Tail resorption observed at the time of amphibian
metamorphosis is controlled by thyroid hormone.... more Tail resorption observed at the time of amphibian metamorphosis is controlled by thyroid hormone. The inherent regulation network is complex and involves an important number of different factors. Consequently, the global understanding of this biological process needs elaborated experiments. However, these experiments can prove to be difficult to realize because of the need to manipulate in space and in time gene expression and hormonal treatments. Hence, we first modelled and simulated this biological process using Hybrid Functional Petri Nets. This powerful formalism offers a number of features and flexibility. Curves obtained in silico by simulations are in agreement with those observed in vivo and in vitro. Our modelling approach led us to ask pertinent biological questions. From these questions, new hypothesis which stay to be experimentally tested have followed.
Background: Accurate interpretation of transcriptome profiling by quantitative PCR requires the e... more Background: Accurate interpretation of transcriptome profiling by quantitative PCR requires the establishment of species-specific standards. However, the selection of reference genes for assessing RNA expression profiles in Xenopus laevis and Xenopus tropicalis was mostly based on historical reasons and they often only reflect the traditions of a laboratory. Results: We investigated the expression stability of 10 genes (dicer1, drosha, eef1a1, elavl3, gsc, h4, odc1, rpl8, smn2, tbp), 8 of which are commonly used as internal controls in published RT-qPCR experiments. We defined specific primer pairs and evaluated their suitability as reference genes by performing RT-qPCR expression profiling in Xenopus tropicalis. Gene expression stability was assayed in a set of 15 developmental stages from the egg to the froglet, and in dissected embryos. Conclusions: Overall, we determined a set of qualified reference genes for distinct developmental periods. We recommend the use of dicer1, drosha, eef1a1, and smn2 from early embryonic development up to the end of metamorphosis. During early embryogenesis drosha, eef1a1, smn2 are suitable. For the whole post-embryonic development and for metamorphic stages including pro-metamorphosis and metamorphic climax, we recommend the use of drosha and smn2. These reference genes should prove their usefulness for data comparison across studies. Developmental Dynamics 000:000–000, 2013. VC 2013 Wiley Periodicals, Inc.
Acoustic communication is widespread in animals. According to
the sensory drive hypothesis [Endle... more Acoustic communication is widespread in animals. According to the sensory drive hypothesis [Endler JA (1993) Philos Trans R Soc Lond B Biol Sci 340(1292):215–225], communication signals and perceptual systems have coevolved. A clear illustration of this is the evolution of the tetrapod middle ear, adapted to life on land. Here we report the discovery of a bone conduction–mediated stimulation of the ear by wave propagation in Sechellophryne gardineri, one of the world’s smallest terrestrial tetrapods, which lacks a middle ear yet produces acoustic signals. Based on X-ray synchrotron holotomography, we measured the biomechanical properties of the otic tissues and modeled the acoustic propagation. Our models show how bone conduction enhanced by the resonating role of the mouth allows these seemingly deaf frogs to communicate effectively without a middle ear.
Atelopus franciscus is a diurnal bufonid frog that lives in South-American tropical rain forests.... more Atelopus franciscus is a diurnal bufonid frog that lives in South-American tropical rain forests. As in many other frogs, males produce calls to defend their territories and attract females. However, this species is a so-called ‘‘earless’’ frog lacking an external tympanum and is thus anatomically deaf. Moreover, A. franciscus has no external vocal sac and lives in a sound constraining environment along river banks where it competes with other calling frogs. Despite these constraints, male A. franciscus reply acoustically to the calls of conspecifics in the field. To resolve this apparent paradox, we studied the vocal apparatus and middle-ear, analysed signal content of the calls, examined sound and signal content propagation in its natural habitat, and performed playback experiments. We show that A. franciscus males can produce only low intensity calls that propagate a short distance (,8 m) as a result of the lack of an external vocal sac. The species-specific coding of the signal is based on the pulse duration, providing a simple coding that is efficient as it allows discrimination from calls of sympatric frogs. Moreover, the signal is redundant and consequently adapted to noisy environments. As such a coding system can be efficient only at short-range, territory holders established themselves at short distances from each other. Finally, we show that the middle-ear of A. franciscus does not present any particular adaptations to compensate for the lack of an external tympanum, suggesting the existence of extra-tympanic pathways for sound propagation.
