Geach TJ et al. (2014), An essential role for LPA signalling in telence...

XB-ART-48518

Development 2014 Feb 01;1414:940-9. doi: 10.1242/dev.104901.

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An essential role for LPA signalling in telencephalon development.

Geach TJ , Faas L , Devader C , Gonzalez-Cordero A , Tabler JM , Brunsdon H , Isaacs HV , Dale L .

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Lysophosphatidic acid (LPA) has wide-ranging effects on many different cell types, acting through G-protein-coupled receptors such as LPAR6. We show that Xenopus lpar6 is expressed from late blastulae and is enriched in the mesoderm and dorsal ectoderm of early gastrulae. Expression in gastrulae is an early response to FGF signalling. Transcripts for lpar6 are enriched in the neural plate of Xenopus neurulae and loss of function caused forebrain defects, with reduced expression of telencephalic markers (foxg1, emx1 and nkx2-1). Midbrain (en2) and hindbrain (egr2) markers were unaffected. Foxg1 expression requires LPAR6 within ectoderm and not mesoderm. Head defects caused by LPAR6 loss of function were enhanced by co-inhibiting FGF signalling, with defects extending into the hindbrain (en2 and egr2 expression reduced). This is more severe than expected from simple summation of individual defects, suggesting that LPAR6 and FGF have overlapping or partially redundant functions in the anterior neural plate. We observed similar defects in forebrain development in loss-of-function experiments for ENPP2, an enzyme involved in the synthesis of extracellular LPA. Our study demonstrates a role for LPA in early forebrain development.

