DEV008524 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Microcephaly with preserved body mass in cyclin D2-null mice. (A) Stereomicrograph of an adult cD2^−/− brain next to a WT age-matched sibling shows the smaller cortex and cerebellum typical of cD2 nulls. (B) Significant, gene dose-dependent differences in surface area between WT and cD2^+/− or cD2^−/− cerebral cortex were found by tracing the perimeter of the cortex and calculating area using Metamorph software. (C) Body mass of gender-matched mice did not vary among genotypes. Their overall lengths were equivalent among WT, cD2^+/− or cD2^−/− genotypes.

• Supplemental Figure S2 -

  Fig. S2. Cortical thinning of superficial cortical lamina is similar in cD2^−/− and cD1^−/− adult brain. Nissl-stained paraffin-embedded sections. Stereological analysis revealed no difference in packing density of neurons between cD2^−/− and WT cortex barrel fields (total neurons/mm³), WT (n=5) versus cD2^−/− (n=5): layers II-III, 1.83×10⁵±2.3×10⁴ versus 1.86×10⁵±1.9×10⁴, P=0.9; layers IV-VI, 1.65×10⁵±2.1×10⁴ versus 1.57×10⁵±1.9×10⁴, P=0.8.

• Supplemental Figure S3 -

  Fig. S3. Reductions in parvalbumin (PV) immunoreactivity are observed in 10-μm cortical sections from adolescent and young adult mice lacking cD2, but not cD1. (A,B) Compared with wildtype (WT) (A) littermates, reductions in PV-immunoreactive neurons in the somatosensory cortex are pronounced in cD2^−/− mice (B) at postnatal day 25 (P25), a time at which the PV expression and numbers of PV-immunolabeled neurons are known to peak in WT mice. (C,D) Reduced numbers of PV-immunoreactive interneurons are evident in the somatosensory cortex of 6-week-old cD2^−/− mice (D) compared with WT littermate (C). (E,F) The density and distribution of PV-immunoreactive neurons is similar in WT (E) and cD1^−/− homozygous null (F) littermates. Therefore, loss of PV-immunolabeled neurons is specific to mice lacking the cD2 protein and appears attributable to a selective PV interneuron deficit. (G,H) The distribution of SSN-immunoreactive neurons is comparable in WT (G) and cD1^−/− (H) littermates. Both PV and SSN interneuron densities thus appear unaffected in cD1^−/− mice.

• Supplemental Figure S4 -

  Fig. S4. Interneuron deficits in cD2^−/− mice do not appear attributable to increased apoptotic cell death in the MGE. (A,B,E,F) At E12.5 (A,B) and E14.5 (E,F), TUNEL-positive nuclei were equally scant in WT and cD2^−/− MGE. (C,D,G,H) A second apoptotic cell marker, activated caspase 3, also failed to reveal enhanced apoptotic cell death at E12.5 (C,D) or E14.5 (G,H) in the MGE of mice lacking cD2, as compared to WT littermates. (I-P) Expression of activated caspase 3 in the cortices of cD2^+/+ and cD2^−/− littermates at E17 (I,J), P3 (K,L), P7 (M,N) and P14 (O,P). No differences in anti-activated caspase 3 staining were observed between genotypes at any age. This supports reduced proliferation as having a more substantive role than apoptosis in the resultant interneuron deficits in the cD2^−/− cortex.

• Supplemental Figure S5 -

  Fig. S5. The density of Gad67-eGFP neurons, which in the cortex only co-labels a subpopulation of parvalbuminergic (PV) interneurons, is consistently reduced in cD2-null as compared with wild-type neocortex. (A-D) Double-labeling for GFP (green) and PV (A), or somatostatin (SSN) (B), calbindin (CB) (C) or calretinin (CALR, red) (D) reveals that only a subgroup of PVergic interneurons exhibit GFP expression in this transgenic mouse model. (E) The numbers of GFP-immunoreactive neurons was stereologically analyzed in the barrel fields of the somatosensory cortex. The density of GFP-immunopositive neurons was calculated for superficial (I-III), deep (IV-VI) or all cortical layers (I-VI) and compared between genotypes and presented with reference to density measures ascertained for PV-immunolabeled interneurons measured (calculated using an identical stereological approach). Mean (±s.e.m.) densities of PV-IR (n=5-8 per genotype) or GFP-IR neurons (n=5 per genotype) per mm³ are plotted for null and WT. The cD2^+/+ versus cD2^−/− % change is provided above each bar set. The densities of GFP- and PV-immunolabeled cells were reduced to a similar degree in the neocortex.

