DEV041129 Supplementary Material

Files in this Data Supplement:

• Supplemental Table S1 -

• Supplemental Table S2 -

• Supplemental Figure S1 -

  Fig. S1. FoxJ1^EGFP expression in the hippocampus. FoxJ1^EGFP expression in the hippocampus is mostly in a subset of Gfap⁺ astrocytes (red) in the dentate gyrus. pGfap, polyclonal Gfap antibody; mGfap, monoclonal Gfap antibody.

• Supplemental Figure S2 -

  Figure S2. FoxJ1^EGFP expression in the embryonic brain. (A-D) In wholemount embryos, FoxJ1^EGFP expression is visible in the primordial cerebellum and choroid plexus (A, arrowhead). Expression was also found in periventricular structures of the caudal midline, including the rostral-most rhombomeres, the ventricular aspect of the Isthmus organizer and the caudal-most prosomeres (A, arrow). FoxJ1^EGFP is heavily expressed around the midline in caudal coronal sections stained for the mitotic marker PH3 that reveals the ventricular zone (B). A rostral coronal section reveals FoxJ1^EGFP expression at the lateral base of the developing forebrain (asterisks). Here, FoxJ1^EGFP is expressed in clusters of radially oriented cells resembling radial glia (D). (E) A sagittal section of an E14 brain reveals region-specific FoxJ1^EGFP expression in the choroid plexus (CP), the lateral ganglionic eminence (LGE), primordial rostral migratory stream (RMS), and the olfactory bulb (OB). (F) Coronal section at the same age reveals additional expression in the septum (Sept), but not in the medial ganglionic eminence (MGE, labeled with MGE-specific marker, Lhx6). (G) FoxJ1^EGFP expression in the dorsolateral aspect of the LGE overlaps with Blbp⁺ radial glia (red) in both the ventricular (VZ) and the subventricular (SVZ) zones. (H) The dorsolateral LGE contains progenitor domains that express Gsh2, Dlx2 and Pax6. Bottom panels are high-magnification images of their respective top panels showing the overlap with FoxJ1^EGFP cells in the VZ and SVZ of the LGE. Only a few Dlx2⁺ progenitors co-express FoxJ1^EGFP in the dorsolateral SVZ of the LGE (arrow).

• Supplemental Figure S3 -

  Figure S3. Volumetric changes in the FoxJ^−/− forebrain. Volumetric estimates in serially reconstructed tissues show a reduction in volume of structures that depend on postnatal neurogenesis in FoxJ1^-/- brains. OB, olfactory bulb; CBL, cerebellum; Hipp CA, CA fields of the hippocampus; Hipp Den, dentate gyrus of the hippocampus; CTX, cortex; DIEN, diencephalon.

• Supplemental Figure S4 -

  Figure S4. FoxJ1 is not required for prenatal patterning of the LGE. (A-C) Patterning of progenitor domains in the dorsal LGE is mostly preserved in the absence of FoxJ1 expression during embryogenesis. Subtle density alterations are noted in the population of Gsh2⁺ cells (A, arrow) and ectopic presence of Pax6⁺ clusters in the FoxJ1^−/− dorsal LGE (C, arrowhead). (D) Transmission electron micrographs (TEM) of the surface of the wild-type and FoxJ1^−/− LGE reveal normal presence of primary cilia. Cross-sections through the cilia reveal 9+0 axonemes. (E-F) Acute administration of BrdU did not reveal any significant difference in proliferation of Pax6⁺ cells in the E14 LGE or olfactory ventricles. BrdU⁺ cell density in F equals the number of cells × 10⁴/mm³. Data are expressed as mean±s.e.m.; n=6; asterisk, P<0.01, Student’s t-test.

• Supplemental Figure S5 -

  Figure S5. FoxJ1 is required for postnatal expression of ependymal cells in the SVZ. Despite the relatively normal embryonic patterning, S100β⁺ cells fail to occupy the P6 ventricular walls, suggesting that ependymal cell differentiation fails to progress in the FoxJ1^−/− brain.