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Species referenced: Xenopus laevis
Genes referenced: cdx4 egr2 emx1 emx1l en2 enpp2 fgf4 fgf8 fgfr1 foxg1 krt12.4 lpar6 mapk1 myc myod1 nkx2-1 odc1 otx2 pax6 rax snai2 sox2 tbxt
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	Fig. 1. Temporal expression of lpar6. (A) RT-PCR analysis for lpar6 and ornithine decarboxylase (odc) in staged Xenopus embryos, showing lpar6 expression from stage 10 (early gastrula) to stage 40 (tadpole). Note the drop in expression at stage 13 (late gastrula). Minus reverse transcriptase control (-RT) was performed at stage 40. (B) Microarray analysis for expression of lpar6, fgf8, bra, cdx4 and a marker for the mid-blastula transition (MBT). Xenopus embryos were collected 0-16 hours post-fertilisation (23°C). (C) RT-PCR analysis for lpar6 and odc in dissected stage 10 embryos. AP, animal pole; DM, dorsal marginal zone; VM, ventral marginal zone; VP, vegetal pole.
	Fig. 2. Spatial expression of lpar6. Whole-mount in situ hybridisation with antisense probe for lapr6. (A) Stage 10, vegetal view, with dorsal blastopore lip (arrowhead). (B) Stage 10, bisected, with dorsal blastopore lip (arrowhead). (C) Stage 10.5, vegetal view. (D) Stage 10.5, lateral view, with dorsal-animal expression (arrowhead). (E) Stage 16, anterior view. (F) Stage 19, anterior view. (G) Stage 24, lateral view with head to the left. (H) Stage 16, dorsal view with head to the left. (I) Stage 28, lateral view with head to the left. (J) Stage 28, trunk section. Scale bars: 200 μm. AP, animal pole; BA, branchial arch; Fb, forebrain; Not, notochord; NS, nervous system; VP, vegetal pole.
	Fig. 3. FGF regulates expression of lpar6. (A) Microarray analysis for lpar6 transcripts in early gastrulae expressing dnFGFR1 or dnFGFR4. (B) RT-PCR analysis for lpar6 and odc in animal caps incubated for 5 hours (18°C) with either FGF4 or Activin. Sibling embryos were used for embryo and -RT controls. (C) RT-PCR analysis for lpar6 and odc in animal caps incubated with FGF4 for up to 180 minutes (18°C). Sibling embryos were used for whole embryo and -RT controls.
	Fig. 4. LPAR6 is required for forebrain development. (A) Sequence (black letters, 5′-3′) of Xenopus lpar6 mRNA (translational start site in red) aligned with sequence for both AMO1 (pink letters 3′-5′) and AMO2 (blue letters 3′-5′). (B) In vitro translation of Xenopus lpar6 and human LPAR6 in the presence of morpholinos. Lane 1, no MO. Lane 2, control MO. Lane 3, AMO1. Lane 4, AMO2. (C-E) Stage 28, lateral view (head to left), injected with 40 ng of morpholino. (C) Normal embryo injected with control MO (100%, n=90). (D) AMO1-injected embryo with head defect (60%, n=95). (E) AMO2-injected embryo with head defect (93%, n=75). Anteroposterior axis length of control-MO-injected embryos was 4.0 mm (s.d.=0.21, n=44) and that of AMO-injected embryos 3.4 mm (s.d.=0.17, n=44). Defects in D and E are statistically significant (Fisher’s exact test, P<0.001). (F-H) Whole-mount in situ hybridisation, with antisense foxg1 probe. Stage 24, lateral view (head to left), injected with 40 ng of morpholino. (F) Normal embryo injected with control MO (100%, n=30). (G) AMO1-injected embryo with reduced foxg1 expression (60%, n=30). (H) AMO2-injected embryo with reduced foxg1 expression (87%, n=30). Defects in G and H are statistically significant (Fisher’s exact test, P<0.001). (I-K) Stage 28, lateral view (head to left), injected with 40 ng of AMO2 and 400 pg of human LPAR6 mRNA. (I) Normal embryo injected with control MO (100%, n=30). (J) AMO2-injected embryo with head defect (80%, n=35). (K) AMO2 plus hLPAR6 mRNA-injected embryo with normal head (66%, n=35). Rescue of head development in K is statistically significant (Fisher’s exact test, P<0.001). Scale bars: 400 μm.
	Fig. 5. LPAR6 is required for neural development. Whole-mount in situ hybridisation analysis of MO injected neurulae. (A-J) Dorsal views of neurulae injected with 20 ng of morpholino into a single blastomere at the two-cell stage. Head at the top and injected side (asterisk) on the right. (A,B) Neural plate marker sox2, with increased width on the AMO2-injected side (70%, n=40). (C,D) Epidermal marker k81a1, with decreased expression on the AMO2-injected side (75%, n=40). (E,F) Neural crest marker snai2, with reduced expression on the AMO2-injected side (69%, n=35). (G,H) Skeletal muscle marker myod1, with no defect (100%, n=38). (I,J) Posterior neural plate marker cdx4, with increased width on the AMO2-injected side (80%, n=40). (K-X) Anterodorsal views of neurulae injected with 40 ng of morpholino. (K,L) Telencephalon marker foxg1, with reduced expression in the AMO2-injected embryo (79%, n=38). (M,N) Hindbrain marker egr2, with normal expression in the AMO2-injected embryo (100%, n=35). (O,P) MHB marker en2, with normal expression in the AMO2-injected embryo (100%, n=35). Expression usually moved anteriorly (74%, n=35). (Q,R) Eyefield marker rax, with reduced expression in the AMO2-injected embryo (70%, n=40). (S,T) Eyefield marker pax6, with reduced expression in the AMO2-injected embryo (60%, n=30). (U,V) Anterior neural plate marker fgf8, with reduced expression in AMO2-injected embryos at the ANR (black arrow) and MHB (white arrow) (100%, n=23). (W,X) Whole-mount immunostaining for dpERK, with reduced ERK activity in AMO2-injected embryos at the ANR (black arrow), MHB (white arrow) and branchial arches (white arrowhead) (92%, n=36). All defects are statistically significant (Fisher’s exact test, P<0.001). Scale bars: 200 μm.
	Fig. 6. LPAR6 is required for telencephalic development. Whole-mount in situ hybridisation of tailbud embryos injected with 40 ng of morpholinos; lateral views of the head. (A,B) Telencephalon marker foxg1, with loss of expression in the AMO2-injected embryo (71%, n=35). (C,D) Dorsal telencephalon marker emx1, with loss of expression in the AMO2-injected embryo (75%, n=32). (E,F) Ventral telencephalon marker nkx2-1, with reduced expression in the AMO2-injected embryo (67%, n=33). (G,H) MHB marker en2, with normal expression in the AMO2-injected embryo (100%, n=32). All defects are statistically significant (Fisher’s exact test, P<0.001). Scale bars: 200 μm.
