Abstract
Hox proteins are key regulators of animal development, providing positional identity and patterning information to cells along the rostrocaudal axis of the embryo. Although their embryonic expression and function are well characterized, their presence and biological importance in adulthood remains poorly investigated. We provide here the first detailed quantitative and neuroanatomical characterization of the expression of the 39 Hox genes in the adult mouse brain. Using RT-qPCR we determined the expression of 24 Hox genes mainly in the brainstem of the adult brain, with low expression of a few genes in the cerebellum and the forebrain. Using in situ hybridization (ISH) we have demonstrated that expression of Hox genes is maintained in territories derived from the early segmental Hox expression domains in the hindbrain. Indeed, we show that expression of genes belonging to paralogy groups PG2-8 is maintained in the hindbrain derivatives at adulthood. The spatial colinearity, which characterizes the early embryonic expression of Hox genes, is still observed in sequential antero-posterior boundaries of expression. Moreover, the main mossy and climbing fibres precerebellar nuclei express PG2-8 Hox genes according to their migration origins. Second, ISH confirms the presence of Hox gene transcripts in territories where they are not detected during development, suggesting neo-expression in these territories in adulthood. Within the forebrain, we have mapped Hoxb1, Hoxb3, Hoxb4, Hoxd3 and Hoxa5 expression in restricted areas of the sensory cerebral cortices as well as in specific thalamic relay nuclei. Our data thus suggest a requirement of Hox genes beyond their role of patterning genes, providing a new dimension to their functional relevance in the central nervous system.
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Change history
19 January 2021
Editor’s Note: Readers are alerted that concerns have been raised regarding Figures 2C and 2D. We will update readers once we have further information and all parties have been given an opportunity to respond in full.
18 March 2021
A Correction to this paper has been published: https://doi.org/10.1007/s00429-021-02252-5
Abbreviations
- AMS:
-
Anterior extramural migrating stream
- AP:
-
Area postrema
- A/P:
-
Antero-posterior
- BS:
-
Brainstem
- Cb:
-
Cerebellum
- CNS:
-
Central nervous system
- Cx:
-
Cerebral cortex
- dmX:
-
Dorsal nucleus of the vagus nerve
- ECU:
-
External cuneate
- Hi:
-
Hippocampus
- Hy:
-
Hypothalamus
- IF5:
-
Interfascicular trigeminal nucleus
- IHC:
-
Immunohistochemistry
- IO:
-
Inferior olive
- ISH:
-
In situ hybridization
- PBS:
-
Phosphate-buffered saline
- IMS:
-
Intramural migrating stream
- LGd:
-
Dorsal lateral geniculate nucleus
- LRN:
-
Lateral reticular nucleus
- MG:
-
Medial geniculate nucleus
- MNs:
-
Motoneurons
- MO:
-
Medulla oblongata
- NTS:
-
Nucleus of the solitary tract
- OA:
-
Olfactory areas
- PCs:
-
Purkinje cells
- PMS:
-
Posterior extramural migrating stream
- PG:
-
Paralogy groups
- PN:
-
Pontine nucleus
- Po:
-
Pons
- PrV:
-
Principal sensory trigeminal nucleus
- r:
-
Rhombomere
- RO:
-
Raphe obscurus
- RPA:
-
Raphe pallidus
- SC:
-
Spinal cord
- SpV:
-
Spinal trigeminal nucleus
- Th:
-
Thalamus
- TRN:
-
Tegmental reticular nucleus
- VPL:
-
Ventral posterolateral nucleus of the thalamus
- VPM:
-
Ventral posteromedial nucleus of the thalamus