Unexpectedly severe radiation damage, showing up through deformation of the saccule,was encounter... more Unexpectedly severe radiation damage, showing up through deformation of the saccule,was encountered during a synchrotron radiation high-resolution (700nm pixel size) tomographic observation of an inner ear, fixed in a formaldehyde solution, of the frog Rana esculenta. The visible displacement of the edge of the otoconia-filled part of the saccule amounted to about 100mafter an irradiation with 20.5 keV X-ray photons corresponding to a dose of 1.5 kGy for the protein matrix. The close-knit coexistence of organic and mineralcomponents in the biological tissuemay be linked to the dramatic increase of radiation dosage sensitivity.
Using the Sleeping Beauty (SB) transposon system, we have developed a simple method for the gener... more Using the Sleeping Beauty (SB) transposon system, we have developed a simple method for the generation of Xenopus laevis transgenic lines. The transgenesis protocol is based on the co-injection of the SB transposase mRNA and a GFP-reporter transposon into one-cell stage embryos. Transposase-dependent reporter gene expression was observed in cell clones and in hemi-transgenic animals. We determined an optimal ratio of transposase mRNA versus transposon-carrying plasmid DNA that enhanced the proportion of hemi-transgenic tadpoles. The transgene is integrated into the genome and may be transmitted to the F1 offspring depending on the germline mosaicism. Although the transposase is necessary for efficient generation of transgenic Xenopus, the integration of the transgene occurred by an non-canonical transposition process. This was observed for two transgenic lines analysed. The transposon-based technique leads to a high transgenesis rate and is simple to handle. For these reasons, it could present an attractive alternative to the classical Restriction Enzyme Mediated Integration (REMI) procedure.
Backgrounds & Aims: Mutations in the JAGGED1 gene are responsible for the Alagille syndrome, an a... more Backgrounds & Aims: Mutations in the JAGGED1 gene are responsible for the Alagille syndrome, an autosomal dominant disorder characterized by neonatal jaundice, intrahepatic cholestasis, and developmental disorders affecting the liver, heart, vertebrae, eyes, and face. We screened a large group of patients for mutations in JAGGED1 and studied transmission of the mutations. Methods: The coding sequence of the JAGGED1 gene was searched by single-strand conformation polymorphism and sequence analysis for mutations in 109 unrelated patients with the Alagille syndrome and their family if available. Results: Sixty-nine patients (63%) had intragenic mutations, including 14 nonsense mutations, 31 frameshifts, 11 splice site mutations, and 13 missense mutations. We identified 59 different types of mutation of which 54 were previously undescribed; 8 were observed more than once. Mutations were de novo in 40 of 57 probands. Conclusions: Most of the observed mutations other than the missense mutations in JAGGED1 are expected to give rise to truncated and unanchored proteins. All mutations mapped to the extracellular domain of the protein, and there appeared to be regional hot spots, although no clustering was observed. Thus, the sequencing of 7 exons of JAGGED1 would detect 51% of the mutations. Transmission analysis showed a high frequency of sporadic cases (70%).GASTROENTEROLOGY 1999;116:1141-1148
Alagille syndrome (AGS) is a well-defined genetic entity assigned to the short arm of Chromosome ... more Alagille syndrome (AGS) is a well-defined genetic entity assigned to the short arm of Chromosome (Chr) 20 by a series of observations of AGS patients associated with microdeletions in this region. By fusing lymphoblastoid cells of an AGS patient that exhibited a microdeletion in the short arm of Chr 20 encompassing bands p11.23 to p12.3 with rodent thermosensitive mutant cells (CHOtsH1-l) deficient in-leucyl-tRNA synthetase, we isolated a somatic cell hybrid segregating the deleted human Chr 20. This hybrid clone, designated NR2, was characterized by several methods, including PCR, with eight pairs of oligonucleotides mapped to Chr 20: D20S5, D20S41, D20S42, D20S56, D20S57, D20S58, adenosine deaminase (ADA), and Prion protein (PRIP); Restriction Fragment Length Polymorphism (RFLP) analyses with four genomic anonymous probes (D20S5, cD3H12, D20S17, D20S18); and fluorescent in situ hybridization (FISH) with total human DNA and D20Z1, a sequence specific to the human Chr 20 centromere, as probes. The NR2 hybrid allowed us to exclude three candidate genes for AGS: hepatic nuclear factor 3 β (HNF3β), paired box 1 (PAX1), and cystatin C (CST3) as shown by their localization outside of the deletion. The NR2 hybrid is a powerful tool for the mapping of new probes of this region, as well as for obtaining new informative probes specific for the deletion by subtractive cloning of the region. Such markers will be useful for linkage analysis and screening of cDNA libraries.
