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. 2017 Mar 14;12(3):e0173258.
doi: 10.1371/journal.pone.0173258. eCollection 2017.

Unique spatiotemporal requirements for intraflagellar transport genes during forebrain development

John Snedeker  1 , Elizabeth N Schock  2   3 , Jamie N Struve  2   3 , Ching-Fang Chang  2   3 , Megan Cionni  1 , Pamela V Tran  4 , Samantha A Brugmann  2   3 , Rolf W Stottmann  1   2
Affiliations

Unique spatiotemporal requirements for intraflagellar transport genes during forebrain development

John Snedeker et al. PLoS One. .

Abstract

Primary cilia are organelles extended from virtually all cells and are required for the proper regulation of a number of canonical developmental pathways. The role in cortical development of proteins important for ciliary form and function is a relatively understudied area. Here we have taken a genetic approach to define the role in forebrain development of three intraflagellar transport proteins known to be important for primary cilia function. We have genetically ablated Kif3a, Ift88, and Ttc21b in a series of specific spatiotemporal domains. The resulting phenotypes allow us to draw several conclusions. First, we conclude that the Ttc21b cortical phenotype is not due to the activity of Ttc21b within the brain itself. Secondly, some of the most striking phenotypes are from ablations in the neural crest cells and the adjacent surface ectoderm indicating that cilia transduce critical tissue-tissue interactions in the developing embryonic head. Finally, we note striking differences in phenotypes from ablations only one embryonic day apart, indicating very discrete spatiotemporal requirements for these three genes in cortical development.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Recombination pattern of Cre transgenics used to ablate cilia genes.
The pattern of Cre-mediated recombination with the R26R-lacZ reporter is shown for all lines used. (A-D) EIIa-Cre is expressed at high levels throughout the embryo with some mosaicism, including the entire nervous system at early organogenesis stages (D). (E-H) Foxg1-Cre is highly expressed in the developing telencephalon from the earliest stages of formation but not in the overlying surface ectoderm (H). (I-L) Emx1-Cre is specific to the dorsal telencephalon with recombination evident between 31 and 34 somite (~E10.5). Note the later onset and more specific recombination as compared to Foxg1-Cre. (M-N) Wnt1-Cre activity is seen in the midbrain and dorsal midline of the neural tube and in the emerging neural crest cells populating the craniofacial tissues (N,O). Note expression is not seen in the telencephalon (P). Ap2-Cre recombination is detected in the dorsal midline and neural crest like Wnt1-Cre, but also in the dorsal telencephalon (T). (t = telencephalon)
Fig 2
Fig 2. Deletion of Ttc21b from solely the developing forebrain does not lead to cortical malformation.
(A-N) Emx1-Cre was used to delete a conditional allele of Ttc21b but does not lead to morphological (A,B,G,H,I,J), histological (C,D,K,L) or neural differentiation (immunohistochemistry for TuJI in E,F) phenotypes. Control and Emx1-Cre; Ttc21bflox/aln embryos are shown at E14.5 (A-F) and E18.5 (G-N).. Foxg1-Cre deletions also do not cause cortical phenotypes at E14.5 (O-T) or E18.5 (U-Z). Cre recombination patterns for each genotype are shown with the ROSAdTom/EGFP reporter allele (M,N,AA,BB). All paired images are at the same magnification. (t = telencephalon) (CC) Quantification for brain sizes are normalized to control for each respective experiment. Center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, outliers are represented by dots; data points are plotted as open circles. n = 11, 4, 4, 3, 4, 4, 6, 3, respectively. Grey = wt, Red = mut
Fig 3
Fig 3. Deletion of Ttc21b with EIIa-Cre phenocopies homozygous Ttc21baln/aln embryos.
Genetic ablation of Ttc21b with the EIIa-Cre creates microcephalic brains (D,G) which are similar to that seen in homozygous null embryos (L,M). The ROSAdTom/EGFP reporter allele shows the mosaic nature of some EIIa-Cre embryos where EGFP expression (B,E,H) marks recombined tissue and dTom expression (C,F,I) indicates tissue without Cre activity. All paired images are at the same magnification. (t = telencephalon)
