of 5' Hoxb genes in posterior hindbrain.
EMBO J. 22, 262-269, 2003.

16. Vermot, J., K. Niederreither, J.M. Garnier, P. Chambon and P. Dollé.
Decreased embryonic retinoic acid synthesis results in a DiGeorge syndrome phenotype in newborn mice.
Proc. Natl. Acad. Sci. U.S.A. 100, 1763-1768, 2003.

17. Abu-Abed, S., P. Dollé, D. Metzger, C. Wood, G.MacLean, P. Chambon and M. Petkovich.
Developing with lethal RA levels: genetic ablation of Rarg can restore the viability of mice lacking Cyp26a1.
Development 130, 1449-1459, 2003.

18. Niederreither, K., J. Vermot, I. Le Roux, B. Schuhbaur, P. Chambon and P. Dollé.
The regional pattern of retinoic acid synthesis by RALDH2 is essential for the development of posterior pharyngeal arches and enteric nervous system.
Development 130, 2525-2534, 2003.

19. Vermot, J., B. Schuhbaur, H. Le Mouellic, P. McCaffery, J.M. Garnier, D. Hentsch, P. Brûlet, K. Niederreither, P. Chambon, P. Dollé and I. Le Roux.
Retinaldehyde dehydrogenase 2 and Hoxc8 are required in the murine brachial spinal cord for the specification of Lim1+ motoneurons and the correct distrubution of Islet1+ motoneurons.
Development 132, 1611-1621, 2005.

20. Vermot, J., J. Gallego-Llamas, V. Fraulob, K. Niederreither, P. Chambon and P. Dollé.
Retinoic acid controls the bilateral symmetry of somite formation in the mouse embryo.
Science 308, 563-567, 2005.