Background As an alternative to the frequently used "reference design" for two-channel microarray... more Background As an alternative to the frequently used "reference design" for two-channel microarrays, other designs have been proposed. These designs have been shown to be more profitable from a theoretical point of view (more replicates of the conditions of interest for the same number of arrays). However, the interpretation of the measurements is less straightforward and a reconstruction method is needed to convert the observed ratios into the genuine profile of interest (e.g. a time profile). The potential advantages of using these alternative designs thus largely depend on the success of the profile reconstruction. Therefore, we compared to what extent different linear models agree with each other in reconstructing expression ratios and corresponding time profiles from a complex design. Results On average the correlation between the estimated ratios was high, and all methods agreed with each other in predicting the same profile, especially for genes of which the expression profile showed a large variance across the different time points. Assessing the similarity in profile shape, it appears that, the more similar the underlying principles of the methods (model and input data), the more similar their results. Methods with a dye effect seemed more robust against array failure. The influence of a different normalization was not drastic and independent of the method used. Conclusion Including a dye effect such as in the methods lmbr_dye, anovaFix and anovaMix compensates for residual dye related inconsistencies in the data and renders the results more robust against array failure. Including random effects requires more parameters to be estimated and is only advised when a design is used with a sufficient number of replicates. Because of this, we believe lmbr_dye, anovaFix and anovaMix are most appropriate for practical use.
Fibroblast growth factors (FGFs) signal through high-affinity tyrosine kinase receptors to regula... more Fibroblast growth factors (FGFs) signal through high-affinity tyrosine kinase receptors to regulate a diverse range of cellular processes, including cell growth, differentiation and migration, as well as cell death. Here we identify XFLRT3, a member of a leucine-rich-repeat transmembrane protein family, as a novel modulator of FGF signalling. XFLRT3 is co-expressed with FGFs, and its expression is both induced after activation and downregulated after inhibition of FGF signalling. In gain- and loss-of function experiments, FLRT3 and FLRT2 phenocopy FGF signalling in Xenopus laevis. XFLRT3 signalling results in phosphorylation of ERK and is blocked by MAPK phosphatase 1, but not by expression of a dominant-negative phosphatidyl inositol 3-OH kinase (PI(3)K) mutant. XFLRT3 interacts with FGF receptors (FGFRs) in co-immunoprecipitation experiments in vitro and in bioluminescence resonance energy transfer assays in vivo. The results indicate that XFLRT3 is a transmembrane modulator of FGF-MAP kinase signalling in vertebrates.
In 1960, Jacob and Monod described the bacterial operon, a cluster of functionally interacting ge... more In 1960, Jacob and Monod described the bacterial operon, a cluster of functionally interacting genes whose expression is tightly coordinated. Global expression analysis has shown that the highly coordinate expression of genes functioning in common processes is also a widespread phenomenon in eukaryotes. These sets of co-regulated genes, or 'synexpression groups', show a striking parallel to the operon, and may be a key determinant facilitating evolutionary change leading to animal diversity.
In 1960, Jacob and Monod described the bacterial operon, a cluster of functionally interacting ge... more In 1960, Jacob and Monod described the bacterial operon, a cluster of functionally interacting genes whose expression is tightly coordinated. Global expression analysis has shown that the highly coordinate expression of genes functioning in common processes is also a widespread phenomenon in eukaryotes. These sets of co-regulated genes, or 'synexpression groups', show a striking parallel to the operon, and may be a key determinant facilitating evolutionary change leading to animal diversity.
