Hox gene and development of the auditory circuit

Sound vibration is sensed by hair cells in the inner ear. The information is transmitted to the cochlear nucleus in the brainstem via spiral ganglion neurons. The information is further transmitted to higher relaying centers in the brain such as superior olivary complex and inferior colliculus. The connectivity between these components is topographically organized in a frequency-specific manner. It is known that the organization is well-established from the beginning of the circuit development. However, little is still known about the molecular mechanisms underlying the development of connectivity in the auditory circuit. Homeobox transcription factors of the Hox gene family are known for their involvement in early anterior-posterior axis patterning of neuronal progenitors in the hindbrain. Recent evidence indicates that they also play important roles in late aspects of neuronal development and establishment of topographic circuitry. Moreover, a mutation in the HOXA2 gene has been recently shown to be responsible for hearing deficits in humans. By means of spatiotemporally controlled Hoxa2 and Hoxb2 conditional mutations in the mouse we analyzed the involvement of these factors in auditory circuit development and connectivity

Human teneurin-1 is a direct target of the homeobox transcription factor EMX2 at a novel alternate promoter

<p>Abstract</p> <p>Background</p> <p>Teneurin-1 is a member of a family of type II transmembrane proteins conserved from <it>C.elegans </it>to vertebrates. Teneurin expression in vertebrates is best studied in mouse and chicken, where the four members teneurin-1 to -4 are predominantly expressed in the developing nervous system in area specific patterns. Based on their distinct, complementary expression a possible function in the establishment of proper connectivity in the brain was postulated. However, the transcription factors contributing to these distinctive expression patterns are largely unknown. Emx2 is a homeobox transcription factor, known to be important for area specification in the developing cortex. A study of Emx2 knock-out mice suggested a role of Emx2 in regulating patterned teneurin expression.</p> <p>Results</p> <p>5'RACE of human teneurin-1 revealed new alternative untranslated exons that are conserved in mouse and chicken. Closer analysis of the conserved region around the newly identified transcription start revealed promoter activity that was induced by EMX2. Mutation of a predicted homeobox binding site decreased the promoter activity in different reporter assays <it>in vitro </it>and <it>in vivo </it>in electroporated chick embryos. We show direct <it>in vivo </it>binding of EMX2 to the newly identified promoter element and finally confirm that the endogenous alternate transcript is specifically upregulated by EMX2.</p> <p>Conclusions</p> <p>We found that human teneurin-1 is directly regulated by EMX2 at a newly identified and conserved promoter region upstream of the published transcription start site, establishing teneurin-1 as the first human EMX2 target gene. We identify and characterize the EMX2 dependent promoter element of human teneurin-1.</p

The Expression Pattern of the Mouse Receptor Tyrosine Kinase GeneMDK1Is Conserved through Evolution and RequiresHoxa-2for Rhombomere-Specific Expression in Mouse Embryos

AbstractSegmentation of the hindbrain has been conserved throughout the vertebrate species and results in the transient formation of rhombomeres, which are lineage-restricted compartments. Studies on the molecular mechanisms underlying the segmentation process have revealed that rhombomeric boundaries coincide with the expression limits of several evolutionary conserved genes such as the zinc-finger transcription factorKrox-20and homeobox genes which are expressed in a specific spatial and temporal order and have been shown to be important regulators of segmental identity. In addition toKrox-20and Hox genes, several members of the Eph subfamily of receptor protein tyrosine kinase (RTK) genes are also expressed in a segment-restricted manner in the hindbrain, suggesting that these receptors may act in concert with Hox genes to establish regional identity. In the cascade of regulatory interactions leading to segmental identity,Krox-20appears to act “upstream” of Hox genes, but the identity of the “downstream” effectors has not yet been identified. We report here the isolation of the zebrafish orthologue of the mouse RTK geneMDK1which belongs to the Eph receptor subfamily and show that the major expression domains of the mouse and the zebrafish genes have been conserved through evolution. Since the coincident spatial and temporal expression ofHoxa-2andMDK1in the mouse hindbrain suggested a possible regulatory link between them, we analyzed the expression of theMDK1inHoxa-2null mutant embryos. A selective lack ofMDK1expression in rhombomere 3 ofHoxa-2mutant hindbrains together with an overall altered expression pattern in the other rhombomeres was observed, thus demonstrating thatMDK1lies downstream ofHoxa-2in the morphogenetic signaling cascade

Distinct roles of Hoxa2 and Krox20 in the development of rhythmic neural networks controlling inspiratory depth, respiratory frequency, and jaw opening

