Investigating Embryonic Expression Patterns and Evolution of AHI1 and CEP290 Genes, Implicated in Joubert Syndrome

Joubert syndrome and related diseases (JSRD) are developmental cerebello-oculo-renal syndromes with phenotypes including cerebellar hypoplasia, retinal dystrophy and nephronophthisis (a cystic kidney disease). We have utilised the MRCWellcome Trust Human Developmental Biology Resource (HDBR), to perform in-situ hybridisation studies on embryonic tissues, revealing an early onset neuronal, retinal and renal expression pattern for AHI1. An almost identical pattern of expression is seen with CEP290 in human embryonic and fetal tissue. A novel finding is that both AHI1 and CEP290 demonstrate strong expression within the developing choroid plexus, a ciliated structure important for central nervous system development. To test if AHI1 and CEP290 may have co-evolved, we carried out a genomic survey of a large group of organisms across eukaryotic evolution. We found that, in animals, ahi1 and cep290 are almost always found together; however in other organisms either one may be found independent of the other. Finally, we tested in murine epithelial cells if Ahi1 was required for recruitment of Cep290 to the centrosome. We found no obvious differences in Cep290 localisation in the presence or absence of Ahi1, suggesting that, while Ahi1 and Cep290 may function together in the whole organism, they are not interdependent for localisation within a single cell. Taken together these data support a role for AHI1 and CEP290 in multiple organs throughout development and we suggest that this accounts for the wide phenotypic spectrum of AHI1 and CEP290 mutations in man

Recessive gene disruptions in autism spectrum disorder

International audienceAutism spectrum disorder (ASD) affects up to 1 in 59 individuals1. Genome-wide association and large-scale sequencing studies strongly implicate both common variants2-4 and rare de novo variants5-10 in ASD. Recessive mutations have also been implicated11-14 but their contribution remains less well defined. Here we demonstrate an excess of biallelic loss-of-function and damaging missense mutations in a large ASD cohort, corresponding to approximately 5% of total cases, including 10% of females, consistent with a female protective effect. We document biallelic disruption of known or emerging recessive neurodevelopmental genes (CA2, DDHD1, NSUN2, PAH, RARB, ROGDI, SLC1A1, USH2A) as well as other genes not previously implicated in ASD including FEV (FEV transcription factor, ETS family member), which encodes a key regulator of the serotonergic circuitry. Our data refine estimates of the contribution of recessive mutation to ASD and suggest new paths for illuminating previously unknown biological pathways responsible for this condition

<i>CEP290</i> is expressed in the developing cerebellum, spinal cord and eye.

<p>(A, G) Negative control hybridisation with <i>CEP290</i> sense RNA probe and (B–F) hybridisation with <i>CEP290</i> antisense RNA probe to transverse sections at CS22. <i>CEP290</i> transcripts are abundant in the developing telencephalon (B, C, E), especially the cortical neuroepithelium (C), and neural retina (D). <i>CEP290</i> expression is demonstrated within the epithelium of the choroid plexus (CP) (E, F) at CS22. Prominent expression is seen in metencephalon (including cerebellum), myelencephalon and mesencephalon at CS19 (H, I, J). Strong expression is detected at the rhombic lip in the developing cerebellum (H, J), the mesencephalic (I) and cerebellar neuroepithelium (J). Prominent staining is demonstrated within the alar plate of spinal cord at CS16 (K) and CS22 (L). Ap, alar plate; Bp, basal plate; CP: choroid plexus; CPE, choroid plexus epithelium; Mes, mesencephalon (midbrain); Met, metencephalon; Mye, myelencephalon; NEP, neuroepithelium; Cor.NEP: cortical neuroepithelium; Cere.NEP: cerebellar neuroepithelium; Mes.NEP: mesencephalic (midbrain) neuroepithelium; NR, neural retina; RPE, retinal pigment epithelium, Sc, spinal cord; SG: spinal ganglion; SyG: sympathetic ganglia; T, telencephalon, Ton, tongue; LRhoL: lower rhombic lip; URhoL: upper rhombic lip; Scale bar: A, B, G, H = 2 mm, C, D, F, K = 500 µm; E = 1 mm; L = 500 µm; I, J = 250 µm.</p

Cep290 does not require Ahi1 for localisation to centrosomes.

<p>Immunofluorescence of IMCD3 monolayers co-transfected with siGLO and either negative-control siRNA (A and B), or siRNA against <i>Ahi1</i> (A and C). (A) Graph to show centrosome position (gamma tubulin immunofluorescence, graph expressed as mean +/− s.e.m.) from apical to basal in control and <i>Ahi1</i>-silenced cells. Apical refers to the upper 1.5–2 µm of the cell (the distance between the nuclear envelope and the plasma membrane in these cells; this is unchanged following silencing of <i>Ahi1</i>, data not shown). Arrows denote the apical and mid-cell positions used in B and C. (B and C) Confocal maximum intensity projections of control (B) and <i>Ahi1</i>-silenced (C) cells showing Cep290 (red), gamma tubulin (green) and DNA (blue). Transfected cells were identified using siGLO (yellow). In each case, the top panel represents an apically-oriented centrosome (position 1 on the graph, A) and the bottom panel represents a centrosome from the mid-cell region (position 2 on the graph). Scale bar: 5 µm.</p

<i>AHI1</i> expression during nephrogenesis.