Tail resorption observed at the time of amphibian
metamorphosis is controlled by thyroid hormone.... more Tail resorption observed at the time of amphibian metamorphosis is controlled by thyroid hormone. The inherent regulation network is complex and involves an important number of different factors. Consequently, the global understanding of this biological process needs elaborated experiments. However, these experiments can prove to be difficult to realize because of the need to manipulate in space and in time gene expression and hormonal treatments. Hence, we first modelled and simulated this biological process using Hybrid Functional Petri Nets. This powerful formalism offers a number of features and flexibility. Curves obtained in silico by simulations are in agreement with those observed in vivo and in vitro. Our modelling approach led us to ask pertinent biological questions. From these questions, new hypothesis which stay to be experimentally tested have followed.
Background: Accurate interpretation of transcriptome profiling by quantitative PCR requires the e... more Background: Accurate interpretation of transcriptome profiling by quantitative PCR requires the establishment of species-specific standards. However, the selection of reference genes for assessing RNA expression profiles in Xenopus laevis and Xenopus tropicalis was mostly based on historical reasons and they often only reflect the traditions of a laboratory. Results: We investigated the expression stability of 10 genes (dicer1, drosha, eef1a1, elavl3, gsc, h4, odc1, rpl8, smn2, tbp), 8 of which are commonly used as internal controls in published RT-qPCR experiments. We defined specific primer pairs and evaluated their suitability as reference genes by performing RT-qPCR expression profiling in Xenopus tropicalis. Gene expression stability was assayed in a set of 15 developmental stages from the egg to the froglet, and in dissected embryos. Conclusions: Overall, we determined a set of qualified reference genes for distinct developmental periods. We recommend the use of dicer1, drosha, eef1a1, and smn2 from early embryonic development up to the end of metamorphosis. During early embryogenesis drosha, eef1a1, smn2 are suitable. For the whole post-embryonic development and for metamorphic stages including pro-metamorphosis and metamorphic climax, we recommend the use of drosha and smn2. These reference genes should prove their usefulness for data comparison across studies. Developmental Dynamics 000:000–000, 2013. VC 2013 Wiley Periodicals, Inc.
Acoustic communication is widespread in animals. According to
the sensory drive hypothesis [Endle... more Acoustic communication is widespread in animals. According to the sensory drive hypothesis [Endler JA (1993) Philos Trans R Soc Lond B Biol Sci 340(1292):215–225], communication signals and perceptual systems have coevolved. A clear illustration of this is the evolution of the tetrapod middle ear, adapted to life on land. Here we report the discovery of a bone conduction–mediated stimulation of the ear by wave propagation in Sechellophryne gardineri, one of the world’s smallest terrestrial tetrapods, which lacks a middle ear yet produces acoustic signals. Based on X-ray synchrotron holotomography, we measured the biomechanical properties of the otic tissues and modeled the acoustic propagation. Our models show how bone conduction enhanced by the resonating role of the mouth allows these seemingly deaf frogs to communicate effectively without a middle ear.
Atelopus franciscus is a diurnal bufonid frog that lives in South-American tropical rain forests.... more Atelopus franciscus is a diurnal bufonid frog that lives in South-American tropical rain forests. As in many other frogs, males produce calls to defend their territories and attract females. However, this species is a so-called ‘‘earless’’ frog lacking an external tympanum and is thus anatomically deaf. Moreover, A. franciscus has no external vocal sac and lives in a sound constraining environment along river banks where it competes with other calling frogs. Despite these constraints, male A. franciscus reply acoustically to the calls of conspecifics in the field. To resolve this apparent paradox, we studied the vocal apparatus and middle-ear, analysed signal content of the calls, examined sound and signal content propagation in its natural habitat, and performed playback experiments. We show that A. franciscus males can produce only low intensity calls that propagate a short distance (,8 m) as a result of the lack of an external vocal sac. The species-specific coding of the signal is based on the pulse duration, providing a simple coding that is efficient as it allows discrimination from calls of sympatric frogs. Moreover, the signal is redundant and consequently adapted to noisy environments. As such a coding system can be efficient only at short-range, territory holders established themselves at short distances from each other. Finally, we show that the middle-ear of A. franciscus does not present any particular adaptations to compensate for the lack of an external tympanum, suggesting the existence of extra-tympanic pathways for sound propagation.
