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A systems-level approach reveals new gene regulatory modules in the developing ear - PubMed

  • ️Sun Jan 01 2017

. 2017 Apr 15;144(8):1531-1543.

doi: 10.1242/dev.148494. Epub 2017 Mar 6.

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A systems-level approach reveals new gene regulatory modules in the developing ear

Jingchen Chen et al. Development. 2017.

Abstract

The inner ear is a complex vertebrate sense organ, yet it arises from a simple epithelium, the otic placode. Specification towards otic fate requires diverse signals and transcriptional inputs that act sequentially and/or in parallel. Using the chick embryo, we uncover novel genes in the gene regulatory network underlying otic commitment and reveal dynamic changes in gene expression. Functional analysis of selected transcription factors reveals the genetic hierarchy underlying the transition from progenitor to committed precursor, integrating known and novel molecular players. Our results not only characterize the otic transcriptome in unprecedented detail, but also identify new gene interactions responsible for inner ear development and for the segregation of the otic lineage from epibranchial progenitors. By recapitulating the embryonic programme, the genes and genetic sub-circuits discovered here might be useful for reprogramming naïve cells towards otic identity to restore hearing loss.

Keywords: Auditory system; Cell fate; Chick; Embryo; Hearing; Placode; Transcription factor.

© 2017. Published by The Company of Biologists Ltd.

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Conflict of interest statement

Competing interests

The authors declare no competing or financial interests.

Figures

Fig. 1.
Fig. 1.

Otic-enriched transcripts. (A,B) Diagrams showing the location of OEPs at 5ss (A,A′, graded pink-blue) and the otic and epibranchial placodes at 11-12ss (B,B′; otic: purple; epibranchial: blue). OEPs are induced by mesoderm-derived FGFs (green in A′). Later, FGFs activate Wnt ligands in the neural tube, which cooperate with Notch to promote otic identity (B′), while FGFs and BMPs from the endoderm promote epibranchial fate. (C,D) RNAseq was performed on dissected otic placodes from 5-6ss, 8-9ss and 11-12ss and otic-enriched transcripts enriched were identified by comparison to the whole embryo (3ss); see also

Tables S1 and S2

. (C) Genes enriched in the otic placode at 5-6ss (blue; fold-change >1.5). (D) Venn diagram showing the number of otic-enriched genes at 5-6ss, 8-9ss and 11-12ss and their intersection. (E) Disease-association of otic-enriched genes. (F-H) Biological processes and signalling pathways over-represented in otic-enriched genes at each stage showing at most the five top over-represented terms for which P<0.01 (Fisher’s Exact test) for each category. Epi, epibranchial domain; OEPD, otic-epibranchial progenitor domain.

Fig. 2.
Fig. 2.

Expression of transcription factors in the otic placode. (A-R) Lmx1a (A,B), Sox13 (C,D) and Zbtb16 (E,F) are expressed in OEPs and in the otic placode (OP). Rere (G,H), Tcf4 (I,J) and Zfhx3 (K,L) expression starts at placode stages, whereas Prdm1 is expressed in OEPs (M) but later restricted to the epibranchial territory (Epi) (N). At 12-13ss, Nr2f2 (O,P) and Vgll2 (Q,R) are absent from the otic placode, but present in epibranchial cells and the ventral ectoderm (Vgll2).

Fig. 3.
Fig. 3.

Temporal changes in otic gene expression. Pairwise comparison of the otic transcriptome at consecutive developmental stages: 5-6ss compared with 0ss (PPR; A-C), 8-9ss compared with 5-6ss (D-G) and 11-12ss compared with 8-9ss (H-J); see also

Table S3

. (A,D,H) Differentially expressed genes with a fold change >1.5; blue indicates upregulated transcripts; orange indicates downregulated transcripts. (B,E,I) Gene ontology analysis of up- and downregulated genes showing the five top over-represented biological processes or signalling pathway (P<0.01; Fisher’s Exact test). There is no significant association for the downregulated genes shown in H. (B′) At 5-6ss terms related to anterior structures are significantly under-represented relative to 0ss. (C,F,G,J) Changes of transcripts associated with signalling pathways over the entire time course. Asterisk indicates that the gene expression level is indicated by the y-axis on the right.

Fig. 4.
Fig. 4.

Clusters of otic transcription factors. (A) Otic transcription factors from the enrichment and time course analysis cluster into five clusters (TFC1-5) based on the row z-score of fold change relative to the PPR at 0ss. (B-F) Expression level of the top 50% transcription factors in each cluster. Line in the top right of each cluster represents the overall expression profiles across the three time points. See also

Table S4

.

Fig. 5.
Fig. 5.

