Gere-Becker MB et al. (2018), Retinoic acid-induced expression of Hnf1b and F...

XB-ART-54918

Development 2018 Jun 08;14512:. doi: 10.1242/dev.161372.

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Retinoic acid-induced expression of Hnf1b and Fzd4 is required for pancreas development in Xenopus laevis.

Gere-Becker MB , Pommerenke C , Lingner T , Pieler T .

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Retinoic acid (RA) is required for pancreas specification in Xenopus and other vertebrates. However, the gene network that is directly induced by RA signalling in this context remains to be defined. By RNA sequencing of in vitro-generated pancreatic explants, we identified the genes encoding the transcription factor Hnf1β and the Wnt-receptor Fzd4/Fzd4s as direct RA target genes. Functional analyses of Hnf1b and Fzd4/Fzd4s in programmed pancreatic explants and whole embryos revealed their requirement for pancreatic progenitor formation and differentiation. Thus, Hnf1β and Fzd4/Fzd4s appear to be involved in pre-patterning events of the embryonic endoderm that allow pancreas formation in Xenopus.

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Species referenced: Xenopus laevis
Genes referenced: aldh1a2 bmp4 cebpd cer1 chrd cyp26a1 darmin dhrs3 dusp5 fabp2 foxh1 fst fzd4 gbx2.1 gcg hhex hnf1b hnf4a hoxa1 hoxb1 hoxb3 hoxd1 hoxd4 igf3 ins kremen2 krt12.4 lhx1 mcf2 meis3 nkx2-1 nkx6-2 nog odc1 pdia2 pdx1 prph psmd6 ptf1a rara sox17a sox2 tesk1 vegt znf703
GO keywords: pancreas development [+]
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???displayArticle.gses??? GSE112718: Xenbase, NCBI

