Gassié L et al. (2015), Hspa9 is required for pronephros specification ...

XB-ART-51226

Dev Dyn 2015 Dec 01;24412:1538-49. doi: 10.1002/dvdy.24344.

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Hspa9 is required for pronephros specification and formation in Xenopus laevis.

Gassié L , Lombard A , Moraldi T , Bibonne A , Leclerc C , Moreau M , Marlier A , Gilbert T .

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Development of the pronephros in Xenopus laevis is largely dependent on retinoic acid signaling at the time of kidney field specification with the simultaneous occurrence of a necessary calcium signaling. At the crossroads of these two signaling pathways, we studied the role of Hspa9 (heat shock 70 kDa protein 9) encoding a mitochondrial chaperone in pronephros development. We first showed that Hspa9 is highly expressed in the pronephros territory and elongating nephric duct. We then observed that upon reduced retinoic acid signaling hspa9 expression was reduced as pax8 and pax2. Overexpression of hspa9 enlarged the pax8 positive pronephros territory, leading to a larger pronephric tubule. Loss of function of hspa9 in the kidney field using morpholino approach severely reduced pax8 expression and pronephros formation. Phenotypic rescue was achieved by co-injection of the full-length murine Hspa9 mRNA. However, no rescue was observed when Hspa9 mRNA lacking the mitochondrial-targeting sequence was injected, as this truncated form is able to interfere with pronephros formation when injected solely. Hspa9 is an important mediator for pronephros development through modulation of pax8. Mitochondrial functions of hspa9 are likely to be involved in specification of pronephric cell fate.

