The mechanisms that determine whether a precursor cell re-enters the cell cycle or exits and differentiates are crucial in determining the types and numbers of cells that constitute a particular organ. Here, we report that Id4 is required for normal brain size, and regulates lateral expansion of the proliferative zone in the developing cortex and hippocampus. In its absence, proliferation of stem cells in the ventricular zone (VZ) is compromised. In early cortical progenitors, Id4 is required for the normal G1-S transition. By contrast, at later ages, ectopically positioned proliferating cells are found in the mantle zone of the Id4-/- cortex. These observations, together with evidence for the premature differentiation of early cortical stem cells, indicate that Id4 has a unique and complex function in regulating neural stem cell proliferation and differentiation.

Introduction

The increased cortical surface area of the human brain is associated with its ability to perform higher order cognitive functions, and hence understanding the molecular mechanisms that regulate cortical progenitor proliferation and differentiation is of great importance. The mammalian telencephalon develops from the rostral-most region of the neural plate. The dorsal part of the telencephalon gives rise to the hippocampus, neocortex,lateral cortex, and parts of the amygdala, along with other smaller structures. These different structures arise from distinguishable progenitor regions in the ventricular zone (VZ) called the medial pallium (MP), dorsal pallium (DP), lateral pallium (LP) and ventral pallium (VP), which are identifiable by combinatorial patterns of homeobox gene expression(Puelles et al., 2000; Yun et al., 2001). From within each zone, there is a sequential generation of neurons and glia in the telencephalon, such that neurogenesis precedes gliogenesis. Most neurons are generated from the proliferative zone between E11.5 and E17.5, whereas glial cells are generated during early postnatal days (P0-P20)(Sauvageot and Stiles,2002).

During early telencephalic development, there is an exponential expansion of neuroepithelial progenitors in the VZ as the result of rapid, symmetric cell divisions. The nuclei of these neuroepithelial cells go through characteristic movements within the VZ, known as the interkinetic nuclear movements. M-phase nuclei are found at the ventricular surface, while S-phase nuclei are found away from the ventricle in the distal region of the VZ(Takahashi et al., 1993; Takahashi et al., 1995a). These early VZ cells (E11.5-E12.5) divide continuously – their cell cycle time is estimated to be about 8 hours(Takahashi et al., 1993; Takahashi et al., 1992)– leading to rapid lateral expansion of the neuroepithelium. From the VZ emerge the cells that give rise to the secondary proliferative population(SPP) in the subventricular zone (SVZ)(Takahashi et al., 1995b) and differentiating neurons of the telencephalon.

Many genes known to affect proliferation and differentiation of the developing mammalian nervous system are transcription factors. In particular,members of the basic helix-loop-helix (bHLH) family have received much attention because of the potency with which they are able to instruct cellular differentiation (Guillemot,1999). Consistent with this role in cell fate determination and differentiation, many bHLH genes are expressed in a tissue-specific manner(class B bHLH proteins). A sub-class of HLH genes, which lack the basic DNA-binding domain, is known as Inhibitors of DNA binding or ID genes. The proteins encoded by these genes act as dominant-negative regulators of bHLH proteins by forming inactive heterodimeric complexes(Benezra et al., 1990; Norton, 2000; Yokota, 2001).

In mammals, there are four known ID gene family members. Id1, Id2,Id3 and Id4 are expressed in progenitors of the central nervous system (CNS) in overlapping but distinctive patterns(Andres-Barquin et al., 2000; Jen et al., 1997). Consistent with their expression patterns, the activities of ID1 and ID3 seem functionally redundant in the nervous system. Deletion of either Id1or Id3 individually does not lead to an observable phenotype, but when they are both inactivated, telencephalic neurogenesis and angiogenesis are greatly perturbed (Lyden et al.,1999). Mice lacking Id2 are viable, although they have an abnormal immune system and a lactation defect(Mori et al., 2000; Yokota et al., 1999). Id1,Id2 and Id3 have been shown to interact with cell cycle regulatory molecules (Zebedee and Hara,2001). For example, they negatively regulate the expression of cyclin D1, p16Ink4a and p21CIP1/WAF1/SDI1(Alani et al., 2001; Lasorella et al., 1996; Prabhu et al., 1997). In addition, both genetic and biochemical evidence indicates that ID2 inhibits RB(Retinoblastoma) protein function to enhance the G1 to S transition in proliferating cells (Iavarone et al.,1994; Toma et al.,2000).