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	Fig. 1. Comparative analysis of the temporal profile for pancreatic marker gene expression in vivo and in programmed explants (A) Ectodermal explants isolated from stage 8/9 embryos and injected as indicated were treated with 5µM RA and cultivated until the equivalent of stage 30. Detection of marker gene expression for the genes indicated was done by RT-PCR. (B) and (C) Transcript quantification of whole embryos (5 embryos per condition) and explants (approximately 50 explants per condition) from two biological replicates with untreated and treated embryos grown to the equivalent of the developmental stages indicated using Nanostring analysis (B) and RT-PCR (C). Average values are given as mean and error bars as s.e.m.. E, embryo; C, un-programmed explants; PE, pancreatic explants (VegT/Noggin/RA); øRA, programmed explants with blocked endogenous RA-signalling (VegT/Noggin/Cyp26a1).
	Fig. 2. Identification of Hnf1β and Fzd4 as direct endodermal RA-target genes (A) Experimental procedure for the identification of early direct RA-target genes in the context of pancreas development. (B) Venn diagram comparing genes differentially expressed within two hours after RA-addition in the absence or presence of CHX. 46 putative direct RA-target genes were induced under both conditions. (C) RNA-sequencing results for RA-mediated Hnf1β and Fzd4 induction. The number of mapped reads two hours after the addition of RA in the presence or absence of CHX is indicated. The data result from two biological replicates with approximately 50 explants per condition. Average values are given as mean and error bars as s.e.m.. (D) WMISH for Hnf1β and Fzd4 in gastrula stage embryos. Whole embryos are depicted on the left-hand side and bisected embryos on the right-hand side. dbl, dorsal blastopore lip; e, endoderm; m, mesoderm; ne, neuro-ectoderm
	Fig. 3. Hnf1β is required for pancreas development in vitro and in vivo (A) Morpholino-mediated knockdown of Hnf1β in pancreatic explants. In order to demonstrate the specificity of the morpholino-effect, RNA for a hormone-inducible version of Hnf1β (Hnf1b-GR) was co-injected and explants were treated with the GR inducer dexamethasone (DEX) together with RA at the equivalent of gastrula stage. At the equivalent of stages 31 and 39, total RNA was isolated from approximately 30 explants each condition and subjected to RT-PCR. Detection was for endogenous (endo) and injected Hnf1β (inj.), as well as for the marker genes indicated. The Hnf1β loss-of function phenotype and its rescue was observed for four independent biological replicates. (B) 4-cell-stage embryos were injected with RNA coding for β-galactosidase (glb1) and either Hnf1β-morpholino or a control-morpholino. At stage 32, embryos from two independent biological replicates were used for WMISH against Pdx1 and Ptf1a and a real-time PCR analysis for Pdx1, Ptf1a and Insulin. The graph indicates the fold change of tested markers in relation to Odc (ornithine decarboxylase). ctr, uninjected embryos. (C) 4-cell-stage embryos were injected with RNA coding for β-galactosidase alone or in combination with Hnf1β -GR RNA. At gastrula stage, embryos were treated with dexamethasone (DEX) to induce Hnf1β function. WMISH against Pdx1 and Ptf1a at stage 32 is shown. Boxplots display the range of the area percentage of Pdx1 and endodermal Ptf1a domains in the endoderm observed in embryos from two independent biological replicates (see Fig. S7). By the use of ImageJ (https://imagej.net), Pdx1 and Ptf1a positive areas were measured (orange dotted line) in ratio to the area of the whole endoderm (green dotted line). Values above the upper whisker, which is set at 1.5 x interquartile range above the third quartile, are indicated as maximum outliers (°). (P-values in an unpaired Student´s t-test <0.01, *<0.001).
	Fig. 4. Fzd4/Fzd4s is required for pancreas development in vitro and in vivo (A) Fzd4-morpholino (mo) or the corresponding mismatch-morpholino (mmo) were coinjected along with Vegt and Noggin encoding RNAs. At the equivalent of stage 28, total RNA was isolated from the programmed explants and subjected to RT-PCR as indicated. (B) Fzd4-gRNA was co-injected along with RNAs encoding Cas9, Vegt and Noggin into one-cell stage embryos. Explants were cultivated until the equivalent of stage 35. RT-PCR was for the genes indicated. Mutation rate is given for Cas9 only or for Cas9 in combination with Fzd4-gRNA. For both loss of function approaches, approximately 30 explants per condition from two independent biological replicates were used. (C) Downregulation of Fzd4/Fzd4s by Fzd4-morpholino injection. 4-cell-stage embryos were injected with RNA coding for β- galactosidase (glb1) and either Fzd4/Fzd4s-morpholino or the corresponding mismatchmorpholino. At stage 35/39, embryos from two independent biological replicates were used for WMISH against indicated pancreatic markers and a real-time PCR analysis for Pdx1, Ptf1a and Insulin. The graph indicates the fold change of tested markers in relation to Odc (ornithine decarboxylase). Average values are given as mean and error bars as s.e.m.. ctr, uninjected embryos.
	Fig. 5. Diagrammatic representation reflecting the role of RA signalling in pancreas specification during early Xenopus embryogenesis During gastrulation, the expression of Fzd4 and Hnf1β is directly induced by RA. The overlapping activity of Fzd4 and Hnf1b establishes a pre-pancreatic domain within the dorsal endoderm. Fzd4/Fzd4s is a regulator of Wnt-signalling within the dorsal endoderm, which modulates Wnt-signalling to a level that allows the specification of pancreatic progenitors characterized by the co-expression of Ptf1a and Pdx1. Pancreatic progenitors subsequently proliferate and differentiate into endocrine and exocrine tissue.