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Species referenced: Xenopus laevis
Genes referenced: cyp26a1 hspa9 lhx1 myod1 odc1 pax2 pax8 rgn st14
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	Figure 1. Expression pattern of Hspa9 in Xenopus laevis embryo. A: RT-PCR hspa9 expression in Xenopus laevis embryos from stages 3 to 40 of development with odc1 as house-keeping gene. B–H: Whole-mount ISH of hspa9. B: Early gastrula, arrow indicates the position of the blastoporal lip. C: Early neurula stage (anterior view, dorsal side up) shows hspa9 expression in neural and mesodermal territories. D,E: hspa9 is expressed in somite (s), mesoderm (m) and intermediate mesoderm (im). F: hspa9 labels the pronephros anlage (pa). G: At early tail bud stage, strong expression of hspa9 in eye (e), otic vesicle (ov), branchial arches (ba), somites and pronephros, from proximal (red arrow) to distal part (black arrow). G′: Section through the plan of dotted line in G. H,H′: Tadpole stage shows persistent hspa9 expression in the pronephros tubule. I–L: hspa9 pronephros expression was compared with pax8 and pax2 renal markers. Arrows show similar hspa9 and pax expression domains, up to the tip of the nephric duct. A strong hspa9 expression is present in the proctodeum (asterisk in I, K). Abbreviations: dt, distal tubule; g, glomus; it, intermediate tubule; n, notochord; pt, proximal tubule. Scale bars = 0.5 mm in B–H; 1 mm in I–L.Download figure to PowerPoint
	Figure 2. Decreased expression of hspa9 following in vivo reduction of RA signaling. A: Analysis of the GFP tracer distribution on one side of cyp26-injected embryos at neurula stage. B–I: pax8 ISH of cyp26-injected embryos at 0.15 ng (B,D,F,H) or 0.50 ng (C,E,G,I) dose was performed at st14. The otic territory (ot) and kidney field (kf) are specifically labeled by pax8 (D–G) and show a strongly reduced (F) or absent (G) pax8 positive renal field as already noticed on dorsal views (red arrows in B and C). Similar observation is made at st25 on the side of cyp26-injected embryos (H, I). J–Q: Expression of hspa9, pax8 and pax2 upon reduced RA signaling. At st24, hspa9 and pax8 are significantly reduced on the cyp26-injected side in the pronephros area (arrows, K,M). Hspa9 expression is reduced in the most anterior somites areas (white bracket) and branchial arch (*) as well (K). Similar results are obtained when compared with pax2 at late tadpole stage (O,Q). The nephric duct elongation was unaffected at the 0.15 ng dose of cyp26 injection (arrows in O,Q).Download figure to PowerPoint
	Figure 3. Overexpression of hspa9 enlarges the size of the pronephric tubule. A–I: pax8 whole-mount ISH at early neurula stage following injection of 0, 0.3 or 3.0 ng of hspa9. Dorsal and lateral views show the otic territory (ot) and kidney field (kf) specifically labeled by pax8. Delineations of both areas are depicted. Red-Gal staining indicates the side of injection. J: Quantitative analysis of overexpression of hspa9 on otic and renal territory, expressed as percent of the uninjected side. K,L: At late tail bud stage, pax8 expression seems stronger in the proximal part of the pronephros derived from the injected side, as shown by the Red-Gal staining. M–P: smp30, specific proximal tubular marker, confirms the findings at late tail bud stage (M,N) and tadpole stage (O,P). Q,R: 3G8 immunofluorescence in st32 embryos. S,T: Double-immunofluorescence with 3G8 (black arrows) and 4A6 (red arrows) at st.42 illustrates a longer tubule on the injected (X-Gal stained) than on the noninjected side. U: Histograms represent the summary and the frequency of the enlarged pronephric tubule observed in hspa9-injected embryos, at 0.3 (n = 32) and 3 ng (n = 68).Download figure to PowerPoint
	Figure 4. Loss of function of hspa9 interferes with early pronephros determination. A–I: Loss of function experiments using control (MoC) and hspa9-targetted morpholino oligonucleotides at 17 ng (Mo17) were analyzed by pax8 ISH at early neurula stage. Delineations of otic (ot) and renal (rf) areas are depicted. G: Quantitative analysis of both otic and renal territory areas, expressed as percent of the uninjected side, following Mo17 injection (MoC, n=14; Mo17: n=13, p<0.01). pax8-positive otic territory was of the same area on both sides. H,I: Dorsal views of whole-mount pax8 ISH at the end of neurula. A strong decrease was observed on the injected side in Mo17 as compared to the unaffected Mo controls. J,K: Dorsal view of embryos injected with 34 ng of Mo (Mo34) and processed for lhx1or myod ISH. Scale bars 0.5 mm.Download figure to PowerPoint
	Figure 5. Impaired hspa9 expression in Xenopus laevis disturbs tubular pronephric development. A–F: Whole-mount smp30 ISH in MoC (A–C) and Mo-injected (D–F) tadpole embryos at the dose of 17 ng. G,H: At tail bud stage, smp30 expression is decreased upon Mo34 injection (34 ng). I–L: pax8 (I,J) and pax2 (K,L) ISH confirm the impaired development of the proximal pronephric tubule after reduced hspa9 expression. M–P: Double-immunofluorescence with 3G8 (red) and 4A6 (blue) antibodies (M,O) and corresponding drawings (N,P) illustrate a simplified pronephric tubule on the Mo17-injected side of the embryo.Download figure to PowerPoint
	Figure 6. Rescue of Mo-induced reduction of the kidney field requires hspa9 mitochondrial sequence. A–L: pax8 whole-mount ISH at early neurula stage following injection of MmHspa9 (A–C), Mo17+MmHspa9 (D–F), Mo17+MmHspa9(-MTS) (G–I), or MmHspa9(-MTS) (J,L). Delineations of both otic (ot) and renal (rf) areas are depicted. Side of injection was determined by GFP transillumination before ISH. M: Quantitative analysis of pax8-positive otic and renal territories following Mm, Mo+Mm, Mo+Mm(-MTS) and Mm(-MTS) experiments, expressed as percent of the uninjected side. A weak but significant increase of the renal field is measured upon MmHspa9 injection (n = 15; P < 0.01), and reversal of the phenotype in Mo17+MmHspa9 embryos was complete (n = 14). In the Mo17+MmHspa9(-MTS), the renal field was still severely reduced (n = 13; P < 0.001). Injection of Mm(-MTS) significantly altered pronephric field determination (n = 14; P < 0.001).Download figure to PowerPoint
	Figure 7. Characterization of the rescued-Mo, failed-rescued and altered (-MTS)-induced pronephros development. A,B: GFP observation illustrates the correct targeting of mesodermal derivatives up to the end of tadpole stage. Arrows indicates gfp-positive tubules. C–F: pax8 whole-mount ISH at early tail bud stage in embryos injected with MmHspa9 alone or in combination with Mo17. G–J: pax2 whole-mount ISH at early tadpole stage in control and Mo17+MmHspa9 injected embryos. K,L: smp30 whole-mount ISH in early tadpole embryos injected with Mo17+MmHspa9. A stronger signal is present on all rescued embryos (arrows). M,N: pax2 whole-mount ISH in early tadpole embryos injected with Mo17+MmHspa9(-MTS). Arrows point to a lack of pax2 positive pronephros and arrowheads to a simplified proximal tubule. O,P: GFP signals were missing in pronephros area of MmHspa9(-MTS) injected tadpoles. Q,R: pax8 expression is severely reduced in MmHspa9(-MTS) injected embryos at stage 21. S–V: Reduced or absent pax2 signals are observed in MmHspa9(-MTS) injected tadpoles. W: Histograms represent the frequency of normal, weakly and severely modified pronephros in all groups (Mo, n = 78; Mm, n = 38; Mo+Mm, n = 50; Mm (-MTS), n = 40; Mo+Mm(-MTS), n = 41).Download figure to PowerPoint
	hspa9 (heat shock protein family A (Hsp70) member 9 ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 10, vegetal view, dorsal up.
	hspa9 (heat shock protein family A (Hsp70) member 9 ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 14, dorsal view, anterior down.
	hspa9 (heat shock protein family A (Hsp70) member 9 ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 24, lateral view, anterior left, dorsal up.
	hspa9 (heat shock protein family A (Hsp70) member 9 ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 36, lateral view, anterior left, dorsal up.