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First published online 3 July 2006
doi: 10.1242/dev.02458

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The Notch-effector HRT1 gene plays a role in glomerular development and patterning of the Xenopus pronephros anlagen

¹ Laboratoire d'Embryologie Moléculaire, Université Libre de Bruxelles, Institut de Biologie et de Médecine Moléculaires (IBMM), rue des Profs. Jeener et Brachet 12, B-6041 Gosselies, Belgium.
² DFG-Center of Molecular Physiology of the Brain, Department of Developmental Biochemistry, University of Göttingen, Justus-von-Liebig Weg 11, 37077 Göttingen, Germany.

View larger version (48K): [in a new window]   Fig. 1. Whole-mount in situ analysis of XHRT1, esr9, esr10, Xhairy1 and Xhairy2b expression in comparison with X-Delta-1 and xWT1 in the pronephros region (arrowheads) of early tailbud stage embryos. Nieuwkoop-Faber stages are indicated (Nieuwkoop and Faber, 1967). (A-D) Comparison of the expression of XHRT1, X-Delta-1 and xWT1 in stage 20 embryos. (A) Whole embryo, lateral view, anterior right. (B-D) Transversal sections. XHRT1 expression coincides with that of xWT1 around the pronephros anlagen and surrounds that of X-Delta-1. (E-N) Comparison of the expression of XHRT1, esr9, esr10, Xhairy1 and Xhairy2b in stage 22-23 embryos. (E,G,I,K,M) Whole embryos, lateral views, anterior right. (F,H,J,L,N) Transversal sections. Note that esr9 and esr10 are co-expressed with XHRT1 around the dorsal border of the pronephros anlagen, and that Xhairy1 and Xhairy2b expression is detected in both mesodermal layers and in the ectoderm. e, ectoderm; pn, pronephros; l, lateral intermediate mesodermal layer; m, medial intermediate mesodermal layer.

View larger version (105K): [in a new window]   Fig. 2. Whole-mount in situ analysis of XHRT1, esr9, esr10, Xhairy1 and Xhairy2b in comparison with X-Delta-1 and X-Serrate-2 in the pronephros region (arrowheads) of late tailbud to tadpole stage embryos. Nieuwkoop-Faber stages are indicated. (A) At stage 25, XHRT1 is expressed in the most dorsoanterior portion of the pronephros anlagen. (B) Transversal section of the embryo shown in A at the level indicated. (C-P) Expression of XHRT1, esr9, esr10, Xhairy1, Xhairy2b, X-Delta-1 and X-Serrate-2 in stage 28 embryos. (A,C,E,G,I,K,M,O) Whole embryos, lateral views; (B,D,F,H,J,L,N,P) transversal sections of the corresponding embryos at the level indicated. Note that esr9 and esr10 staining appears slightly ventral compared with XHRT1. While X-Delta-1 expression is restricted to a band just below XHRT1, Serrate2 expression is broader. (Q,R) Double labeling of XHRT1 and X-Delta-1 or Xhairy2b. (S,T) At stage 35, Xhairy2b is co-expressed with X-Serrate-1 in the proximal and distal tubules. dt, distal tubules; pn, pronephros; pt, proximal tubules; m, medial intermediate mesodermal layer.

View larger version (78K): [in a new window]   Fig. 3. XHRT1, esr9, esr10, Xhairy1 and Xhairy2b are responsive to Notch signaling in the pronephric mesoderm (arrowheads). Whole-mount in situ analysis of stage 23-30 embryos injected with hGR/Su(H)/Ank or Su(H)DBM mRNA. The inducible construct was activated at stage 18. (A, parts a-t) Control and injected sides of embryos, anterior right. Note the posterior expansion of the expression of the different bHLH-O genes on the injected side of hGR/Su(H)/Ank-injected embryos (arrowheads). Note the inhibition of their expression in Su(H)DBM-injected embryos (arrowheads). Except in d,f,h and j, the injected side is revealed by ß-gal staining (red). (B, parts a-e) Transversal sections of the embryos shown in b,d,f,h,j at the level indicated. Note that the strongest esr9, esr10 and XHRT1 ectopic staining is detected in the lateral part of the intermediate mesoderm, while Xhairy1 and Xhairy2b expression is found in both layers (arrowheads). Xhairy2b is also strongly activated in the ectoderm (arrow). (B, part f) Transversal sections in stage 23 embryos in the posterior portion of the pronephros showing that XHRT1 ectopic expression in response to activation of Notch is already detected at that stage in both layers. (B, part g) Transversal section in the posterior portion of the pronephros of stage 23 embryos stained with xWT1. Ectopic staining of xWT1 is restricted to the medial layer.

View larger version (92K): [in a new window]   Fig. 4. XHRT1 early expression in the developing glomus is affected by translational inhibition of xWT1. (A) Design of the xWT1 MOs that target both pseudoalleles. (Bottom) In vivo translation of 500 pg of xWT1-eGFP is specifically inhibited by 15 ng of xWT1 MOs. (B-I) Control and injected sides of xWT1-depleted embryos stained with the indicated probes. xWT1 MOs abolished the expression of nephrin but had no effect on XSMP-30, Pax8 and Evi1. (J,K) Transversal sections of Evi1- and Pax8-stained xWT1-depleted embryos. Expression of both markers is unaffected by xWT1 knockdown. (L-O) Control and injected sides of xWT1-depleted embryos stained with XHRT1. Note that xWT1 knockdown decreases early but not late XHRT1 pronephric expression.