Unexpectedly severe radiation damage, showing up through deformation of the saccule,was encounter... more Unexpectedly severe radiation damage, showing up through deformation of the saccule,was encountered during a synchrotron radiation high-resolution (700nm pixel size) tomographic observation of an inner ear, fixed in a formaldehyde solution, of the frog Rana esculenta. The visible displacement of the edge of the otoconia-filled part of the saccule amounted to about 100mafter an irradiation with 20.5 keV X-ray photons corresponding to a dose of 1.5 kGy for the protein matrix. The close-knit coexistence of organic and mineralcomponents in the biological tissuemay be linked to the dramatic increase of radiation dosage sensitivity.
Using the Sleeping Beauty (SB) transposon system, we have developed a simple method for the gener... more Using the Sleeping Beauty (SB) transposon system, we have developed a simple method for the generation of Xenopus laevis transgenic lines. The transgenesis protocol is based on the co-injection of the SB transposase mRNA and a GFP-reporter transposon into one-cell stage embryos. Transposase-dependent reporter gene expression was observed in cell clones and in hemi-transgenic animals. We determined an optimal ratio of transposase mRNA versus transposon-carrying plasmid DNA that enhanced the proportion of hemi-transgenic tadpoles. The transgene is integrated into the genome and may be transmitted to the F1 offspring depending on the germline mosaicism. Although the transposase is necessary for efficient generation of transgenic Xenopus, the integration of the transgene occurred by an non-canonical transposition process. This was observed for two transgenic lines analysed. The transposon-based technique leads to a high transgenesis rate and is simple to handle. For these reasons, it could present an attractive alternative to the classical Restriction Enzyme Mediated Integration (REMI) procedure.
Backgrounds & Aims: Mutations in the JAGGED1 gene are responsible for the Alagille syndrome, an a... more Backgrounds & Aims: Mutations in the JAGGED1 gene are responsible for the Alagille syndrome, an autosomal dominant disorder characterized by neonatal jaundice, intrahepatic cholestasis, and developmental disorders affecting the liver, heart, vertebrae, eyes, and face. We screened a large group of patients for mutations in JAGGED1 and studied transmission of the mutations. Methods: The coding sequence of the JAGGED1 gene was searched by single-strand conformation polymorphism and sequence analysis for mutations in 109 unrelated patients with the Alagille syndrome and their family if available. Results: Sixty-nine patients (63%) had intragenic mutations, including 14 nonsense mutations, 31 frameshifts, 11 splice site mutations, and 13 missense mutations. We identified 59 different types of mutation of which 54 were previously undescribed; 8 were observed more than once. Mutations were de novo in 40 of 57 probands. Conclusions: Most of the observed mutations other than the missense mutations in JAGGED1 are expected to give rise to truncated and unanchored proteins. All mutations mapped to the extracellular domain of the protein, and there appeared to be regional hot spots, although no clustering was observed. Thus, the sequencing of 7 exons of JAGGED1 would detect 51% of the mutations. Transmission analysis showed a high frequency of sporadic cases (70%).GASTROENTEROLOGY 1999;116:1141-1148
Alagille syndrome (AGS) is a well-defined genetic entity assigned to the short arm of Chromosome ... more Alagille syndrome (AGS) is a well-defined genetic entity assigned to the short arm of Chromosome (Chr) 20 by a series of observations of AGS patients associated with microdeletions in this region. By fusing lymphoblastoid cells of an AGS patient that exhibited a microdeletion in the short arm of Chr 20 encompassing bands p11.23 to p12.3 with rodent thermosensitive mutant cells (CHOtsH1-l) deficient in-leucyl-tRNA synthetase, we isolated a somatic cell hybrid segregating the deleted human Chr 20. This hybrid clone, designated NR2, was characterized by several methods, including PCR, with eight pairs of oligonucleotides mapped to Chr 20: D20S5, D20S41, D20S42, D20S56, D20S57, D20S58, adenosine deaminase (ADA), and Prion protein (PRIP); Restriction Fragment Length Polymorphism (RFLP) analyses with four genomic anonymous probes (D20S5, cD3H12, D20S17, D20S18); and fluorescent in situ hybridization (FISH) with total human DNA and D20Z1, a sequence specific to the human Chr 20 centromere, as probes. The NR2 hybrid allowed us to exclude three candidate genes for AGS: hepatic nuclear factor 3 β (HNF3β), paired box 1 (PAX1), and cystatin C (CST3) as shown by their localization outside of the deletion. The NR2 hybrid is a powerful tool for the mapping of new probes of this region, as well as for obtaining new informative probes specific for the deletion by subtractive cloning of the region. Such markers will be useful for linkage analysis and screening of cDNA libraries.