<p>(A, C, E) <i>AHI1</i> sense RNA probe and (B, D, F) <i>AHI1</i> antisense RNA probe hybridised to sections from (A, B) CS16; (C, D) CS22 and (E, F) 9 PCW. <i>AHI1</i> transcripts are abundant in the mesonephros (Meso) and metanephros (Meta) of the developing kidney as well as the spinal cord (Sc), liver and embryonic gonad. (G, H). A series of sections at different stages of mesonephric and metanephric development. <i>AHI1</i> expression is seen in (G) mesonephric excretory unit - a mesonephric tubule, glomerulus and duct at CS14, CS16 and CS22. By CS23 expression is visible in degenerating glomeruli (DG). By 9 PCW expression is detectable in the mesonephric tubule, ducts and paramesonephric duct. <i>AHI1</i> expression is seen in (H) early permanent metanephric kidney with intense staining in the metanephric cap and ureteric bud (CS14 and CS16). By CS22 and later developing glomeruli, tubules and collecting ducts are seen to strongly express <i>AHI1</i> (CS22, CS23 and 9 PCW). In 9 PCW human fetal sections, <i>AHI1</i> transcripts are abundant in the developing nephrons and collecting ducts of renal cortex and there is weak expression in medulla/renal pelvis. CD, collecting duct; DG, degenerating glomerulus; Gl, glomerulus; H, hindgut; LL, lower limb; Mc, metanephric cap; Md, mesonephric duct; Med, renal medulla; Meso, mesonephros; Meta, metanephros; Mt, mesonephric tubule; P, renal pelvis; Pmeso, paramesonephric duct; Sc, spinal cord; St, stomach; UB, ureteric bud; UL, upper limb. Scale bars: A–F = 2 mm; G: CS16 = 500 µm; CS14, CS23 & 9WPC = 250 µm; CS22 = 125 µm; H: CS22 & 9WPC = 500 µm; CS14, CS16 & CS23 = 250 µm.</p

<i>CEP290</i> is expressed in the developing kidney.

<p>Hybridisation with <i>CEP290</i> antisense RNA probe (A–F) to transverse sections. (A) CS12 and (B) CS14; (C) CS15; (D) CS16. <i>CEP290</i> transcripts are abundant in the mesonephros (Meso) and metanephros (Meta) of the developing kidney as well as the spinal cord (Sc). (E, F) A series of sections through mesonephric and metanephric development, respectively, from CS14 to 9 PCW. Hybridisations were also carried out with <i>CEP290</i> sense probe as a negative control and no signals were detected (data not shown). CD, collecting duct; Gl, glomerulus; H, hindgut; Meso, mesonephros; DMeso, degenerating mesonephros; Meta, metanephros; Sc, spinal cord; St, stomach; UL, upper limb; LL, lower limb. P, renal pelvis. Scale bar: A–D = 1 mm; E: CS12–CS16 = 500 µm; CS23 = 125 µm F: CS 14 = 250 µm; CS17 = 125 µm; CS20 = 250 µm; CS22 and 9 PCW = 125 µm.</p

Spatial transcriptomics reveals novel genes during the remodelling of the embryonic human arterial valves.

Abnormalities of the arterial valves, including bicuspid aortic valve (BAV) are amongst the most common congenital defects and are a significant cause of morbidity as well as predisposition to disease in later life. Despite this, and compounded by their small size and relative inaccessibility, there is still much to understand about how the arterial valves form and remodel during embryogenesis, both at the morphological and genetic level. Here we set out to address this in human embryos, using Spatial Transcriptomics (ST). We show that ST can be used to investigate the transcriptome of the developing arterial valves, circumventing the problems of accurately dissecting out these tiny structures from the developing embryo. We show that the transcriptome of CS16 and CS19 arterial valves overlap considerably, despite being several days apart in terms of human gestation, and that expression data confirm that the great majority of the most differentially expressed genes are valve-specific. Moreover, we show that the transcriptome of the human arterial valves overlaps with that of mouse atrioventricular valves from a range of gestations, validating our dataset but also highlighting novel genes, including four that are not found in the mouse genome and have not previously been linked to valve development. Importantly, our data suggests that valve transcriptomes are under-represented when using commonly used databases to filter for genes important in cardiac development; this means that causative variants in valve-related genes may be excluded during filtering for genomic data analyses for, for example, BAV. Finally, we highlight "novel" pathways that likely play important roles in arterial valve development, showing that mouse knockouts of RBP1 have arterial valve defects. Thus, this study has confirmed the utility of ST for studies of the developing heart valves and broadens our knowledge of the genes and signalling pathways important in human valve development