References
Ahn Y, Mullan HE, Krumlauf R (2014) Long-range regulation by shared retinoic acid response elements modulates dynamic expression of posterior Hoxb genes in CNS development. Dev Biol 388(1):134–144. doi:10.1016/j.ydbio.2014.01.027
Alexander T, Nolte C, Krumlauf R (2009) Hox genes and segmentation of the hindbrain and axial skeleton. Annu Rev Cell Dev Biol 25:431–456. doi:10.1146/annurev.cellbio.042308.113423
Angerer LM, Angerer RC (1992) In situ Hybridization to cellular RNA with radiolabelled RNA probes. In: Wilkinson DJ (ed) In situ Hybridization. Oxford University Press, A practical Approach, pp 15–32
Bami M, Episkopou V, Gavalas A, Gouti M (2011) Directed neural differentiation of mouse embryonic stem cells is a sensitive system for the identification of novel Hox gene effectors. PLoS ONE 6(5):e20197. doi:10.1371/journal.pone.0020197PONE-D-11-04362
Batista PJ, Chang HY (2013) Long noncoding RNAs: cellular address codes in development and disease. Cell 152(6):1298–1307. doi:10.1016/j.cell.2013.02.012
Blockus H, Chedotal A (2014) The multifaceted roles of Slits and Robos in cortical circuits: from proliferation to axon guidance and neurological diseases. Curr Opin Neurobiol 27C:82–88. doi:10.1016/j.conb.2014.03.003
Brock HW, Hodgson JW, Petruk S, Mazo A (2009) Regulatory noncoding RNAs at Hox loci. Biochem Cell Biol 87(1):27–34. doi:10.1139/O08-108
Bustin SA (2000) Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinol 25(2):169–193 (JME00927)
Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55(4):611–622. doi:10.1373/clinchem.2008.112797
Calonge WM, Martinez L, Lacadena J, Fernandez-Dumont V, Matesanz R, Tovar JA (2007) Expression of homeotic genes Hoxa3, Hoxb3, Hoxd3 and Hoxc4 is decreased in the lungs but not in the hearts of adriamycin-exposed mice. Pediatr Surg Int 23(5):419–424. doi:10.1007/s00383-006-1865-7
Cang J, Feldheim DA (2013) Developmental mechanisms of topographic map formation and alignment. Annu Rev Neurosci 36:51–77. doi:10.1146/annurev-neuro-062012-170341
Chen H, Rubin E, Zhang H, Chung S, Jie CC, Garrett E, Biswal S, Sukumar S (2005) Identification of transcriptional targets of HOXA5. J Biol Chem 280(19):19373–19380. doi:10.1074/jbc.M413528200
Chotteau-Lelievre A, Dolle P, Gofflot F (2006) Expression analysis of murine genes using in situ hybridization with radioactive and nonradioactively labeled RNA probes. Methods Mol Biol 326:61–87
Condie BG, Capecchi MR (1993) Mice homozygous for a targeted disruption of Hoxd-3 (Hox-4.1) exhibit anterior transformations of the first and second cervical vertebrae, the atlas and the axis. Development 119(3):579–595
Coulombe Y, Lemieux M, Moreau J, Aubin J, Joksimovic M, Berube-Simard FA, Tabaries S, Boucherat O, Guillou F, Larochelle C, Tuggle CK, Jeannotte L (2010) Multiple promoters and alternative splicing: Hoxa5 transcriptional complexity in the mouse embryo. PLoS One 5(5):e10600. doi:10.1371/journal.pone.0010600
Dasen JS, Jessell TM (2009) Hox networks and the origins of motor neuron diversity. Curr Top Dev Biol 88:169–200. doi:10.1016/S0070-2153(09)88006-X
Dasen JS, Tice BC, Brenner-Morton S, Jessell TM (2005) A Hox regulatory network establishes motor neuron pool identity and target-muscle connectivity. Cell 123(3):477–491. doi:10.1016/j.cell.2005.09.009