The role of the most recently identified member of the ID family, Id4, in nervous system development has not been explored. In vitro studies suggest that ectopic expression of Id4 prevents oligodendrocytic differentiation (Kondo and Raff, 2000). However, its role in regulating neural stem cell proliferation and differentiation is not known. Here, we identify a role of Id4 during cortical development in vivo and reveal a novel function for Id4 in regulating cortical neurogenesis.

Materials and methods

Generation of Id4 mutant animals

A NcoI-HindIII fragment, including the EGFP cDNA from the pEGFP-N1 plasmid vector (Clontech) ligated to a 1.1 kbp HindIII-NcoI Id4-5′ flanking genomic fragment and a 4 kb NotI-PstI fragment(Id4-3′-flanking genomic fragment), was inserted into the neo-tk cassette (Qiu et al.,1995) and introduced into JM-1 ES cells by electroporation. Of the 24 clones examined, 4 clones were found to contain a 6.5 kb band corresponding to the mutant allele. PCR primers specific for the wild-type and targeted alleles that were used included:

  • Id4-wild-type sense, 5′-CTGCAGTGCGATATGAACG-3′;

  • Id4-wild-type antisense, 5′-GCGGCTGTCTCAGCAAAG-3′;

  • Id4-mutant sense, 5′-GTCGATTTCTGGAGCTCGGA-3′); and

  • Id4-mutant antisense, 5′-AACTTGTGGCCGTTTACGTC-3′.

The PCR cycling conditions were 95°C for 30 seconds, 60°C for 40 seconds and 72°C for 1 minute for 30 cycles. The amplified PCR products were analyzed on gels to separate the wild-type (166 bp) and targeted (220 bp)allele fragments.

Ventricular surface area measurements

Coronal and saggital sections of Id4+/– and Id4-/- tissues at matching levels of the CNS were stained with Cresyl Violet and compared by measuring the VZ surface area using the ImagePro program. Three separate measurements of the same sections were averaged and compared. In each age group, three separate littermate brain pairs, sectioned in coronal and saggital planes at similar angles and matched to comparable levels along the rostrocaudal and mediolateral axis were analyzed.

In situ hybridization

In situ hybridization on frozen sections was carried out using 35S-labeled antisense-riboprobes(Yun et al., 2001). Each probe was examined on three to five different mutant-control littermate pairs from at least two independent crosses at each age shown. Photographs were taken using dark-field optics on an Olympus SZH10 microscope. The cDNAs used in this study included: Wnt3a (A. McMahon, Harvard University, USA); Emx1 (J. Rubenstein, UCSF, USA); Dbx1 (T. Jessell, Colombia University, USA); Ngn2 and NeuroD (F. Guillemot, NIMR, UK); Tbr2 (A. Bulfone, TIGEM, Italy); and Id2 and Id4(M. Israel, Dartmouth Medical School, USA).

Immunofluorescence

Labeling of S-phase cells by BrdU incorporation was performed by injecting BrdU (1 mg/10 g body weight) into pregnant females and waiting for 45-60 minutes before sacrificing. A standard immunofluorescence protocol was used. Primary antibodies used in this study included: phosphorylated Histone 3 (PH3)(rabbit IgG; Upstate Biotech), BrdU (rat; Harlen), MAP2 (mouse IgG; Sigma,catalog number M1406), PCNA (mouse IgG; Novocastra), TBR1 (gift of M. Sheng,Harvard Medical School, USA). Confocal images of PCNA staining were collected as 2 μm optical slices.

