Gene expression across mammalian organ development

The evolution of gene expression in mammalian organ development remains largely uncharacterized. Here we report the transcriptomes of seven organs (cerebrum, cerebellum, heart, kidney, liver, ovary and testis) across developmental time points from early organogenesis to adulthood for human, macaque, mouse, rat, rabbit, opossum and chicken. Comparisons of gene expression patterns identified developmental stage correspondences across species, and differences in the timing of key events during the development of the gonads. We found that the breadth of gene expression and the extent of purifying selection gradually decrease during development, whereas the amount of positive selection and expression of new genes increase. We identified differences in the temporal trajectories of expression of individual genes across species, with brain tissues showing the smallest percentage of trajectory changes, and the liver and testis showing the largest. Our work provides a resource of developmental transcriptomes of seven organs across seven species, and comparative analyses that characterize the development and evolution of mammalian organs

The Ciliogenic Transcription Factor RFX3 Regulates Early Midline Distribution of Guidepost Neurons Required for Corpus Callosum Development

The corpus callosum (CC) is the major commissure that bridges the cerebral hemispheres. Agenesis of the CC is associated with human ciliopathies, but the origin of this default is unclear. Regulatory Factor X3 (RFX3) is a transcription factor involved in the control of ciliogenesis, and Rfx3–deficient mice show several hallmarks of ciliopathies including left–right asymmetry defects and hydrocephalus. Here we show that Rfx3–deficient mice suffer from CC agenesis associated with a marked disorganisation of guidepost neurons required for axon pathfinding across the midline. Using transplantation assays, we demonstrate that abnormalities of the mutant midline region are primarily responsible for the CC malformation. Conditional genetic inactivation shows that RFX3 is not required in guidepost cells for proper CC formation, but is required before E12.5 for proper patterning of the cortical septal boundary and hence accurate distribution of guidepost neurons at later stages. We observe focused but consistent ectopic expression of Fibroblast growth factor 8 (Fgf8) at the rostro commissural plate associated with a reduced ratio of GLIoma-associated oncogene family zinc finger 3 (GLI3) repressor to activator forms. We demonstrate on brain explant cultures that ectopic FGF8 reproduces the guidepost neuronal defects observed in Rfx3 mutants. This study unravels a crucial role of RFX3 during early brain development by indirectly regulating GLI3 activity, which leads to FGF8 upregulation and ultimately to disturbed distribution of guidepost neurons required for CC morphogenesis. Hence, the RFX3 mutant mouse model brings novel understandings of the mechanisms that underlie CC agenesis in ciliopathies

Expression pattern of <i>Rfx3</i> in the developing mouse telencephalon from E11.5 to E14.5.

<p>(A–D) <i>In situ</i> hybridization for <i>Rfx3</i> mRNAs on coronal brain sections of wild type (B1–B3) and <i>Rfx3−/−</i> (A1–A3) embryos at E11.5. <i>In situ</i> hybridizations for <i>Rfx3</i> (in red) combined with immunohistochemical staining for calretinin (C1–C3 and D1–D3) (in green) on coronal brain sections of wild type embryos at E12.5 (C1–C3) and E14.5 (D1–D3). A1, B1, C1 and D1 are coronal sections at the corticoseptal boundary (CSB, *) level, while A3, B3, C3 and D3 are caudal coronal sections at the level of the cortical hem (CH). A2, B2 and C2 are higher power views of the CSB seen in A1, B1 and C1 respectively. D2 is a lateral view of the telencephalon. (A and B) At E11.5, <i>Rfx3</i> is strongly expressed in wild type mice throughout the entire neuroepithelium of the CSB, and at more caudal levels in the cortical hem (B1–B3). The <i>Rfx3</i> hybridization signal is specific since no signal is visible in the same brain area of <i>Rfx3−/−</i> (A1–A3). (C–D) From E12.5 to E14.5, <i>Rfx3</i> expression is restricted to the cingulate cortex (CCi) that contains pioneer callosally projecting neurons, and throughout the CSB at the midline where the CC will form (C1–C2 to D1–D2). In addition, <i>Rfx3</i> is detected within the glial wedge (GW, open arrows) and the septum. (C3 to D3) On more caudal sections, <i>Rfx3</i> mRNAs are expressed in the cortical hem (CH), choroid plexus, ventral pallium (VP) and preoptic area (POA) (open arrows). Bar = 435 µm in A1, A3, B1, B3, C1, C3, D1, D2, D3 and 60 µm in A2, B2, C2.</p

Aberrant localization of midline neurons before CC formation at E14.5.