Background As an alternative to the frequently used "reference design" for two-channel microarray... more Background As an alternative to the frequently used "reference design" for two-channel microarrays, other designs have been proposed. These designs have been shown to be more profitable from a theoretical point of view (more replicates of the conditions of interest for the same number of arrays). However, the interpretation of the measurements is less straightforward and a reconstruction method is needed to convert the observed ratios into the genuine profile of interest (e.g. a time profile). The potential advantages of using these alternative designs thus largely depend on the success of the profile reconstruction. Therefore, we compared to what extent different linear models agree with each other in reconstructing expression ratios and corresponding time profiles from a complex design. Results On average the correlation between the estimated ratios was high, and all methods agreed with each other in predicting the same profile, especially for genes of which the expression profile showed a large variance across the different time points. Assessing the similarity in profile shape, it appears that, the more similar the underlying principles of the methods (model and input data), the more similar their results. Methods with a dye effect seemed more robust against array failure. The influence of a different normalization was not drastic and independent of the method used. Conclusion Including a dye effect such as in the methods lmbr_dye, anovaFix and anovaMix compensates for residual dye related inconsistencies in the data and renders the results more robust against array failure. Including random effects requires more parameters to be estimated and is only advised when a design is used with a sufficient number of replicates. Because of this, we believe lmbr_dye, anovaFix and anovaMix are most appropriate for practical use.
Fibroblast growth factors (FGFs) signal through high-affinity tyrosine kinase receptors to regula... more Fibroblast growth factors (FGFs) signal through high-affinity tyrosine kinase receptors to regulate a diverse range of cellular processes, including cell growth, differentiation and migration, as well as cell death. Here we identify XFLRT3, a member of a leucine-rich-repeat transmembrane protein family, as a novel modulator of FGF signalling. XFLRT3 is co-expressed with FGFs, and its expression is both induced after activation and downregulated after inhibition of FGF signalling. In gain- and loss-of function experiments, FLRT3 and FLRT2 phenocopy FGF signalling in Xenopus laevis. XFLRT3 signalling results in phosphorylation of ERK and is blocked by MAPK phosphatase 1, but not by expression of a dominant-negative phosphatidyl inositol 3-OH kinase (PI(3)K) mutant. XFLRT3 interacts with FGF receptors (FGFRs) in co-immunoprecipitation experiments in vitro and in bioluminescence resonance energy transfer assays in vivo. The results indicate that XFLRT3 is a transmembrane modulator of FGF-MAP kinase signalling in vertebrates.
In 1960, Jacob and Monod described the bacterial operon, a cluster of functionally interacting ge... more In 1960, Jacob and Monod described the bacterial operon, a cluster of functionally interacting genes whose expression is tightly coordinated. Global expression analysis has shown that the highly coordinate expression of genes functioning in common processes is also a widespread phenomenon in eukaryotes. These sets of co-regulated genes, or 'synexpression groups', show a striking parallel to the operon, and may be a key determinant facilitating evolutionary change leading to animal diversity.
In 1960, Jacob and Monod described the bacterial operon, a cluster of functionally interacting ge... more In 1960, Jacob and Monod described the bacterial operon, a cluster of functionally interacting genes whose expression is tightly coordinated. Global expression analysis has shown that the highly coordinate expression of genes functioning in common processes is also a widespread phenomenon in eukaryotes. These sets of co-regulated genes, or 'synexpression groups', show a striking parallel to the operon, and may be a key determinant facilitating evolutionary change leading to animal diversity.
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Papers by Nicolas Pollet
metamorphosis is controlled by thyroid hormone.
The inherent regulation network is complex and
involves an important number of different factors.
Consequently, the global understanding of this
biological process needs elaborated experiments.