FACS scan analysis

E12.5 dorsal telencephalon from Id4-/- and control littermates was dissected in cold PBS. Cells were dissociated in 0.05%trypsin, rinsed, and resuspended in 100 μl of PBS. Cells were fixed in 70%ethanol, rinsed, resuspended in 1 ml of solution containing 100 μg/ml RNaseA/0.2% Triton X-100/50 μg/ml Propidium Iodide, and incubated at 37°C for 30 minutes. Stained cells were scanned in a Becton Dickinson (San Jose, CA) FACScan flow cytometer, and the cell cycle profile was quantitated using ModFit software.

Results

Smaller brain size in Id4-/- animals

To understand the in vivo function of Id4, we used homologous recombination to generate a mutant mouse in which the Id4 locus was disrupted (Fig. 1A). Exons 1 and 2 of Id4 were replaced by the coding regions of GFP(green fluorescent protein) and the neomycin-resistance gene. Following transfection into embryonic stem cells, recombination events were confirmed by PCR and Southern blot analysis (Fig. 1A). Two independent lines of recombinant animals carrying an altered Id4 locus were identified. In both lines, animals lacking functional Id4 survived postnatally in clean environments, but their brain (Fig. 1B) size was consistently smaller than that of their littermates (not shown).

Fig. 1.

(A) The recombinant targeting construct used to generate a mutant mouse with a disrupted Id4 locus, and a Southern blot and PCR analysis demonstrating the successful targeting event. (B) E15.5 Id4 mutant brain (right) and a littermate wild-type control brain (left). (C,C′)Cresyl violet-stained coronal sections through E12.5 wild-type and mutant telencephalon. Measurements of neocortex and hippocampal primordium shown in Fig. 2 were made between the arrows shown. Also, the thickening of the neocortex in the mutant (C′)is indicated by a comparison of the length of the black bar (wild-type thickness) to the yellow bar (mutant thickness). Basal ganglia (LGE and MGE)of the mutant appear normal in size. (D,D′) E18.5 saggital sections of control (D) and Id4-/- (D′) telencephalon stained with cresyl violet. (E-H) Quantitation of the cortical VZ surface areas at E11.5, E12.5 and E15.5, in control and Id4-/- littermates at matched levels on coronal sections (E,F) and saggital sections (G,H),normalized to littermate controls. Yellow bar, Id4+/–; blue bar, Id4-/-; wt, wild type; mt, mutant; LGE, lateral ganglionic eminence; MGE, medial ganglionic eminence; hp, hippocampus; ctx, neocortex;OB, olfactory bulb; st, striatum; LV, lateral ventricle.

Fig. 1.

(A) The recombinant targeting construct used to generate a mutant mouse with a disrupted Id4 locus, and a Southern blot and PCR analysis demonstrating the successful targeting event. (B) E15.5 Id4 mutant brain (right) and a littermate wild-type control brain (left). (C,C′)Cresyl violet-stained coronal sections through E12.5 wild-type and mutant telencephalon. Measurements of neocortex and hippocampal primordium shown in Fig. 2 were made between the arrows shown. Also, the thickening of the neocortex in the mutant (C′)is indicated by a comparison of the length of the black bar (wild-type thickness) to the yellow bar (mutant thickness). Basal ganglia (LGE and MGE)of the mutant appear normal in size. (D,D′) E18.5 saggital sections of control (D) and Id4-/- (D′) telencephalon stained with cresyl violet. (E-H) Quantitation of the cortical VZ surface areas at E11.5, E12.5 and E15.5, in control and Id4-/- littermates at matched levels on coronal sections (E,F) and saggital sections (G,H),normalized to littermate controls. Yellow bar, Id4+/–; blue bar, Id4-/-; wt, wild type; mt, mutant; LGE, lateral ganglionic eminence; MGE, medial ganglionic eminence; hp, hippocampus; ctx, neocortex;OB, olfactory bulb; st, striatum; LV, lateral ventricle.