	Fig. S1. Distribution of pancreatic cells in programmed explants (A) Co-injection of vegt and noggin mRNAs into the animal pole of two cell stage embryos. Explants were treated with three different concentrations of RA (5, 15 and 30µM) at the equivalent of stage 11 for 1h and used for WMISH at the equivalent of stage 35. (B) Different categories according to the intensity of pancreatic marker gene expression are presented. (C) Diagram reflecting the percentage of explants positive for pancreatic marker gene expression in the different categories. The total number of explants analyzed is indicated in brackets.
	Fig. S2. Verification of RA-responsiveness (A) Ectodermal explants from vegt/nog/cyp26a1-injected embryos (in vitro) or whole embryos (in vivo) were treated with RA or left untreated. Analysis of candidate gene expression was by use of the Nanostring technology. (B) RA-signalling was either impaired by Cyp26a1 RNA injection into the two dorsal blastomers at the four-cell stage and dorsal tissue dissected at stage 11 (red dotted line), or whole embryos were treated with BMS453 from stage 9 to 12/14 and analyzed using Nanostring. (C) Venn diagram illustrating the number of genes verified for their RA-inducibility in vitro and in vivo as well as for their RA-dependence in vivo.
	Fig. S3. Expression characteristics of RA-responsive genes at gastrula stage Schematic overview for the expression patterns of 22 RA-responsive genes by WMISH in gastrula stage embryos. Candidate genes were grouped according to their expression domains. Dots indicate direct RA-target genes. The upmost scheme describes the expression domains of Raldh2 (RA generating enzyme), Cyp26a1 (RA-degrading enzyme) and the localization of prospective pancreatic progenitor cells. The color code reflects expression domains as indicated.
	Fig. S4. Nanostring analysis for the spatial expression characteristics of RAresponsive genes at gastrula stage Four-cell stage embryos were injected into the two dorsal blastomeres with GFP mRNA. At stage 10, embryos exhibiting a GFP signal on the dorsal side were selected for further cultivation. At stage 11, either dorsal and ventral endoderm or the whole tissue surrounding the dorsal blastoporus and the corresponding ventral tissue were dissected. Nanostring counts are mean values from two independent experiments. DE = dorsal endoderm, VE = ventral endoderm, D = dorsal part, V = ventral part
	Fig. S5. Embryonic expression of Hnf1β and Fzd4 is RA-dependent (A) WMISH for Hnf1β and Fzd4 at gastrula stage in untreated, RA-treated and cyp26a1- injected embryos. Images on the left display whole embryos (dorsal side up) and images on the right show bisected embryos (dorsal side on the right). dbl, dorsal blastopore lip; e, endoderm; m, mesoderm; ne, neuro-ectoderm. (B) Nanostring analysis of untreated, RAand BMS-treated embryos collected at indicates stages.
	Fig. S6. Specificity of the Hnf1β morpholino antisense oligonucleotide (A) The Hnf1β morphilono antisense oligonucleotide (Hnf1b-mo) targets the intron1/exon2 boundary (E1/E2) of the Hnf1β pre-mRNA, resulting in the loss of exon 2, leading to a shortened open reading frame (ORF) lacking functional DNA-binding domains (POUs and POUh) and also lacking the transactivation domain. (B) The specificity of the Morpholino was tested in the explant system. RT-PCR with oligonucleotides bind to exon 1 and exon 3. A smaller Hnf1β amplicon is detected in the presence of the morpholino. (C) Sequence analysis of the shorter Hnf1β amplicon upon Hnf1b-mo application confirms the loss of exon 2.
	Fig. S7. Quantification of endodermal expression domains for Pdx1 and Ptf1a upon overexpression of Hnf1ß Area size of Pdx1 and endodermal Ptf1a expression domains (orange dotted lines) were estimated by ImageJ and the ratio to the whole endoderm (green dotted line) was calculated. A series of control and Hnf1ß injected embryos from two independent experiments (A and B) is displayed.
	Fig. S8. Hnf1β is not a sufficient substitute for the induction of pancreatic gene expression by RA (A) Programmed explants were from embryos co-injected with Hnf1β-GR RNA at the two-cell stage. Treatment with dexamethasone (DEX) and RA was at the equivalent of gastrula stage. At the equivalent of stage 28, total RNA was isolated and analyzed by RT-PCR. (B) RT-PCR analysis for pancreatic and endodermal genes as well as for the known direct Hnf1β target Hnf4α.
	Fig. S9. Fzd4 and the splice variant Fzd4s are direct RA-target genes (A) RT-PCR analysis with explants programmed as indicated making use of oligonucleotides distinguishing between Fzd4 and Fzd4s transcripts. (B) Transcript-specific determination of Fzd4/Fzd4s abundances. RNA sequencing reads mapping to the whole fzd4 gene region (Fzd4/Fzd4s) and the annotated fzd4 intron only (Fzd4s) were estimated. The fold change of transcript numbers in the absence or presence of RA is shown.
	Fig. S10. Mutation analysis for the genomic locus of Fzd4 exon1 and the putative offtarget Kremen2 in CRISPR/Cas-injected pancreatic explants DNA sequences of (A) Fzd4 and (B) Kremen2 amplicons from CRISPR/Cas injected pancreatic explants were aligned to genomic X.leavis Fzd4 and Kremen2 sequences. The Fzd4-gRNA target sequence is highlighted in yellow and the number of deleted nucleotides indicated.
	Fig. S11. Effects of Hnf1β down- or upregulation on the expression of various endodermal organ marker genes Four-cell stage embryos were injected with RNA encoding β-galactosidase and either (A) Hnf1β-morpholino or control-morpholino, or (B) RNA coding for Hnf1β-GR, vegetally into the two dorsal blastomeres. At stage 36/37 embryos were used for WMISH against Pdx1 (pancreas, duodenum), hHex (liver), Nkx2.1 (thyroid, lung) and (B) Darmin (posterior endoderm).
	Fig. S12 Effects of Fzd4/Fzd4s downregulation on the expression of various endodermal organ marker genes Eight-cell stage embryos were injected with RNA coding for β-galactosidase and either Fzd4/ Fzd4s-morpholino or the corresponding mismatch-morpholino. At stage 38/39, embryos were used for WMISH against Pdx1 (pancreas, duodenum), hHex (liver), Nkx2.1 (thyroid, lung) and IFABP (stomach, intestine).

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