Figures and data in Ciliary Hedgehog signaling regulates cell survival to build the facial midline

Version of Record: November 15, 2021
Accepted Manuscript: October 21, 2021

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Shaun R Abrams

Jeremy F Reiter

(2021)
Ciliary Hedgehog signaling regulates cell survival to build the facial midline

eLife 10:e68558.

https://doi.org/10.7554/eLife.68558
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Figures
Tables
Additional files

6 figures, 1 table and 2 additional files

Figures

Figure 1 with 2 supplements

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The ciliary Meckel syndrome (MKS) transition zone complex is essential for midline facial development.

Frontal view images of *Tctn2* (A), *Cc2d2a* (B), *Tmem231* (C) wild-type and null embryos at embryonic day (E)10.5 and E11.5. *Tmem67* null embryos (D) display normal midface width at E11.5. Quantification …

Figure 1—figure supplement 1

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The ciliary Meckel syndrome (MKS) transition zone complex is essential for midline facial development.

Reproduced frontal view images from Figure 1 of *Tctn2* (A), *Cc2d2a* (B), *Tmem231* (C), wild-type and null embryos at embryonic day (E)10.5 and E11.5 with the addition of heterozygous embryos. (D) *Tmem67*…

Figure 1—figure supplement 2

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Removing TCTN2 in the neural crest does not result in hypotelorism.

(A) Frontal view images of embryonic day (E)11.5 *Tctn2* control (*Tctn2*^lox/-) and neural crest deletion (*Wnt1*^Cre;*Tctn2*^fl/-) embryos with corresponding midface width quantification. (B) Brightfield and …

Figure 2

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*Tctn2* mutants exhibit defects in prechordal plate differentiation soon after gastrulation.

(A) Whole mount in situ hybridization (WM-ISH) of embryonic day (E)8.0 embryos for axial mesendoderm markers *Shh* and *Brachyury* (T). (B) WM-ISH of E8.0 embryos for prechordal plate marker *Goosecoid* (G…

Figure 3

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*Tctn2* mutants display decreased Hedgehog (HH) signaling in the ventral telencephalon.

Whole mount in situ hybridization (WM-ISH) for *Shh* in *Tctn2* control and mutant embryos at embryonic day (E)8.75 and E9.5 (A) show reduced expression in the ventral telencephalons (arrowheads) of …

Figure 4 with 2 supplements

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TCTN2 protects the neurectoderm and facial ectoderm from apoptosis.

Immunostaining for proliferation marker phospho-histone H3 (pHH3) in transverse sections of *Tctn2* control and mutant embryonic day (E9.5) embryos (A) with corresponding quantification (B). (C) TUNEL …

Figure 4—figure supplement 1

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Deletion of *Tctn2* in the prechordal plate by *Isl1*^Cre, neurectoderm by *Sox1*^Cre, facial ectoderm by *Tcfap2a*^Cre, or forebrain and facial ectoderm by *Foxg1*^Cre does not result in hypotelorism.

(A) Analysis of *Isl1*^Cre recombination pattern at E8.5 and E11.5 using the *R26*^mTmG reporter shows robust recombination in the prechordal plate and endodermal derivatives at respective timepoints. (B) …

Figure 4—figure supplement 2

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Residual TCTN2 function through persistent ARL13B ciliary localization in TCTN2 conditional mutants.

(A) Analysis of *Tctn2* deletion in the prechordal plate of embryonic day (E)8.5 *Isl1*^Cre;*Tctn2*^fl/- embryos through ciliary localization of ARL13B indicates residual TCTN2 function remains at this …

Figure 5

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Reducing *Ptch1* gene dosage rescues the facial midline defect in transition zone mutants.

(A) Frontal images of *Tctn2* wild-type, mutant, and *Ptch1*^+/- rescue embryonic day (E)11.5 embryos with corresponding midline width quantification (B). (C) Frontal images of *Cc2d2a* wild-type, mutant, …

Figure 6

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Model for transition zone coordination of facial midline development.

(A) In wild-type embryos, the transition zone complex mediates signaling in the prechordal plate and Hedgehog (HH) pathway activation in the adjacent neurectoderm to at allow for cell survival and …