Id4 is widely expressed in the developing mouse embryo nervous system (Andres-Barquin et al.,2000; Jen et al.,1997). Because we found that animals lacking Id4 had smaller brains, we examined telencephalic development in the Id4mutant animal to better understand the physiological role of Id4. Although we did not observe a phenotype in the Id4 heterozygote, the reduced brain size shown in Fig. 1B is apparent in homozygous animals from E11.5 onwards. When littermate brains were sectioned and compared at E12.5(Fig. 1C,C′), the ventricular surface of the mutant telencephalon was found to be approximately 30% smaller than that of the wild type along the mediolateral axis (compare the distance between the arrows in Fig. 1C,C′,F). In particular, the dorsal and medial pallial regions, primordia for the neocortex and the hippocampus, respectively,appeared much smaller than the more ventral regions in both the cortical(lateral and ventral pallium) and subcortical (ganglionic eminences) tissues(Fig. 1C,C′,E,F). Saggital sections at E15.5 and E18.5 showed smaller lateral ventricles and reduced ventricular surface along the rostrocaudal axis as well(Fig. 1D,D′,G). Concurrently, the mutant cortical plates are consistently thicker than those of their littermates (Fig. 1D,D′,H). From this analysis, we conclude that Id4function is required for the development of a normal size brain, and especially for the lateral expansion of the cortical neuroepithelium.

Reduced dorsomedial progenitor zones in the Id4-/- mutant

To determine whether the smaller cortex in Id4-/-animals reflected the loss of a specific region or an overall reduction in all cortical regions, we examined the expression of molecular markers that distinguish different progenitor zones. Genes expressed at the dorsal and ventral extremes of the pallial (cortical) progenitor zone appeared normal. For example, Wnt3a expression in the cortical hem(Lee et al., 2000) was unchanged in mutant animals (Fig. 2A,A′). In addition, no consistent differences were found in the expression patterns of genes marking the ventral and lateral pallium progenitors (Dbx1, Emx1, Tbr2 and Wnt7b; Fig. 2B-D′, and not shown), or the mantle (not shown) (Puelles et al., 2000; Yun et al.,2001) in Id4-/- animals. To quantitate the difference in the surface area of the cortical proliferative zone, we measured the cortical VZ width on matched coronal sections from littermate control and Id4-/- animals. We found progressive shortening of the VZ surface area (Fig. 2G) and consistent reduction in the hippocampal primordium (approximately 50%, Fig. 2H) in Id4-/- animals. These findings, in the context of the histological analysis shown in Fig. 1, indicate that the hypoplasia of the Id4-/-telencephalon most severely affects dorsomedial structures.

Fig. 2.

E11.5 coronal sections through wild-type (A-F) and Id4-/- (A′-F′) telencephalon. Wnt3a, Dbx1,Emx1 and Tbr2 expression in LP and VP progenitor zones appears normal (A-D′). (A-B′) The limits of the Wnt3a and Dbx1 expression domains are indicated by the arrowheads. (C,C′) Arrowheads indicate mantle expression of Emx1 in the lateral pallium. (D′)Arrowhead indicates ectopic Tbr2-expressing cells in the VZ; arrow points to the increased number of Tbr2-expressing postmitotic cells in the dorsomedial mantle of the Id4-/- animal. (E)Endogenous Id2 expression shows a ventral-to-dorsal gradient of expression and is undetectable between the arrowheads. (E′) The boundaries of the reduced Id2-negative region in the Id4-/- brain is indicated by arrowheads. (F) Id4expression is uniformly high in all cortical areas. (F′) GFP expression from the Id4 locus is detectable in the Id4-/-brain (arrowhead). LGE, lateral ganglionic eminence; VP, ventral pallium; LP,lateral pallium; DP, dorsal pallium; MP, medial pallium; ctx, neocortex; LV,lateral ventricle.