However, these experiments can prove to be difficult
to realize because of the need to manipulate in
space and in time gene expression and hormonal
treatments. Hence, we first modelled and simulated
this biological process using Hybrid Functional Petri
Nets. This powerful formalism offers a number of
features and flexibility. Curves obtained in silico by
simulations are in agreement with those observed
in vivo and in vitro. Our modelling approach led
us to ask pertinent biological questions. From
these questions, new hypothesis which stay to be
experimentally tested have followed.
of species-specific standards. However, the selection of reference genes for assessing RNA
expression profiles in Xenopus laevis and Xenopus tropicalis was mostly based on historical reasons and
they often only reflect the traditions of a laboratory. Results: We investigated the expression stability of
10 genes (dicer1, drosha, eef1a1, elavl3, gsc, h4, odc1, rpl8, smn2, tbp), 8 of which are commonly used as internal
controls in published RT-qPCR experiments. We defined specific primer pairs and evaluated their
suitability as reference genes by performing RT-qPCR expression profiling in Xenopus tropicalis. Gene
expression stability was assayed in a set of 15 developmental stages from the egg to the froglet, and in dissected
embryos. Conclusions: Overall, we determined a set of qualified reference genes for distinct developmental
periods. We recommend the use of dicer1, drosha, eef1a1, and smn2 from early embryonic
development up to the end of metamorphosis. During early embryogenesis drosha, eef1a1, smn2 are suitable.
For the whole post-embryonic development and for metamorphic stages including pro-metamorphosis
and metamorphic climax, we recommend the use of drosha and smn2. These reference genes should prove
their usefulness for data comparison across studies. Developmental Dynamics 000:000–000, 2013. VC 2013
Wiley Periodicals, Inc.
the sensory drive hypothesis [Endler JA (1993) Philos Trans R Soc
Lond B Biol Sci 340(1292):215–225], communication signals and
perceptual systems have coevolved. A clear illustration of this is
the evolution of the tetrapod middle ear, adapted to life on land.
Here we report the discovery of a bone conduction–mediated
stimulation of the ear by wave propagation in Sechellophryne
gardineri, one of the world’s smallest terrestrial tetrapods, which
lacks a middle ear yet produces acoustic signals. Based on X-ray
synchrotron holotomography, we measured the biomechanical
properties of the otic tissues and modeled the acoustic propagation.
Our models show how bone conduction enhanced by the
resonating role of the mouth allows these seemingly deaf frogs
to communicate effectively without a middle ear.
produce calls to defend their territories and attract females. However, this species is a so-called ‘‘earless’’ frog lacking an
external tympanum and is thus anatomically deaf. Moreover, A. franciscus has no external vocal sac and lives in a sound
constraining environment along river banks where it competes with other calling frogs. Despite these constraints, male A.
franciscus reply acoustically to the calls of conspecifics in the field. To resolve this apparent paradox, we studied the vocal
apparatus and middle-ear, analysed signal content of the calls, examined sound and signal content propagation in its
natural habitat, and performed playback experiments. We show that A. franciscus males can produce only low intensity calls
that propagate a short distance (,8 m) as a result of the lack of an external vocal sac. The species-specific coding of the
signal is based on the pulse duration, providing a simple coding that is efficient as it allows discrimination from calls of
sympatric frogs. Moreover, the signal is redundant and consequently adapted to noisy environments. As such a coding
system can be efficient only at short-range, territory holders established themselves at short distances from each other.
Finally, we show that the middle-ear of A. franciscus does not present any particular adaptations to compensate for the lack
of an external tympanum, suggesting the existence of extra-tympanic pathways for sound propagation.
during a synchrotron radiation high-resolution (700nm pixel size) tomographic observation of an inner
ear, fixed in a formaldehyde solution, of the frog Rana esculenta. The visible displacement of the edge of
the otoconia-filled part of the saccule amounted to about 100mafter an irradiation with 20.5 keV X-ray
photons corresponding to a dose of 1.5 kGy for the protein matrix. The close-knit coexistence of organic
and mineralcomponents in the biological tissuemay be linked to the dramatic increase of radiation dosage
sensitivity.
metamorphosis is controlled by thyroid hormone.
The inherent regulation network is complex and
involves an important number of different factors.