Dasen JS, De Camilli A, Wang B, Tucker PW, Jessell TM (2008) Hox repertoires for motor neuron diversity and connectivity gated by a single accessory factor, FoxP1. Cell 134(2):304–316. doi:10.1016/j.cell.2008.06.019
De Block M, Debrouwer D (1993) RNA-RNA in situ hybridization using digoxigenin-labeled probes: the use of high-molecular-weight polyvinyl alcohol in the alkaline phosphatase indoxyl-nitroblue tetrazolium reaction. Anal Biochem 215(1):86–89
Derveaux S, Vandesompele J, Hellemans J (2009) How to do successful gene expression analysis using real-time PCR. Methods. doi:10.1016/j.ymeth.2009.11.001
Deschamps J (2007) Ancestral and recently recruited global control of the Hox genes in development. Curr Opin Genet Dev 17(5):422–427. doi:10.1016/j.gde.2007.07.008
Di Bonito M, Glover JC, Studer M (2013a) Hox genes and region-specific sensorimotor circuit formation in the hindbrain and spinal cord. Dev Dyn 242(12):1348–1368. doi:10.1002/dvdy.24055
Di Bonito M, Narita Y, Avallone B, Sequino L, Mancuso M, Andolfi G, Franze AM, Puelles L, Rijli FM, Studer M (2013b) Assembly of the auditory circuitry by a Hox genetic network in the mouse brainstem. PLoS Genet 9(2):e1003249. doi:10.1371/journal.pgen.1003249PGENETICS-D-12-00505
Di Meglio T, Kratochwil CF, Vilain N, Loche A, Vitobello A, Yonehara K, Hrycaj SM, Roska B, Peters AH, Eichmann A, Wellik D, Ducret S, Rijli FM (2013) Ezh2 orchestrates topographic migration and connectivity of mouse precerebellar neurons. Science 339(6116):204–207. doi:10.1126/science.1229326
Dickson BJ (2002) Molecular mechanisms of axon guidance. Science 298(5600):1959–1964. doi:10.1126/science.1072165298/5600/1959
Erzurumlu RS, Gaspar P (2012) Development and critical period plasticity of the barrel cortex. Eur J Neurosci 35(10):1540–1553. doi:10.1111/j.1460-9568.2012.08075.x
Erzurumlu RS, Murakami Y, Rijli FM (2010) Mapping the face in the somatosensory brainstem. Nat Rev Neurosci 11(4):252–263. doi:10.1038/nrn2804
Evsyukova I, Plestant C, Anton ES (2013) Integrative Mechanisms of Oriented Neuronal Migration in the Developing Brain. Annu Rev Cell Dev Biol. doi:10.1146/annurev-cellbio-101512-122400
Farago AF, Awatramani RB, Dymecki SM (2006) Assembly of the brainstem cochlear nuclear complex is revealed by intersectional and subtractive genetic fate maps. Neuron 50(2):205–218. doi:10.1016/j.neuron.2006.03.014
Frohman MA, Martin GR (1992) Isolation and analysis of embryonic expression of Hox-4.9, a member of the murine labial-like gene family. Mech Dev 38(1):55–67 (0925-4773(92)90038-L)
Fu Y, Tvrdik P, Makki N, Machold R, Paxinos G, Watson C (2013) The interfascicular trigeminal nucleus: a precerebellar nucleus in the mouse defined by retrograde neuronal tracing and genetic fate mapping. J Comp Neurol 521(3):697–708. doi:10.1002/cne.23200
Geisen MJ, Di Meglio T, Pasqualetti M, Ducret S, Brunet JF, Chedotal A, Rijli FM (2008) Hox paralog group 2 genes control the migration of mouse pontine neurons through slit-robo signaling. PLoS Biol 6(6):e142. doi:10.1371/journal.pbio.0060142
Gofflot F, Chartoire N, Vasseur L, Heikkinen S, Dembele D, Le Merrer J, Auwerx J (2007) Systematic gene expression mapping clusters nuclear receptors according to their function in the brain. Cell 131(2):405–418. doi:10.1016/j.cell.2007.09.012
Gould A, Itasaki N, Krumlauf R (1998) Initiation of rhombomeric Hoxb4 expression requires induction by somites and a retinoid pathway. Neuron 21(1):39–51 (S0896-6273(00)80513-9[pii])