<p>Abstract</p> <p>Background</p> <p>Little is known about the involvement of molecular determinants of segmental patterning of rhombomeres (r) in the development of rhythmic neural networks in the mouse hindbrain. Here, we compare the phenotypes of mice carrying targeted inactivations of <it>Hoxa2</it>, the only <it>Hox </it>gene expressed up to r2, and of <it>Krox20</it>, expressed in r3 and r5. We investigated the impact of such mutations on the neural circuits controlling jaw opening and breathing in newborn mice, compatible with Hoxa2-dependent trigeminal defects and direct regulation of <it>Hoxa2 </it>by Krox20 in r3.</p> <p>Results</p> <p>We found that <it>Hoxa2 </it>mutants displayed an impaired oro-buccal reflex, similarly to <it>Krox20 </it>mutants. In contrast, while <it>Krox20 </it>is required for the development of the rhythm-promoting parafacial respiratory group (pFRG) modulating respiratory frequency,<it> Hoxa2 </it>inactivation did not affect neonatal breathing frequency. Instead, we found that <it>Hoxa2</it>^-/-but not <it>Krox20</it>^-/-mutation leads to the elimination of a transient control of the inspiratory amplitude normally occurring during the first hours following birth. Tracing of r2-specific progenies of <it>Hoxa2 </it>expressing cells indicated that the control of inspiratory activity resides in rostral pontine areas and required an intact r2-derived territory.</p> <p>Conclusion</p> <p>Thus, inspiratory shaping and respiratory frequency are under the control of distinct <it>Hox</it>-dependent segmental cues in the mammalian brain. Moreover, these data point to the importance of rhombomere-specific genetic control in the development of modular neural networks in the mammalian hindbrain.</p

Postmitotic Hoxa5 Expression Specifies Pontine Neuron Positional Identity and Input Connectivity of Cortical Afferent Subsets

The mammalian precerebellar pontine nucleus (PN) has a main role in relaying cortical information to the cerebellum. The molecular determinants establishing ordered connectivity patterns between cortical afferents and precerebellar neurons are largely unknown. We show that expression of Hox5 transcription factors is induced in specific subsets of postmitotic PN neurons at migration onset. Hox5 induction is achieved by response to retinoic acid signaling, resulting in Jmjd3-dependent derepression of Polycomb chromatin and 3D conformational changes. Hoxa5 drives neurons to settle posteriorly in the PN, where they are monosynaptically targeted by cortical neuron subsets mainly carrying limb somatosensation. Furthermore, Hoxa5 postmigratory ectopic expression in PN neurons is sufficient to attract cortical somatosensory inputs regardless of position and avoid visual afferents. Transcriptome analysis further suggests that Hoxa5 is involved in circuit formation. Thus, Hoxa5 coordinates postmitotic specification, migration, settling position, and subcircuit assembly of PN neuron subsets in the cortico-cerebellar pathway.Peer reviewe

Dynamic interplay between thalamic activity and Cajal-Retzius cells regulates the wiring of cortical layer 1

Cortical wiring relies on guidepost cells and activity-dependent processes that are thought to act sequentially. Here, we show that the construction of layer 1 (L1), a main site of top-down integration, is regulated by crosstalk between transient Cajal-Retzius cells (CRc) and spontaneous activity of the thalamus, a main driver of bottom-up information. While activity was known to regulate CRc migration and elimination, we found that prenatal spontaneous thalamic activity and NMDA receptors selectively control CRc early density, without affecting their demise. CRc density, in turn, regulates the distribution of upper layer interneurons and excitatory synapses, thereby drastically impairing the apical dendrite activity of output pyramidal neurons. In contrast, postnatal sensory-evoked activity had a limited impact on L1 and selectively perturbed basal dendrites synaptogenesis. Collectively, our study highlights a remarkable interplay between thalamic activity and CRc in L1 functional wiring, with major implications for our understanding of cortical development.We thank the IBENS Imaging Facility (France BioImaging, supported by ANR-10-INBS-04, ANR-10-LABX-54 MEMO LIFE, and ANR-11-IDEX-000-02 PSL∗ Research University, “Investments for the Future”). This work was supported by grants from the Spanish Ministry of Science, Innovation, and Universities (PGC2018-096631-B-I00) and the European Research Council (ERC-2014-CoG-647012) to G.L.-B. N.C. received funding from the Marie Skłodowska-Curie individual fellowship under the European Union’s Horizon 2020 research and innovation program (AXO-MATH, grant agreement no. 798326). F.G. received funding from the Agence Nationale de la Recherche (SyTune, ANR-21-CE37-0010), the European Research Council under the European Union’s Horizon 2020 research and innovation program (NEUROGOAL, grant agreement no.677878), the Region Nouvelle-Aquitaine, and the University of Bordeaux. The Garel laboratory is supported by INSERM, CNRS, ANR-15-CE16-0003, ANR-19-CE16-0017-02, Investissements d’Avenir implemented by ANR-10-LABX-54 MEMO LIFE, ANR-11-IDEX-0001-02 PSL∗ Research University, and the European Research Council (ERC-2013-CoG-616080, NImO). I.G. is a recipient of a fellowship from the French Ministry of Research and postdoctoral funding from Labex MemoLife, and S.G. is part of the Ecole des Neurosciences de Paris Ile-de-France network.Peer reviewe

