The RNA-binding protein HuD regulates neuronal cell identity and maturation

Abstract

Neural Hu proteins (HuB/C/D) are RNA-binding proteins that have been shown to induce neuronal differentiation activity when overexpressed in immature neural progenitor cells or undifferentiated neuronal tumors. Newly generated HuD-deficient mice exhibited a transient impaired-cranial-nerve-development phenotype at an early embryonic stage. Adult HuD-deficient mice exhibited an abnormal hind-limb reflex and poor rotarod performance. Analysis of neurosphere formation revealed that the number and self-renewal capacity of the neural stem/progenitor cells were increased in HuD-deficient mice. HuD-deficient primary neurospheres also generated a smaller number of neurons. Cohort analysis of the cellular proliferative activity by using BrdUrd and iododeoxuridine labeling revealed that the number of differentiating quiescent cells in the embryonic cerebral wall was decreased. Long-term administration of BrdUrd revealed that the number of slowly dividing stem cells in the adult subventricular zone was increased in the HuD-deficient mice. Taken together, the results suggest that HuD is required at multiple points during neuronal development, including negative regulation of proliferative activity and neuronal cell-fate acquisition of neural stem/progenitor cells.

Fig. 1.

Targeting strategy, germ-line transmission, and expression analysis of the HuD gene. (A) Organization of the target vector, the mouse HuD gene, and the allele resulting from homologous recombination. An exon (black box) of the HuD allele containing the initiation codon was replaced with a PGK-neocassette. A 0.7-kb BamHI-EcoRI fragment (5′ probe) was used to screen for recombinant alleles, and the sizes of the recombinant and wild-type fragments after SacI digestion are shown (bidrectional arrows). (B) Germ-line transmission was confirmed by Southern blot analysis of SacI-digested tail DNA from a litter of F₁ mice, using a 5′ probe. (C) The deletion of the HuD mRNA from the adult brains of homozygous animals was confirmed by RT-PCR analysis. No amplified bands were observed from the RT samples of the HuD–/– brains, using either of the primer pairs (Exon 1-Exon 2 and Exon 2-Exon 3). (D) Immunoblots of adult brain or liver lysates with anti-HuD 16C12 monoclonal antibody (Upper) or anti-HuB/C/D serum (Lower). The bands indicated by a double arrowhead represent the HuD protein (Upper) and are missing in the liver lysates from the wild-type mice. The bands indicated by the double asterisk (Upper Left) represent nonspecific background immunoreactivity, which was also observed in negative control using wild-type liver lysates (Upper Right).

Fig. 2.

Histological analysis of the HuD-deficient mouse embryos. (A–D) Lateral views of E10.5 embryos of HuD–/– mice (A and C) and their wild-type littermates (B and D) stained with 2H3 antineurofilament-M antibody. In the wild-type embryos, axons of the glossopharyngeal nerve (IX) and hypoglossal nerve (XII) are seen extending from the caudal hindbrain and rostral spinal cord. On the other hand, the axons of these cranial nerves are not visualized in the HuD–/– embryos. Development of other cranial nerves, including the trigeminal (V) and acousticofacial (VII/VIII) nerves, also seems to be impaired in the HuD-deficient embryos as compared with that in their wild-type littermates. (E and F) The hind-feet-clenching phenotype in adult HuD–/– mice at 24 weeks of age. The HuD–/– mice displayed hind-feet-clenching behavior when picked up by the tail from 4 to 8 weeks of age (E). In wild-type littermates, however, the angles of the hind feet were close to a right angle (F). (G) Rotarod analysis. HuD–/– and their wild-type littermates at 20–26 weeks of age were used for the analysis. In HuD–/– mice, improvement of retention time by learning could scarcely be seen. The retention time was significantly shorter in the HuD–/– mice than in the wild-type littermates in the fifth and sixth trials.

Fig. 3.

Examination of neurosphere formation and differentiation in HuD-deficient embryos. (A) The number of neurospheres generated from 12,500 cells derived from the ganglionic eminence (GE) and cerebral cortex (Cortex) of E14.5 embryos, in 500 μl of the proliferation medium. (B) The number of secondary spheres derived from a single dissociated primary neurosphere is shown. (C) After allowing 72 h of differentiation of the primary neurospheres, the cell sheets generated from the neurospheres were fixed and triple-immunostained by using anti-TuJ1 (red), GFAP (blue), and O4 (green) antibodies. (Bar, 300 μm.) (D) The differentiation capacity of each primary neurosphere was determined based on the cell types contained in each clone. The clone types were analyzed for 100 spheres from each genotype.

Fig. 4.

Cohort analysis of cellular proliferation in the embryonic cerebral wall. (A and B) The distributions of the cells in the P + Q(A) or Q(B) fractions of the E14 HuD–/– and wild-type embryos. The analysis was undertaken in a coronal sector of the dorsomedial cerebral wall, ≈100 μm thick in its medial–lateral dimension and 4 μm thick (corresponding to the section thickness) in its rostral–caudal dimension. The sector was divided in its radial dimension into bins (x axis) 10 μm in height and numbered 1, 2, 3, and so on, from the ventricular margin. The numbers of cells in the Q or P + Q fraction [N(Q) or N(P+Q)] for each bin (y axis) were determined by counting only cells labeled by IdUrd in each protocol. IZ, intermediate zone; VZ, ventricular zone; and CS, cortical surface. (C) Total number of N(P + Q) and N(Q) cells in 5–16 bins is shown. Q fraction is calculated as N(Q)/N(P + Q). (D) Apoptotic cells in 4-μm-thick paraffin-embedded coronal sections of HuD–/– and wild-type embryos were detected by using the TUNEL method. The number of TUNEL-positive cells present along the lateral ventricular wall in the medial cerebral wall was counted. The apoptotic cells were then classified according to their location, based on the histological structure of the sections (IZ, intermediate zone; and CP, cortical plate).

Fig. 5.

Slowly dividing cells in the SVZ of adult HuD-deficient mice. (A and B) BrdUrd-labeled cells in the SVZ of HuD–/– mice (B) and their wild-type littermates were detected by immunostaining by using anti-BrdUrd monoclonal antibody. BrdUrd-positive cells are indicated by arrows. (Bar, 100 μm.) (C) The number of BrdUrd-positive cells in each of the frontal levels of the lateral SVZ in the animals was calculated. Three different frontal levels were analyzed. From rostral to caudal, slices were classified in three levels, SVZ1, SVZ2, and SVZ3 (also see Fig. 8).