Greer JM, Capecchi MR (2002) Hoxb8 is required for normal grooming behavior in mice. Neuron 33(1):23–34
Greig LC, Woodworth MB, Galazo MJ, Padmanabhan H, Macklis JD (2013) Molecular logic of neocortical projection neuron specification, development and diversity. Nat Rev Neurosci 14(11):755–769. doi:10.1038/nrn3586
Guo T, Mandai K, Condie BG, Wickramasinghe SR, Capecchi MR, Ginty DD (2011) An evolving NGF-Hoxd1 signaling pathway mediates development of divergent neural circuits in vertebrates. Nat Neurosci 14(1):31–36. doi:10.1038/nn.2710
Guthrie S (2007) Patterning and axon guidance of cranial motor neurons. Nat Rev Neurosci 8(11):859–871. doi:10.1038/nrn2254
Hatten ME (2002) New directions in neuronal migration. Science 297(5587):1660–1663. doi:10.1126/science.1074572297/5587/1660
Hensch TK (2005) Critical period plasticity in local cortical circuits. Nat Rev Neurosci 6(11):877–888. doi:10.1038/nrn1787
Hevner RF, Daza RA, Rubenstein JL, Stunnenberg H, Olavarria JF, Englund C (2003) Beyond laminar fate: toward a molecular classification of cortical projection/pyramidal neurons. Dev Neurosci 25(2–4):139–151 (72263)
Hof PR, Young WG, Bloom FE, Belichenko PV, Celio MR (2000) Comparative cytoarchitectonic atlas of C57BL/6 and 129/SV Mouse Brains. Elsevier
Horan GS, Kovacs EN, Behringer RR, Featherstone MS (1995) Mutations in paralogous Hox genes result in overlapping homeotic transformations of the axial skeleton: evidence for unique and redundant function. Dev Biol 169(1):359–372. doi:10.1006/dbio.1995.1150
Hruska M, Dalva MB (2012) Ephrin regulation of synapse formation, function and plasticity. Mol Cell Neurosci 50(1):35–44. doi:10.1016/j.mcn.2012.03.004
Jones EG (1998) Viewpoint: the core and matrix of thalamic organization. Neuroscience 85(2):331–345 (S0306-4522(97)00581-2)
Kiecker C, Lumsden A (2005) Compartments and their boundaries in vertebrate brain development. Nat Rev Neurosci 6(7):553–564. doi:10.1038/nrn1702
Killackey HP, Koralek KA, Chiaia NL, Rhodes RW (1989) Laminar and areal differences in the origin of the subcortical projection neurons of the rat somatosensory cortex. J Comp Neurol 282(3):428–445. doi:10.1002/cne.902820309
Kmita M, Duboule D (2003) Organizing axes in time and space; 25 years of colinear tinkering. Science 301(5631):331–333. doi:10.1126/science.1085753301/5631/331
Legg CR, Mercier B, Glickstein M (1989) Corticopontine projection in the rat: the distribution of labelled cortical cells after large injections of horseradish peroxidase in the pontine nuclei. J Comp Neurol 286(4):427–441. doi:10.1002/cne.902860403
Lein ES, Hawrylycz MJ, Ao N et al (2007) Genome-wide atlas of gene expression in the adult mouse brain. Nature 445(7124):168–176. doi:10.1038/nature05453
Mainguy G, Koster J, Woltering J, Jansen H, Durston A (2007) Extensive polycistronism and antisense transcription in the mammalian Hox clusters. PLoS ONE 2(4):e356. doi:10.1371/journal.pone.0000356
Mayer M, Bercsenyi K, Geczi K, Szabo G, Lele Z (2010) Expression of two type II cadherins, Cdh12 and Cdh22 in the developing and adult mouse brain. Gene expression patterns : GEP 10(7–8):351–360. doi:10.1016/j.gep.2010.08.002
Molyneaux BJ, Arlotta P, Menezes JR, Macklis JD (2007) Neuronal subtype specification in the cerebral cortex. Nat Rev Neurosci 8(6):427–437. doi:10.1038/nrn2151