An ultraconserved Hox–Pbx responsive element resides in the coding sequence of Hoxa2 and is active in rhombomere 4

The Hoxa2 gene has a fundamental role in vertebrate craniofacial and hindbrain patterning. Segmental control of Hoxa2 expression is crucial to its function and several studies have highlighted transcriptional regulatory elements governing its activity in distinct rhombomeres. Here, we identify a putative Hox–Pbx responsive cis-regulatory sequence, which resides in the coding sequence of Hoxa2 and is an important component of Hoxa2 regulation in rhombomere (r) 4. By using cell transfection and chromatin immunoprecipitation (ChIP) assays, we show that this regulatory sequence is responsive to paralogue group 1 and 2 Hox proteins and to their Pbx co-factors. Importantly, we also show that the Hox–Pbx element cooperates with a previously reported Hoxa2 r4 intronic enhancer and that its integrity is required to drive specific reporter gene expression in r4 upon electroporation in the chick embryo hindbrain. Thus, both intronic as well as exonic regulatory sequences are involved in Hoxa2 segmental regulation in the developing r4. Finally, we found that the Hox–Pbx exonic element is embedded in a larger 205-bp long ultraconserved genomic element (UCE) shared by all vertebrate genomes. In this respect, our data further support the idea that extreme conservation of UCE sequences may be the result of multiple superposed functional and evolutionary constraints

A unique bipartite Polycomb signature regulates stimulus-response transcription during development

Rapid cellular responses to environmental stimuli are fundamental for development and maturation. Immediate early genes can be transcriptionally induced within minutes in response to a variety of signals. How their induction levels are regulated and their untimely activation by spurious signals prevented during development is poorly understood. We found that in developing sensory neurons, before perinatal sensory-activity-dependent induction, immediate early genes are embedded into a unique bipartite Polycomb chromatin signature, carrying active H3K27ac on promoters but repressive Ezh2-dependent H3K27me3 on gene bodies. This bipartite signature is widely present in developing cell types, including embryonic stem cells. Polycomb marking of gene bodies inhibits mRNA elongation, dampening productive transcription, while still allowing for fast stimulus-dependent mark removal and bipartite gene induction. We reveal a developmental epigenetic mechanism regulating the rapidity and amplitude of the transcriptional response to relevant stimuli, while preventing inappropriate activation of stimulus-response genes.T.K. was supported by a Japan Society for the Promotion of Science fellowship, and O.J. was supported by an EMBO Long-Term fellowship. F.M.R. was supported by the Swiss National Science Foundation (31003A_149573 and 31003A_175776). This project has also received funding from the European Research Council under the European Union’s Horizon 2020 research and innovation programme (grant no. 810111-EpiCrest2Reg). F.M.R. and M.B.S. were also supported by the Novartis Research Foundation.Peer reviewe

Hox Paralog Group 2 Genes Control the Migration of Mouse Pontine Neurons through Slit-Robo Signaling

The pontine neurons (PN) represent a major source of mossy fiber projections to the cerebellum. During mouse hindbrain development, PN migrate tangentially and sequentially along both the anteroposterior (AP) and dorsoventral (DV) axes. Unlike DV migration, which is controlled by the Netrin-1/Dcc attractive pathway, little is known about the molecular mechanisms guiding PN migration along the AP axis. Here, we show that Hoxa2 and Hoxb2 are required both intrinsically and extrinsically to maintain normal AP migration of subsets of PN, by preventing their premature ventral attraction towards the midline. Moreover, the migration defects observed in Hoxa2 and Hoxb2 mutant mice were phenocopied in compound Robo1;Robo2, Slit1;Slit2, and Robo2;Slit2 knockout animals, indicating that these guidance molecules act downstream of Hox genes to control PN migration. Indeed, using chromatin immunoprecipitation assays, we further demonstrated that Robo2 is a direct target of Hoxa2 in vivo and that maintenance of high Robo and Slit expression levels was impaired in Hoxa2 mutant mice. Lastly, the analysis of Phox2b-deficient mice indicated that the facial motor nucleus is a major Slit signaling source required to prevent premature ventral migration of PN. These findings provide novel insights into the molecular control of neuronal migration from transcription factor to regulation of guidance receptor and ligand expression. Specifically, they address the question of how exposure to multiple guidance cues along the AP and DV axes is regulated at the transcriptional level and in turn translated into stereotyped migratory responses during tangential migration of neurons in the developing mammalian brain