• Supplemental Figure S6 -

  Figure S6. Ultrastructural characterization of the FoxJ1^−/− SCN. (A) TEM micrographs reveal the presence of primary cilia in FoxJ1^−/− SVZ (red arrowheads). (B) Junctional complexes are present near the ventricles in the FoxJ1^−/− SVZ (red arrows). Scale bar: 500nm.

• Supplemental Figure S7 -

  Figure S7. shRNA knockdown of FoxJ1 blocks ependymal cell differentiation. (A) To identify effective shRNA sequences for knockdown of FoxJ1 in vivo, unique target sequences (#60-64 located at 423-445 bp, 721-743 bp, 1323-1345 bp, 1989-2011 bp and 2489-2511 bp, respectively, of the FoxJ1 coding region) were subcloned into the pCYLH vector with an internal ribosomal entry site (IRES) upstream of EGFP reporter. The vectors were transfected into immortalized human bronchial epithelial cells (HBE1) with endogenous FoxJ1 expression. Cells were incubated for 24 hours, 48 hours and 10 days. Three of the five constructs (#62, #63 and #64) resulted in varying knockdown levels of total FoxJ1 protein in the transfected cells (actin, green is blotted for the control). As control, a scrambled version of sequence #62 (#62 Scram) did not result in FoxJ1 knockdown. (B) To limit the knockdown quantification to levels of newly synthesized FoxJ1, we used proline-C14 labeling in shRNA-transfected cells. HBE1 cells were incubated with proline-C14 for 3 hours, washed and allowed to incubate for another 24 hours. Cells were then lysed and endogenous FoxJ1 was immunoprecipitated using a monoclonal FoxJ1 antibody. Immunoprecipitated FoxJ1 protein was separated on an SDS/PAGE gel, and C14-labeled FoxJ1 was detected directly in gels and quantified by means of electronic autoradiography (black and white blot). Of the shRNA constructs, expression of #62 resulted in greater than 95% knockdown in newly synthesized FoxJ1 after 24 hours of incubation and was used for the in vivo experiments. (C-E) Lentiviral-mediated expression of a FoxJ1-specific shRNA sequence (shFoxJ1) prevented expression of S100β and CD133 in ventricular zone ependymal cells (C). Aggregates of γ-tubulin were found in shFoxJ1 cells (D, arrowheads). Data shown in the bar chart (E) are mean6s.e.m.; asterisks, P<0.01, Student’s t-test.

• Supplemental Figure S8 -

  Figure S8. Differentiation of ependymal cells and FoxJ1⁺ astrocytes in the SCN is independent of hydrocephaly. The numbering of panels in P6 under ‘FoxJ1^+/+’ is consistent with all other panels: 1, low-magnification coronal view of the brain at the level of the SVZ; 2, S100β⁺ (red) and mGfap⁺ (green) cells in the SVZ; 3, high-magnification view of panel 2. Comparable degree of hydrocephaly (asterisks) in mice missing the intermediate chain of axonemal dynein protein in their motile cilia (Dnaic1^−/−) and kaolin-injected brains. S100β⁺ ependymal cells (red) and mGfap⁺ astrocytes (green) differentiate in the SVZ of wild-type, Dnaic1^−/− and kaolin-induced brains, but are disrupted in the FoxJ1^−/− SVZ. Blue, Nissl stain.

• Supplemental Figure S9 -

  Figure S9. Microtubule-associated proteins with a significant decrease in expression in the FoxJ1^−/− SCN, are expressed in the ependymal layer of the adult wild-type SCN. Images of brain sections were obtained from the Allen Brain Atlas (http://mouse.brain-map.org/), and the SVZ region was cropped in Adobe Photoshop (red box in top cartoon). Blue/brown, presence of mRNA for the labeled genes.