Nolte C, Krumlauf R (2007) Expression of Hox Genes in the Nervous System of Vertebrates. In: Papageorgio S (ed) HOX Gene Expression. Landes Bioscience and Springer Science + Business Media., pp 14–41
Oosterveen T, Niederreither K, Dolle P, Chambon P, Meijlink F, Deschamps J (2003) Retinoids regulate the anterior expression boundaries of 5′ Hoxb genes in posterior hindbrain. EMBO J 22(2):262–269. doi:10.1093/emboj/cdg029
Oosterveen T, Meijlink F, Deschamps J (2004) Expression of retinaldehyde dehydrogenase II and sequential activation of 5′ Hoxb genes in the mouse caudal hindbrain. Gene expression patterns : GEP 4(3):243–247. doi:10.1016/j.modgep.2003.11.007
Oury F, Murakami Y, Renaud JS, Pasqualetti M, Charnay P, Ren SY, Rijli FM (2006) Hoxa2- and rhombomere-dependent development of the mouse facial somatosensory map. Science 313(5792):1408–1413. doi:10.1126/science.1130042
Pasqualetti M, Diaz C, Renaud JS, Rijli FM, Glover JC (2007) Fate-mapping the mammalian hindbrain: segmental origins of vestibular projection neurons assessed using rhombomere-specific Hoxa2 enhancer elements in the mouse embryo. J Neurosci 27(36):9670–9681. doi:10.1523/JNEUROSCI.2189-07.2007
Pasterkamp RJ, Giger RJ (2009) Semaphorin function in neural plasticity and disease. Curr Opin Neurobiol 19(3):263–274. doi:10.1016/j.conb.2009.06.001
Ponting CP, Oliver PL, Reik W (2009) Evolution and functions of long noncoding RNAs. Cell 136(4):629–641. doi:10.1016/j.cell.2009.02.006
Ray RS, Dymecki SM (2009) Rautenlippe Redux – toward a unified view of the precerebellar rhombic lip. Curr Opin Cell Biol 21(6):741–747. doi:10.1016/j.ceb.2009.10.003
Redd KJ, Oberdick J, McCoy J, Denker BM, Luo Y (2002) Association and colocalization of G protein alpha subunits and Purkinje cell protein 2 (Pcp2) in mammalian cerebellum. J Neurosci Res 70(5):631–637. doi:10.1002/jnr.10460
Rodriguez CI, Dymecki SM (2000) Origin of the precerebellar system. Neuron 27(3):475–486 (S0896-6273(00)00059-3)
Sabourin JC, Ackema KB, Ohayon D, Guichet PO, Perrin FE, Garces A, Ripoll C, Charite J, Simonneau L, Kettenmann H, Zine A, Privat A, Valmier J, Pattyn A, Hugnot JP (2009) A mesenchymal-like ZEB1(+) niche harbors dorsal radial glial fibrillary acidic protein-positive stem cells in the spinal cord. Stem Cells 27(11):2722–2733. doi:10.1002/stem.226
Sanlioglu S, Zhang X, Baader SL, Oberdick J (1998) Regulation of a Purkinje cell-specific promoter by homeodomain proteins: repression by engrailed-2 vs. synergistic activation by Hoxa5 and Hoxb7. J Neurobiol 36(4):559–571
Schubert D, Kotter R, Luhmann HJ, Staiger JF (2006) Morphology, electrophysiology and functional input connectivity of pyramidal neurons characterizes a genuine layer va in the primary somatosensory cortex. Cereb Cortex 16(2):223–236. doi:10.1093/cercor/bhi100
Sham MH, Hunt P, Nonchev S, Papalopulu N, Graham A, Boncinelli E, Krumlauf R (1992) Analysis of the murine Hox-2.7 gene: conserved alternative transcripts with differential distributions in the nervous system and the potential for shared regulatory regions. EMBO J 11(5):1825–1836
Shen K, Cowan CW (2010) Guidance molecules in synapse formation and plasticity. Cold Spring Harb Perspect Biol 2(4):a001842. doi:10.1101/cshperspect.a001842
Sugiyama S, Prochiantz A, Hensch TK (2009) From brain formation to plasticity: insights on Otx2 homeoprotein. Dev Growth Differ 51(3):369–377. doi:10.1111/j.1440-169X.2009.01093.x