Fig 4
Fig 4. Expression of Ttc21b.
Ttc21blacZ expression at E6.5-E10.5. Frontal views are shown in B,D,F,H.
Fig 5
Fig 5. Deletion of Ttc21b from neural crest cells and surface ectoderm.
(A-L) Wnt1-Cre mediated deletion of Ttc21b does not lead to morphological (A,B,G,H,I,J), histological (C,D,K,L) or neural differentiation (E,F) phenotypes in the forebrain at E14.5 (A-F) or E18.5 (G-L). The midbrain is enlarged at E18.5 (double arrowheads in J). (M-X) Ap2-Cre; Ttc21bflox/aln embryos at E14.5 and E18.5 have an enlarged forebrain (arrowhead in N, V) with disrupted cortical architecture (P,X) and reduced numbers of differentiated neurons (R). Loss of olfactory bulbs is also noted at E18.5 (asterisk in V). All paired images are at the same magnification. (Y) Quantification for brain sizes. Center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, outliers are represented by dots; data points are plotted as open circles. n = 6, 3, 5, 12, 5, 3, 3, 5, respectively. (**: p <0.005).
Fig 6
Fig 6. Deletion of Kif3a in early stages of forebrain development leads to increased brain size.
(A-L) Foxg1-Cre was used to delete a conditional allele of Kif3a and the forebrain was enlarged at E14.5 (B) and E18.5 (H,J,). Fewer differentiated neurons are noted in medial regions at E14.5 (D,F, arrows show specific areas of decreased TuJI). Olfactory bulbs are absent at E18.5 in mutants (asterisks in J). (M-X) Similar enlargements are seen with Emx1-Cre ablation but the effects are much less severe. All paired images are at the same magnification. (Y) Quantification for brain sizes. Center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, outliers are represented by dots; data points are plotted as open circles. n = 12, 9, 12, 7, 9, 7, 12, 4, respectively. (*:p<0.05, **:p<0.005)
Fig 7
Fig 7. Deletion of Kif3a from neural crest and surface causes cortical malformation.
(A-L) Wnt1-Cre mediated deletion of Kif3a causes morphological (A,B,G,H,I,J), and histological (C,D,K,L) phenotypes in the forebrain at E14.5 (A-F) and E18.5 (G-L). The third ventricle is enlarged at E14.5 (arrow indicates widened base of ventricle in D). (M-X) Ap2-Cre; Kif3aflox/flox embryos at E14.5 and E18.5 have a profoundly enlarged forebrain (N, V) with disrupted cortical architecture (P,R,X). All paired images are at the same magnification. (Y) Quantification for brain sizes. Center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, outliers are represented by dots; data points are plotted as open circles. n = 6, 6, 8, 3, 5, 4, 6, 5, respectively. (*:p<0.05, **:p<0.0005)
Fig 8
Fig 8. Deletion of Ift88 in early stages of forebrain development leads to increased brain size.
(A-L) Foxg1-Cre was used to delete a conditional allele of Ift88 and the forebrain was enlarged at E14.5 (B) and E18.5 (H,J,). Cortical architecture is disrupted at all stages examined (D,L) and fewer differentiated neurons are seen at E14.5 (F). (M-R) Similar, but less severe, phenotypes are seen with Emx1-Cre ablation at E14.5. (S-X) E18.5 mutants appear phenotypically normal. All paired images are at the same magnification. (Y) Quantification for brain sizes. Center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, outliers are represented by dots; data points are plotted as open circles. n = 12, 6, 7, 5, 7, 4, 13, 4, respectively. (*:p = 0.011, **:p<0.005).
Fig 9
Fig 9. Deletion of Ift88 from neural crest and surface causes cortical malformation.
(A-L) Wnt1-Cre mediated deletion of Ift88 causes morphological (A,B,G,H,I,J), and histological (C,D,K,L) phenotypes in the forebrain at E14.5 (A-F) and E18.5 (G-L). The third ventricle is enlarged at E14.5 (arrow indicates widened base of ventricle in D) and cleft at E18.5 (L). (M-X) Ap2-Cre; Ift88flox/flox embryos at E14.5 and E18.5 have a profoundly enlarged forebrain (N, V) with disrupted cortical architecture (P,R,X). All paired images are at the same magnification. (Y) Quantification for brain sizes. Center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, outliers are represented by dots; data points are plotted as open circles. n = 7, 4, 6, 6, 8, 4, 4, 4, respectively. (*:p = 0.013, **:p<0.002)
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