Tables

Key resources table

Reagent type (species) or resource	Designation	Source or reference	Identifiers	Additional information
Antibody	Anti-Arl13b (rabbit polyclonal)	Proteintech	17711–1-AP	(1:1000)
Antibody	Anti-cleaved-caspase-3 (Asp175) (rabbit polyclonal)	Cell Signaling	#9664	(1:400)
Antibody	Anti-cleaved-caspase-9 (Asp353)	Cell Signaling	#9509	(1:100)
Antibody	Anti-phospho-histone H3 (Ser28) (rabbit polyclonal)	Cell Signaling	#9713	(1:400)
Antibody	Anti-acetylated tubulin (mouse monoclonal)	Sigma-Aldrich	T6793	(1:1000)
Antibody	Anti-FoxA2 (rabbit monoclonal)	abcam	Ab108422	(1:400)
Antibody	Anti-Gamma tubulin (goat polyclonal)	Santa Cruz Biotechnology	Sc-7396	(1:200)
Antibody	Anti-GFP (chicken polyclonal)	Aves Labs	GFP-1020	(1:1000)
Antibody	Anti-E-cadherin (rat monoclonal)	Invitrogen	13–1900	(1:1000)
Commercial assay or kit	RNAeasy Micro Kit	QIAGEN	74004
Commercial assay or kit	In Situ Cell Death Detection Kit	Roche	11684795910
Commercial assay or kit	iScript cDNA synthesis kit	BioRad	1708891
Genetic reagent (Mus musculus)	Tctn2^tm1.1Reit	PMID:21565611	MGI:5292130;RRID:MGI:5292219
Genetic reagent (Mus musculus)	Cc2d2a^{Gt(AA0274)Wtsi}	PMID:21725307	MGI:4344514;RRID:MGI:5292228
Genetic reagent (Mus musculus)	Tmem231^{Gt(OST335874)Lex}	PMID:22179047	MGI:4284576;RRID:MGI:5301844
Genetic reagent (Mus musculus)	Tmem67^tm1Dgen	PMID:21725307	MGI:5292220;RRID:MGI:5292226
Genetic reagent (Mus musculus)	Wnt1^Cre: H2az2Tg^{(Wnt1-cre)11Rth}	PMID:9843687	MGI:2386570;RRIDSupplemental:IMSR_JAX:003829	Gift from Brian Black
Genetic reagent (Mus musculus)	Isl1^tm1(cre)Sev	PMID:16556916	MGI:3623159;RRID:IMSR_HAR:3351	Gift from Brian Black
Genetic reagent (Mus musculus)	Sox1^tm1(cre)Take	PMID:17604725	MGI:3807952;RRID:IMSR_RBRC05065	Gift from Jeff Bush
Genetic reagent (Mus musculus)	Foxg1^tm1(cre)Skm	PMID:10837119	MGI:1932522;RRID:IMSR_JAX:004337	Gift from Stavros Lomvardas
Genetic reagent (Mus musculus)	Tg(Tcfap2a-cre)^1Will	PMID:21087601	MGI:4887352;RRID:MGI:4887452	Gift from Trevor Williams
Sequence-based reagent	Shh in situ probe	PMID:7916661		Gift from Andrew McMahon
Sequence-based reagent	Gsc in situ probe	PMID:1352187		Gift from Edward De Robertis
Sequence-based reagent	Ptch1 in situ probe	PMID:10395791		Gift from Lisa Goodrich
Sequence-based reagent	RT-qPCR primers	This paper		*See Supplementary file 1
Sequence-based reagent	Genotyping primers	This paper		*See Supplementary file 1
Software, algorithm	GraphPad Prism	GraphPad Prism (https://graphpad.com)	RRID:SCR_002798
Software, algorithm	ImageJ	ImageJ (http://imagej.nih.gov/ij/)	RRID:SCR_003070

Additional files

Supplementary file 1 Primer sets used for genotyping and quantitative real-time polymerase chain reaction (RT-qPCR). File contains all sequencing primer pairs used for genotyping of all mouse strains used in this study. In addition, file contains primer sets used for RT-qPCR experiments. Inquiries regarding primers should be sent to Jeremy Reiter.: https://cdn.elifesciences.org/articles/68558/elife-68558-supp1-v2.pdf
Download elife-68558-supp1-v2.pdf
Transparent reporting form: https://cdn.elifesciences.org/articles/68558/elife-68558-transrepform1-v2.docx
Download elife-68558-transrepform1-v2.docx

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Figures

The ciliary Meckel syndrome (MKS) transition zone complex is essential for midline facial development.

The ciliary Meckel syndrome (MKS) transition zone complex is essential for midline facial development.

Removing TCTN2 in the neural crest does not result in hypotelorism.

Tctn2 mutants exhibit defects in prechordal plate differentiation soon after gastrulation.

Tctn2 mutants display decreased Hedgehog (HH) signaling in the ventral telencephalon.

TCTN2 protects the neurectoderm and facial ectoderm from apoptosis.

Deletion of Tctn2 in the prechordal plate by Isl1^Cre, neurectoderm by Sox1^Cre, facial ectoderm by Tcfap2a^Cre, or forebrain and facial ectoderm by Foxg1^Cre does not result in hypotelorism.

Residual TCTN2 function through persistent ARL13B ciliary localization in TCTN2 conditional mutants.

Reducing Ptch1 gene dosage rescues the facial midline defect in transition zone mutants.

Model for transition zone coordination of facial midline development.

Tables

Additional files

Supplementary file 1

Transparent reporting form

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Cite this article

The ciliary Meckel syndrome (MKS) transition zone complex is essential for midline facial development.

The ciliary Meckel syndrome (MKS) transition zone complex is essential for midline facial development.

Removing TCTN2 in the neural crest does not result in hypotelorism.

Tctn2 mutants exhibit defects in prechordal plate differentiation soon after gastrulation.

Tctn2 mutants display decreased Hedgehog (HH) signaling in the ventral telencephalon.

TCTN2 protects the neurectoderm and facial ectoderm from apoptosis.

Deletion of Tctn2 in the prechordal plate by Isl1Cre, neurectoderm by Sox1Cre, facial ectoderm by Tcfap2aCre, or forebrain and facial ectoderm by Foxg1Cre does not result in hypotelorism.

Residual TCTN2 function through persistent ARL13B ciliary localization in TCTN2 conditional mutants.

Reducing Ptch1 gene dosage rescues the facial midline defect in transition zone mutants.

Model for transition zone coordination of facial midline development.

Supplementary file 1

Transparent reporting form

Deletion of Tctn2 in the prechordal plate by Isl1^Cre, neurectoderm by Sox1^Cre, facial ectoderm by Tcfap2a^Cre, or forebrain and facial ectoderm by Foxg1^Cre does not result in hypotelorism.