	Fig. 7. LPAR6 is required in the ectoderm. (A) Schematic diagrams of an eight-cell Xenopus embryo, indicating injected blastomere pairs and their normal fate. (B-F) Stage 40 embryos, lateral view (head to left), injected with 5 ng per blastomere of AMO2. (B) Uninjected normal embryo (n=50). (C) DA injected embryo with head defect (86%, n=69). (D) DV injected embryo with dorsal defect but a normal head (61%, n=54). (E) VA injected embryo with tail defect but a normal head (76%, n=66). (F) VV injected embryo with defect in the posterior endoderm (arrowhead) but a normal head (60%, n=60). All defects are statistically significant (Fisher’s exact test, P<0.001). (G) Schematic diagram of animal pole (AP) and dorsal marginal zone grafts. (H) RT-PCR analysis for foxg1, sox2 and odc expression in AP:DMZ grafts. Grafts were made between uninjected (U) and AMO2-injected (M) fragments. Scale bars: 500 μm. -RT, minus reverse transcriptase control, uninjected stage 19 embryos; AP, animal pole; DA, dorsal-animal; DV, dorsal-vegetal; Ep, epidermis; He, heart; M:M, AMO2-injected AP and AMO2-injected DMZ; M:U, AMO2-injected AP and uninjected DMZ; No, notochord; NP, neural plate; U:M, uninjected AP and AMO2-injected DMZ; U:U, uninjected AP and uninjected DMZ; VA, ventral-animal; VV, ventral-vegetal; WE, stage 19 embryo.
	Fig. 8. LPAR6 and FGF co-regulate neural development. (A-D) stage 32, lateral view (head to left), injected with 40 ng per blastomere of MO plus or minus 1 ng of dominant-negative FGFR1 (dnfgfr1) mRNA. (A) Normal embryo injected with control MO (100%, n=27). (B) AMO2-injected embryo with head defect (81%, n=37). (C) Control MO plus dnfgfr1-injected embryo with posterior defect but normal head (92%, n=25). (D) AMO2 plus dnfgfr1-injected embryo with both head and posterior defects (100%, n=28). Note that the head defect is more severe than with AMO alone. All defects are statistically significant (Fisher’s exact test, P<0.001). (E) RT-PCR analysis of MO-injected embryos incubated with or without 10 μM SU5402, from stage 9 to stage 16. Embryos were analysed for expression of the foxg1 and otx2 (forebrain), rax (eyefield), en2 (MHB), egr2 (hindbrain), sox2 (neural plate), myod1 (skeletal muscle) and odc (control). Scale bars: 500 μm. -RT, control-morpholino-injected embryos.
	Fig. 9. ENPP2 is required for forebrain development. (A) Sequence of Xenopus enpp2a (black lettering, 5′-3′) and enpp2b (green lettering, 5-3′), translational start site in red, aligned with sequence for AMO (blue letters, 3′-5′). (B) Western blot analysis of Xenopus embryos injected with 1 μg of enpp2a.myc mRNA and 40 ng of morpholino. (C,D) Stage 28, lateral views (head to right) injected with 20 ng of either control MO or enpp2-AMO. Anteroposterior axis length of control-morpholino-injected embryos was 4.0 mm (s.d.=0.13, n=45) and that of AMO-injected embryos 2.9 mm (s.d.=0.22, n=45). (E-P) Whole-mount in situ hybridisation analysis of neurulae injected with 20 ng of either control (CMO) or enpp2-AMO. All embryos are viewed from anterodorsal perspective. (E,F) Telencephalon marker foxg1, with reduced expression in AMO-injected embryo (57%, n=82) (G,H) Ventral telencephalon marker nkx2-1, with reduced expression in AMO-injected embryo (59%, n=70). (I,J) Dorsal telencephalon marker emx1, with reduced expression in AMO-injected embryo (72%, n=53). (K,L) Eyefield marker rax, with normal expression in AMO-injected embryo (100%, n=77). (M,N) MHB marker en2, with reduced expression in AMO-injected embryo (16%, n=73 - 6% in controls, n=50). (O,P) Anterior neural plate marker fgf8, with reduced expression in AMO-injected embryo in both ANR (black arrow) and MHB (white arrow) (71%, n=21). (Q,R) Whole-mount immunolocalisation for dpERK. Note reduced ERK activity in AMO-injected embryos in the ANR (black arrow), the MHB (white arrow) and the branchial arches (white arrowhead) (96%, n=26). All defects are statistically significant (Fisher’s exact test, P<0.001) except for en2 (M,N). Scale bars: 600 μm in C,D; 200 μm in E-P.
	Supplemental Figure 6. Inhibition of enpp2 inhibits telencephalon development. Xenopus laevis embryos were injected at the 2-cell stage with 10 ng per blastomere of enpp2 AMO and incubated until stage 25. Embryos were analysed by whole mount in situ hybridization using DIG labelled antisense RNA probes. A, B) Head of MO injected embryos stained for expression of the telencephalic marker foxg1 (arrow). Note absence of foxg1 expression in ennp2 AMO injected embryo. C, D) Head of MO injected embryos stained for expression of the ventral telencephalic marker nkx2-1 (arrow). Note strong expression of nkx2-1 in ennp2 AMO injected embryo, despite being greatly reduced in neurulae (see figure 9 of main article). E, F) Head of MO injected embryos stained for expression of the dorsal telencephalic marker emx1 (arrow). Note absence of emx1 expression in ennp2 AMO injected embryo. G, H) Head of MO injected embryos stained for expression of the eyefield marker rax. Note small spot of rax expression in the pineal gland (arrow), which is absent from ennp2 AMO injected embryo. The pineal gland is formed by the diencephalon of the newly formed brain.
	lpar6 (lysophosphatidic acid receptor 6) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 10, bisected view, animal up.
	lpar6 (lysophosphatidic acid receptor 6) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 19, anterior view, dorsal up.
	lpar6 (lysophosphatidic acid receptor 6) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 24, lateral view, anterior left, dorsal up.
	lpar6 (lysophosphatidic acid receptor 6) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anterior left, dorsal up.