Table 1.
Overview of renal and brain development time points in human embryogenesis.
Figure 1.
AHI1 is expressed in the developing cerebellum, spinal cord, choroid plexus and eye.
(A, E) Negative control hybridisation with AHI1 sense RNA probe and (B–D) hybridisation with AHI1 antisense RNA probe to transverse sections at CS22. AHI1 transcripts are abundant in the developing telencephalon, especially the neuroepithelium (C) and neural retina (D). Prominent expression is seen in (F–H) metencephalon (including cerebellum), myelencephalon and mesencephalon at CS19. Strong expression is detected in the neuroepithelium of the developing mesencephalon and cerebellum (G, H). Within the developing ventricles, strong expression is seen in the choroid plexus epithelium (I, J) and the neuroepithelium of the ganglionic eminences at CS23 (I). Prominent staining is demonstrated within (K) the alar plate of spinal cord at CS16 and (L) CS22 and in the spinal ganglia. Ap, alar plate; Bp, basal plate; GE, ganglionic eminence; NEP, neuroepithelium; Cor.NEP: cortical neuroepithelium; Mes.NEP: mesencephalic NEP; Cere.NEP: cerebellar NEP; CP: choroid plexus; CPE, choroid plexus epithelium; Mes, mesencephalon (midbrain); Met, metencephalon; Mye, myelencephalon; NR, neural retina; OS, optic stalk; URhoL, upper rhombic lip; RPE, retinal pigment epithelium; Sc, spinal cord; SG, spinal ganglion; SyG, sympathetic ganglion; T, telencephalon; Ton, tongue; VG, vagal ganglion. Scale bars: A, B, E, F = 2 mm; C, D, G, H, K = 500 µm; I = 1 mm; J = 250 µm; L = 500 µm.
Figure 2.
AHI1 expression during nephrogenesis.
(A, C, E) AHI1 sense RNA probe and (B, D, F) AHI1 antisense RNA probe hybridised to sections from (A, B) CS16; (C, D) CS22 and (E, F) 9 PCW. AHI1 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. AHI1 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. AHI1 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 AHI1 (CS22, CS23 and 9 PCW). In 9 PCW human fetal sections, AHI1 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.
Figure 3.
CEP290 is expressed in the developing cerebellum, spinal cord and eye.
(A, G) Negative control hybridisation with CEP290 sense RNA probe and (B–F) hybridisation with CEP290 antisense RNA probe to transverse sections at CS22. CEP290 transcripts are abundant in the developing telencephalon (B, C, E), especially the cortical neuroepithelium (C), and neural retina (D). CEP290 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.
Figure 4.
CEP290 is expressed in the developing kidney.
Hybridisation with CEP290 antisense RNA probe (A–F) to transverse sections. (A) CS12 and (B) CS14; (C) CS15; (D) CS16. CEP290 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 CEP290 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.
Figure 5.
Cep290 does not require Ahi1 for localisation to centrosomes.
Immunofluorescence of IMCD3 monolayers co-transfected with siGLO and either negative-control siRNA (A and B), or siRNA against Ahi1 (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 Ahi1-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 Ahi1, 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 Ahi1-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.