View larger version (60K): [in a new window]   Fig. 5. XHRT1, but not esr9, esr10 or Xhairy2b inhibits pronephric distal tubule and duct formation. (A-R) Whole-mount in situ hybridization of embryos injected with 500 pg of mRNA encoding the indicated inducible constructs together with ß-galactosidase mRNA analysed at stage 16 for N-tubulin (+Dex, stage 12; A,D,G,J,M,P) or stage 28 for Evi1 expression (+Dex, stage 18; B,C,E,F,H,I,K,L,N,O,Q,R). Embryos at neurula stage are viewed from the dorsal side, injected side downwards. Lateral view of control and injected sides of stage 28 embryos are shown. (A-C) Embryos injected with hGR/Su(H)/Ank mRNA showed an inhibition of N-tubulin and Evi1 expression (C, arrow). (D-R) Although overexpression of all bHLH-O genes inhibited N-tubulin in the neural plate, only XHRT1 overexpression inhibited Evi1 expression (F, arrow) in the pronephros. Lines in A,D,G,J,M,P indicate the injected and uninjected sides.

View larger version (85K): [in a new window]   Fig. 6. Comparison of the ability of hGR/Su(H)/Ank and XHRT1-MT-hGR to affect the expression of proximal tubule and glomus markers. (A) Activation of Notch signaling until stage 25 expands xWT1 efficiently, while later activation had no effect. XSMP-30 expression was increased in some embryos activated between stages 22 and 27. Embryos were co-injected with hGR/Su(H)/Ank and ß-galactosidase mRNA. Injected embryos were treated, or not, with Dex at the indicated times and analysed at stage 32-35. Changes in the expression of xWT1 and XSMP-30 at each stage were scored in individual embryos by comparing the injected and injected sides in at least two different injections. Embryos were classified into three phenotypes (no changes, increase or decrease). n, number of cases analysed. (B-M) Control and injected sides of embryos injected with the indicated mRNA together with ß-galactosidase mRNA, treated with Dex at stage 18 (xWT1, nephrin) or 25 (XSMP-30) and analysed with the indicated probes. Note that hGR/Su(H)/Ank expands xWT1, nephrin and, in a few cases, XSMP-30, while XHRT1-MT-hGR has no effect.

View larger version (35K): [in a new window]   Fig. 7. XHRT1 reverses the effect of Su(H)DBM on Evi1 but is not sufficient to restore XSMP-30 expression. (A-H) Late tailbud/early tadpole stage embryos injected with 500 pg of Su(H)DBM mRNA together with 500 pg of XHRT1a-mut-MT-hGR mRNA, untreated (A-D) or dexamethasone treated (E-H) at stage 22. ß-galactosidase RNA was co-injected to identify the injected side. Note the decrease of XSMP-30 and the increase of Evi1 expression in untreated embryos, and the reduction of both XSMP-30 and Evi1 expression in treated embryos. (I) Quantification of the results. Embryos were classified into two phenotypes (increase Evi1 and decrease XSMP-30). n, number of cases analysed.

View larger version (90K): [in a new window]   Fig. 8. Antisense morpholinos against XHRT1 reduces the expression of glomus and proximal tubule markers. (A) Design of the XHRT1-MO that targets both pseudoalleles. (Bottom) In vivo translation of XHRT1a-eGFP is specifically inhibited by XHRT1-MOs. Embryos were injected with 500 pg of XHRT1-eGFP or eGFP mRNA, alone or in combination with 15 ng of the XHRT1-MO, as indicated. (B-O) Embryos injected with 15 ng XHRT1-MO and ß-galactosidase mRNA analysed with the indicated markers. (B-I) Control and injected sides of XHRT1-MO-injected embryos with decreased XSMP-30, xPDZK1, xWT1 and nephrin expression. (J,K) Transversal sections of XHRT1-MO-injected embryos. (L,M) XHRT1 knockdown has no effect on Evi1 expression. (N,O) Transversal sections of XHRT1-MO-injected embryos. Ep. keratin and N-tubulin expression is unaffected by XHRT1 knockdown. (P) Co-injection of the XHRT1-MO with 500 pg of XHRT1a-mut-MT-hGR mRNA rescues XSMP-30 expression in stage 22 dexamethasone-treated XHRT1-MO-injected embryos. Changes in the expression of XSMP-30 were scored and classified as in Fig. 6A. (Q) Co-injection of 15 ng XHRT1-MO inhibits the effect of overexpression of hGR/Su(H)/Ank (500 pg) on XSMP-30 expression. Injected embryos were dexamethasone treated at stage 22. Changes in the expression of XSMP-30 were classified into two groups (no change or increase, decrease). n, number of embryos analysed; m, medial intermediate mesodermal layer; pn, pronephros; pt, pronephric tubules.

View larger version (30K): [in a new window]   Fig. 9. XHRT1 specific function in the pronephros is dependent on its C-terminal region. (A) Schematic representation of the XHRT1, XHes2 and chimeric proteins. All constructs encode Myc tag and hGR fusion proteins (not represented). The numbers correspond to the amino acids of the protein domains. (B) Western blot analysis of the expression level of XHRT1, XHes2 and chimeric proteins. Extracts prepared from animal caps derived from embryos injected with 250 pg of each construct were immunoblotted with anti-Myc and anti-ß-tubulin antibodies. (C) Comparison of the activity of XHRT1, XHes2 and chimeric proteins. Embryos were injected with 500 pg mRNA of each construct. The embryos were treated with dexamethasone at stage 18 and fixed at stage 26. Changes in Evi1 expression were scored as in Fig. 6A.