Consequently, the global understanding of this
biological process needs elaborated experiments.
However, these experiments can prove to be difficult
to realize because of the need to manipulate in
space and in time gene expression and hormonal
treatments. Hence, we first modelled and simulated
this biological process using Hybrid Functional Petri
Nets. This powerful formalism offers a number of
features and flexibility. Curves obtained in silico by
simulations are in agreement with those observed
in vivo and in vitro. Our modelling approach led
us to ask pertinent biological questions. From
these questions, new hypothesis which stay to be
experimentally tested have followed.
of species-specific standards. However, the selection of reference genes for assessing RNA
expression profiles in Xenopus laevis and Xenopus tropicalis was mostly based on historical reasons and
they often only reflect the traditions of a laboratory. Results: We investigated the expression stability of
10 genes (dicer1, drosha, eef1a1, elavl3, gsc, h4, odc1, rpl8, smn2, tbp), 8 of which are commonly used as internal
controls in published RT-qPCR experiments. We defined specific primer pairs and evaluated their
suitability as reference genes by performing RT-qPCR expression profiling in Xenopus tropicalis. Gene
expression stability was assayed in a set of 15 developmental stages from the egg to the froglet, and in dissected
embryos. Conclusions: Overall, we determined a set of qualified reference genes for distinct developmental
periods. We recommend the use of dicer1, drosha, eef1a1, and smn2 from early embryonic
development up to the end of metamorphosis. During early embryogenesis drosha, eef1a1, smn2 are suitable.
For the whole post-embryonic development and for metamorphic stages including pro-metamorphosis
and metamorphic climax, we recommend the use of drosha and smn2. These reference genes should prove
their usefulness for data comparison across studies. Developmental Dynamics 000:000–000, 2013. VC 2013
Wiley Periodicals, Inc.
the sensory drive hypothesis [Endler JA (1993) Philos Trans R Soc
Lond B Biol Sci 340(1292):215–225], communication signals and
perceptual systems have coevolved. A clear illustration of this is
the evolution of the tetrapod middle ear, adapted to life on land.
Here we report the discovery of a bone conduction–mediated
stimulation of the ear by wave propagation in Sechellophryne
gardineri, one of the world’s smallest terrestrial tetrapods, which
lacks a middle ear yet produces acoustic signals. Based on X-ray
synchrotron holotomography, we measured the biomechanical
properties of the otic tissues and modeled the acoustic propagation.
Our models show how bone conduction enhanced by the
resonating role of the mouth allows these seemingly deaf frogs
to communicate effectively without a middle ear.
produce calls to defend their territories and attract females. However, this species is a so-called ‘‘earless’’ frog lacking an
external tympanum and is thus anatomically deaf. Moreover, A. franciscus has no external vocal sac and lives in a sound
constraining environment along river banks where it competes with other calling frogs. Despite these constraints, male A.
franciscus reply acoustically to the calls of conspecifics in the field. To resolve this apparent paradox, we studied the vocal
apparatus and middle-ear, analysed signal content of the calls, examined sound and signal content propagation in its
natural habitat, and performed playback experiments. We show that A. franciscus males can produce only low intensity calls
that propagate a short distance (,8 m) as a result of the lack of an external vocal sac. The species-specific coding of the
signal is based on the pulse duration, providing a simple coding that is efficient as it allows discrimination from calls of
sympatric frogs. Moreover, the signal is redundant and consequently adapted to noisy environments. As such a coding
system can be efficient only at short-range, territory holders established themselves at short distances from each other.
Finally, we show that the middle-ear of A. franciscus does not present any particular adaptations to compensate for the lack
of an external tympanum, suggesting the existence of extra-tympanic pathways for sound propagation.
during a synchrotron radiation high-resolution (700nm pixel size) tomographic observation of an inner
ear, fixed in a formaldehyde solution, of the frog Rana esculenta. The visible displacement of the edge of
the otoconia-filled part of the saccule amounted to about 100mafter an irradiation with 20.5 keV X-ray
photons corresponding to a dose of 1.5 kGy for the protein matrix. The close-knit coexistence of organic
and mineralcomponents in the biological tissuemay be linked to the dramatic increase of radiation dosage
sensitivity.