<p><i>In situ</i> hybridization for <i>reelin</i> mRNAs (A1–A3 and B1–B3) and DAB staining for calretinin (C1–C3 and D1–D3) and calbindin (E1–E3 and F1–F3) on coronal rostromedial slices from E14.5 WT (A1–A3, C1–C3 and E1–E3) and <i>Rfx3</i>−/− (B1–B3, D1–D3 and F1–F3) mice. A2, A3, B2, B3, C2 and D2 are higher power views of the corticoseptal boundary (CSB,*) seen in A1, B1, C1 and D1 respectively. E2, E3, F2 and F3 are higher power views of the induseum griseum (IG) region seen in E1 and F1, respectively. C3 and D3 are higher power views of the cortical marginal zone (MZ) seen in C1 and D1 respectively. (A–F) As early as E14.5, before inter-hemispheric midline fusion occurs, the organization of reelin+ (B1–B3), calretinin+ (D1–D2) and calbindin+ (F1–F2) midline neurons is severely affected in the CSB and the IG regions of the <i>Rfx3</i>−/− mutant (arrows and open arrowheads). In addition, the neurons lose their tangential organization through the <i>Rfx3</i>−/− cortical MZ (B3, D3 and F2–F3; arrows). Bar = 300 µm in A1, B1, E1, F1; 150 µm in A2, B2, C1, C3, D1, D3, E2, F2 and 60 µm in A3, B3, C2, D2, E3, F3.</p

Abnormal callosal axon pathfinding in <i>Rfx3−/−</i> mice.

<p>(A–F) Immunohistochemistry for calretinin and Npn-1 (A1–A3 and B1–B3), for calbindin and Npn-1 (C1–C3 and D1–D3), and for GFAP and L1 (E1–E3 and F1–F3) in coronal CC sections from E18.5 WT (A1–A3, C1–C3 and E1–E3) or <i>Rfx3</i>−/− (B1–B3, D1–D3 and F1–F3) mice. A2, B2, C2, D2, E2 and F2 are higher magnifications of the lateral CC seen in A1, B1, C1, D1, E1 and F1, respectively. A3, B3, C3, D3, E3 and F3 are higher magnifications of the medial CC seen in A1, B1, C1, D1, E1 and F1, respectively. (A1–A3, C1–C3 and E1–E3) At E18.5, the hemispheres of WT brains have fused. Callosal fibres (in red) cross the midline and project into the contralateral cortex. (B1–B3, D1–D3 and F1–F3) Aberrant callosal axon bundles are observed in <i>Rfx3−/−</i> embryos (arrowheads). (B1–B3 and F1–F3) While the hemispheres have fused, most of callosal fibres do not cross the midline and form large ectopic bundles on the CC border, reminiscent of Probst bundles (PB). (D1–D3) Some <i>Rfx3−/−</i> embryos exhibit a more severe phenotype with an absence of midline hemispheric fusion and absolutely no callosal axons crossing the midline. In this case, a large bulge is observed along the inter-hemispheric fissure at the location where the callosal axons approach the midline (O). In all mutants, axonal defects are accompanied by cellular mis-positioning through the CC and the IG (open arrowheads). While calretinin+ or calbindin+ neurons, as well as, GFAP+ glia, are still present in the CC and the IG of <i>Rfx3−/−</i> mice, there is a midline disorganization and a lateral shift of these cell populations. Bar = 435 µm in A1, B1, C1, D1, E1, F1; 220 µm in A3, B3, C3, D2, D3, E2, F2 and 110 µm in A2, B2, C2, E3, F3.</p

Midline integrity is necessary for pathfinding by callosal axons.