Takahashi Y, Hamada J, Murakawa K, Takada M, Tada M, Nogami I, Hayashi N, Nakamori S, Monden M, Miyamoto M, Katoh H, Moriuchi T (2004) Expression profiles of 39 HOX genes in normal human adult organs and anaplastic thyroid cancer cell lines by quantitative real-time RT-PCR system. Exp Cell Res 293(1):144–153
Tanaka M, Maeda N, Noda M, Marunouchi T (2003) A chondroitin sulfate proteoglycan PTPzeta/RPTPbeta regulates the morphogenesis of Purkinje cell dendrites in the developing cerebellum. J Neurosci 23(7):2804–2814 (23/7/2804)
Tumpel S, Wiedemann LM, Krumlauf R (2009) Hox genes and segmentation of the vertebrate hindbrain. Curr Top Dev Biol 88:103–137. doi:10.1016/S0070-2153(09)88004-6
Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3(7):RESEARCH0034
Watakabe A, Ohsawa S, Hashikawa T, Yamamori T (2006) Binding and complementary expression patterns of semaphorin 3E and plexin D1 in the mature neocortices of mice and monkeys. J Comp Neurol 499(2):258–273. doi:10.1002/cne.21106
Weimann JM, Zhang YA, Levin ME, Devine WP, Brulet P, McConnell SK (1999) Cortical neurons require Otx1 for the refinement of exuberant axonal projections to subcortical targets. Neuron 24(4):819–831
Wise SP, Jones EG (1977) Somatotopic and columnar organization in the corticotectal projection of the rat somatic sensory cortex. Brain res 133(2):223–235
Wolf LV, Yeung JM, Doucette JR, Nazarali AJ (2001) Coordinated expression of Hoxa2, Hoxd1 and Pax6 in the developing diencephalon. Neuro Report 12(2):329–333
Woodworth MB, Custo Greig L, Kriegstein AR, Macklis JD (2012) SnapShot: cortical development. Cell 151(4):918–918e911. doi:10.1016/j.cell.2012.10.004
Zervas M, Blaess S, Joyner AL (2005) Classical embryological studies and modern genetic analysis of midbrain and cerebellum development. Curr Top Dev Biol 69:101–138. doi:10.1016/S0070-2153(05)69005-9
Zhang X, Zhang H, Oberdick J (2002) Conservation of the developmentally regulated dendritic localization of a Purkinje cell-specific mRNA that encodes a G-protein modulator: comparison of rodent and human Pcp 2(L7) gene structure and expression. Brain Res Mol Brain Res 105(1–2):1–10 (S0169328X02003790)
Zimmer C, Tiveron MC, Bodmer R, Cremer H (2004) Dynamics of Cux2 expression suggests that an early pool of SVZ precursors is fated to become upper cortical layer neurons. Cereb Cortex 14(12):1408–1420. doi:10.1093/cercor/bhh102
Acknowledgments
The authors want to thank Coralie Piget for animal husbandry, and Arnaud Van Hecke and Jean-François de Backer for their contribution in setting up the procedures. We are grateful to René Rezsohazy and Bernard Knoops for helpful discussions and comments on the manuscript and Helen Christian for critical reading. This work was supported by the Fonds de la Recherche Scientifique-FNRS under Grant MIS-Ulysse n °F.6003.09, by the Fonds Spéciaux de Recherche of the Université catholique de Louvain, and by the «Communauté française de Belgique-Actions de Recherche Concertées» (12/17-041). B.L. is a FRIA fellow of the Fonds de la Recherche Scientifique-FNRS.
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Hutlet, B., Theys, N., Coste, C. et al. Systematic expression analysis of Hox genes at adulthood reveals novel patterns in the central nervous system. Brain Struct Funct 221, 1223–1243 (2016). https://doi.org/10.1007/s00429-014-0965-8
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DOI: https://doi.org/10.1007/s00429-014-0965-8