• Supplemental Figure S6 -

  Fig. S6. Interneuron deficits in cD2<^b>⁻<^/b>^/<^b>⁻ mice are specific to the PV-immunoreactive subclass. Immunohistochemical labeling of 40-μm sections of the somatosensory cortex with antibodies against calbindin (CB) (A,B), calretinin (CALR) (C,D), neuropeptide Y (NPY) (E,F) and vasoactive intestinal peptide (VIP) (G,H) reveals no difference in the distribution or numbers of immunolabeled interneurons in adult WT (A,C,E,G) or cD2^−/− (B,D,F,H) littermates.

• Supplemental Figure S7 -

  Fig. S7. The cyclin-dependent kinase inhibitory p27Kip1 plays a more significant role than p57Kip2 in the developing GE. (A,B) The expression of p57 is not robust in the MGE at E12 (10× magnification) and is predominantly localized in cells found at the hypothalamic-GE transition area in cD2^+/+ (A) and cD2^−/− (B) brains. (C,D) At E14.5 (20× magnification), p57-immunoreactive neurons are not found in cD2^+/+ (C) or cD2^−/− (D) GE. Immunolabeling for p57 does not differ in the GE between genotypes. (E,F) At E14.5 (and earlier ages, data not shown), p57 immunoreactivity is robust in the septum (E) and hypothalamus (F). These data in the MGE support a more significant Cdk inhibitory role for p27, as compared with another KIP-family CdkI, p57.

• Supplemental Figure S8 -

  Fig. S8. The expression of cyclins D1 and D2 does not overlap with the post-mitotic neuronal marker NeuN and marginally overlaps with Tuj1-immunolabeled neuroblasts, but not Tuj1-labeled postmitotic neurons. (A-B′) The MGE at E14.5 was processed to label cD1 (A) or cD2 (B) immunoreactive neurons using an immunoperoxidase product and subsequently labeled for NeuN immunofluorescence (A′,B′). The immunoperoxidase photomicrographs, once inverted, pseudocolored green and presented with the NeuN overlay, document that the expression of either cyclin does not overlap with NeuN (overlay). (C-D′). Immunoperoxidase labeling for cD1 (C) or cD2 (D) immunoreactive neurons in the E14.5 MGE preceded subsequent Tuj1 immunofluorescent staining (C′,D′). The immunoperoxidase photomicrographs, once inverted, pseudocolored green and presented with the Tuj1 overlay, demonstrate that the expression of both cyclins marginally overlaps with Tuj1 labeling (overlay). Co-labeled cells are those closest to the SVZ-VZ border, which have the lowest levels of Tuj1 immunofluorescence, presumably indicative of their being partially differentiated neuroblasts and not post-mitotic neurons. The numbers of cD2-Tuj1 co-labeled cells exceed that of cD1-Tuj1 co-labeled cells (arrows). (E-G) The expression of NeuN supports that, in comparison with cD2^+/+ mice (E), the non-proliferative zones are increased in cD2^−/− (F) and somewhat reduced in cD1^−/− (G) MGE-mantle regions. (H-J) Tuj1 immunolabeling is enhanced in cD2^−/− (I) compared with cD2^+/+ (H) and cD1^−/− (J) MGE. NeuN and Tuj1 immunolabeling comparisons between mutant and wild-type mice are consistent with findings of increased cell cycle exit and proliferative defects in cD2-null MGE and potentially enhanced proliferation or cell cycle duration in cD1-null MGE.