Regulation of otic transcription factors by Pax2, Etv4 and Lmx1a. Target-specific morpholinos were electroporated at 0-1ss and changes in gene expression were assessed by NanoString with three biological replicates, each of which containing five pieces of otic placode (A,G; see also

Table S5

), in situ hybridization (B-B″,C-C″,D-F,H′-K″,M,M′) or RT-PCR with two biological replicates each containing five pieces of otic placode (L). (A) Etv4 knockdown analysed by NanoString. Green indicates downregulated genes, red indicates upregulated genes. Open triangles represent data points that have a value beyond the axis limit. (B-F) In situ hybridization after Etv4 knockdown for the genes indicated in each panel. A reduction of Pax2 (8/12; B′), Zbtb16 (6/6; C′); Prdm1 (7/11; D), Tcf4 (8/10; E) and Vgll2 (4/5; F) is observed. Asterisks indicate the electroporated side. B and C show morpholino fluorescence of the embryos shown in B′ and C′, respectively; B″ and C″ show sections through the embryos shown in B′ and C′, respectively, at the level marked by the horizontal lines. (D-F) Sections of embryos electroporated with Etv4 morpholino. (G-K″) Pax2 knockdown analysed by NanoString (G). Green indicates downregulated genes, red indicates upregulated genes. Open triangles represent data points that have a value beyond the axis limit. (H-K″) In situ hybridization after Pax2 knockdown for the genes indicated in each panel. Asterisks indicate the electroporated side. Lmx1a (4/4; H′), Zbtb16 (4/4; I′), Prdm1 (3/4; J′) and Vgll2 (4/4; K′) are reduced. H-K show morpholino fluorescence of the embryos shown in H′-K′; H″-K″ show sections through the embryos shown in H′-K′, respectively, at the level marked by the horizontal lines. (L) Lmx1a knockdown analysed by RT-qPCR. The results are presented as fold change ±s.d. and two-tailed Student's t-test was used to calculate P-value. (M) Morpholino fluorescence of the embryo shown in M′ (3/4). (N-P″). In situ hybridization after control morpholino electroporation for the genes indicated in the panels; N′-P′ show sections of the embryos in N-P, respectively, at the level marked by the horizontal lines. In situ hybridization for each gene was performed on four embryos electroporated with control morpholino.

Fig. 6.
Fig. 6.

Regulatory modules during OEP specification. All diagrams summarize data from the literature and from this study (for details see text). (A) Lmx1a and Pax2 mutually activate each other, control common targets and appear to repress alternative fates (see text for details). (B) Pax2 is controlled by the posterior PPR factors Six1, Eya2, Foxi3 and Gbx2, as well as by the FGF mediator Etv4. (C,D) Etv4 and Pax2 could act in a linear pathway (C) or in a feed-forward loop (D) to control other OEP genes (see text for details). Irx5 is regulated by both Etv4 and Pax2; however, because Pax2 is also regulated by Etv4 for simplicity the network in Fig. 7 assumes that Etv4 regulates Irx5 via Pax2: Etv4→Pax2→Irx5.

Fig. 7.
Fig. 7.

Gene regulatory network incorporating new functional data. (A-C) Signalling inputs, gene expression changes and regulatory relationships at the three different stages. Regulator links are based on data from the literature (

Fig. S5

) and our perturbation experiments (Fig. 5;

Fig. S6A-C

). Direct interactions confirmed from literature are indicated with blue diamonds and bold lines. As enhancers for most genes are currently unknown, the network assumes the simplest interactions depending on perturbation data (see also Fig. 6). Epi, epibranchial domain; OEPD, otic-epibranchial progenitor domain; OEPs, otic epibranchial progenitors; OP, otic placode; pEpi, pre-epibranchial domain.

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References

    1. Abelló G., Khatri S., Radosevic M., Scotting P. J., Giráldez F. and Alsina B. (2010). Independent regulation of Sox3 and Lmx1b by FGF and BMP signaling influences the neurogenic and non-neurogenic domains in the chick otic placode. Dev. Biol. 339, 166-178. 10.1016/j.ydbio.2009.12.027 - DOI - PubMed
    1. Abu-Elmagd M., Ishii Y., Cheung M., Rex M., Le Rouëdec D. and Scotting P. J. (2001). cSox3 expression and neurogenesis in the epibranchial placodes. Dev. Biol. 237, 258-269. 10.1006/dbio.2001.0378 - DOI - PubMed
    1. Adam J., Myat A., Le Roux I., Eddison M., Henrique D., Ish-Horowicz D. and Lewis J. (1998). Cell fate choices and the expression of Notch, Delta and Serrate homologues in the chick inner ear: parallels with Drosophila sense-organ development. Development 125, 4645-4654. - PubMed
    1. Adamska M., Herbrand H., Adamski M., Krüger M., Braun T. and Bober E. (2001). FGFs control the patterning of the inner ear but are not able to induce the full ear program. Mech. Dev. 109, 303-313. 10.1016/S0925-4773(01)00550-0 - DOI - PubMed
    1. Ahrens K. and Schlosser G. (2005). Tissues and signals involved in the induction of placodal Six1 expression in Xenopus laevis. Dev. Biol. 288, 40-59. 10.1016/j.ydbio.2005.07.022 - DOI - PubMed

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