<p>(A1) Experimental paradigm used to confirm the growth of E16.5 <i>Rfx3+/+</i> control callosal axons in midline structure transplants from <i>Rfx3+/+</i> control mice. (A2–A3) DiI labeling showing that WT callosal axons grow normally and cross the midline when they are confronted to a WT environment. (B1) Experimental paradigm used to confirm the growth defects of E16.5 <i>Rfx3−/−</i> callosal axons in midline transplants from <i>Rfx3−/−</i> mice. (B2–B3) DiI labeling showing that <i>Rfx3−/−</i> callosal axons are misrouted and do not cross the midline of <i>Rfx3</i> mutants. (C1) Experimental paradigm used to study the growth defects of E16.5 control <i>Rfx3+/+</i> callosal axons in transplants of midline structures from <i>Rfx3−/−</i> mice. (C2–C3) DiI labeling showing that WT callosal axons are misrouted and do not cross the midline of <i>Rfx3</i> mutants. (D1) Experimental paradigm used to test whether the midline integrity is necessary and sufficient to direct the growth of callosal axons. To this end, control <i>Rfx3+/+</i> midline regions are transplanted in <i>Rfx3−/−</i> slices. (D2–D3) DiI labeling showing the complete restoration of <i>Rfx3−/−</i> callosal axon pathfinding. Dashed lines outline the CC transplant localizations. Brain slices in A2–A3, B2–B3, C2–C3 and D2–D3 were counterstained with Hoechst. Bar = 435 µm in A2, B2, C2, D2 and 220 µm in A3, B3, C3 and D3.</p

Abnormal neuron localization and aberrant callosal axon pathfinding at the onset of CC formation.

<p>(A–H) Immunohistochemistry for calretinin and reelin (A1–A2 and B1–B2), for calretinin and neuropilin-1 receptor (Npn-1) (C1–C2 and D1–D2), for calbindin and L1 receptor (E1–E2 and F1–F2) and for GFAP and L1 receptor (G1–G2 and H1–H2), in coronal CC sections from WT (A1–A2, C1–C2, E1–E2 and G1–G2) and <i>Rfx3</i>−/− (B1–B2, D1–D2, F1–F2 and H1–H2) mice. A2, B2, C2, D2, E2, F2, G2 and H2 are higher magnifications of the midline seen in A1, B1, C1, D1, E1, F1, G1 and H1. (A1–A2 to D1–D2) From E15.5 to E16.5, calretinin+ guidepost neurons fail to form a well organized band of neurons at the CSB (*) and are dispersed in the septum of <i>Rfx3−/−</i> mice (B2 and D2, white open arrowheads). Reelin+ and calretinin+ neurons loose their tangential organization through the cortical marginal zone (MZ) (compare B2 to A2, red open arrowheads). (E1–E2 and F1–F2) At E16.5, calbindin+ neurons (green) do not organize appropriately within the indusium griseum (IG) and accumulate at the CC midline in <i>Rfx3</i>−/− mice (compare F2 to E2, open arrowheads). (G1–G2 and H1–H2) At E16.5, the organization of GFAP+ glial cell populations within the CC is indistinguishable between WT and <i>Rfx3</i>−/− mice. (A to H) Axonal misrouting of pioneer callosal axons from E15.5 to E16.5. (A1–A2, C1–C2, and E1–E2) In WT brains, pioneer callosal fibres grow within the CSB and reach the midline. (B1–B2, D1–D2 and F1–F2) In <i>Rfx3−/−</i> brains, most callosal fibres form ectopic bundles of axons in the septum (B2 and D2) and the IG (F2) on either side of the midline (white arrowheads). Bar = 435 µm in C1, D1, E1, F1, G1, H1; 220 µm in A1, B1, C2, D2, E2, F2; 110 µm in G2, H2; 60 µm in A2, B2.</p