Fig. 2.

E11.5 coronal sections through wild-type (A-F) and Id4-/- (A′-F′) telencephalon. Wnt3a, Dbx1,Emx1 and Tbr2 expression in LP and VP progenitor zones appears normal (A-D′). (A-B′) The limits of the Wnt3a and Dbx1 expression domains are indicated by the arrowheads. (C,C′) Arrowheads indicate mantle expression of Emx1 in the lateral pallium. (D′)Arrowhead indicates ectopic Tbr2-expressing cells in the VZ; arrow points to the increased number of Tbr2-expressing postmitotic cells in the dorsomedial mantle of the Id4-/- animal. (E)Endogenous Id2 expression shows a ventral-to-dorsal gradient of expression and is undetectable between the arrowheads. (E′) The boundaries of the reduced Id2-negative region in the Id4-/- brain is indicated by arrowheads. (F) Id4expression is uniformly high in all cortical areas. (F′) GFP expression from the Id4 locus is detectable in the Id4-/-brain (arrowhead). LGE, lateral ganglionic eminence; VP, ventral pallium; LP,lateral pallium; DP, dorsal pallium; MP, medial pallium; ctx, neocortex; LV,lateral ventricle.

As Id4 is highly expressed in all cortical progenitor zones(Fig. 2F), we were surprised to find these regional differences. We hypothesized that ID4 function is unique in the dorsomedial cortex and that other ID genes compensate for Id4loss in other regions of the telencephalon. Consistent with this possibility,we observed elevated levels of Id1, Id2 and Id3 mRNA in the Id4-/- telencephalon by RT-PCR (data not shown). Because Id2 is expressed in a lateral-to-medial gradient in the VZ of the cortex (levels are undetectable between the arrowheads in Fig. 2E), we used in situ hybridization to determine whether an altered pattern of Id2expression could be identified. We found that Id2 expression was increased beyond its normal dorsal boundary in the cortex extending all the way to the midline in Id4′ animals(Fig. 2E′). This observation raises the possibility that Id4 normally represses Id2 expression in the dorsomedial region, or that the hypoplastic region of the Id4-/- mouse cortex corresponds to areas in which Id2 is normally not expressed.

To determine whether the molecular abnormalities associated with the loss of Id4 also contributed to cortical hypoplasia by enhancing progenitor cell death, we examined apoptosis in the developing telencephalon of Id4-/- animals. We did not detect differences in the number of apoptotic cells in the Id4-/- and control telencephalon at E11.5, E12.5 or E15.5 (data not shown). Furthermore, the insertion of DNA encoding GFP into the Id4 locus allowed the identification of cells in which Id4 transcription would occur. GFP-expressing cells were clearly present in Id4-/-mutants at E12.5 (Fig. 2F′), indicating that the loss of Id4 function does not result in extensive cell death.

Precocious differentiation of early cortical progenitors

To determine whether the dorsomedial cortical progenitor zones are reduced as a result of precocious differentiation or compromised proliferation of neural stem cells, we first examined early neurogenesis in the mutant telencephalon. At E11.5, cortical neurogenesis is just beginning in the dorsal and medial pallium, whereas in the more ventral regions of the cortex, the ventral and lateral pallium, neurons are produced from E10.5. Expression of pan-neuronal markers at E11.5 (MAP2 and βIII-tubulin, Fig. 3A,A′, and not shown) was reproducibly increased throughout the postmitotic zone of the Id4-/- cortex, suggesting precocious neurogenesis. Therefore, we examined the expression of other genes that either regulate neurogenesis in cortical progenitors or are markers of cortical neurons. At E11.5, neurogenin 2 (Ngn2; Neurog2 – Mouse Genome Informatics), a proneural bHLH gene (Fode et al., 2000; Gradwohl et al.,1996), was expressed in a similar pattern in both wild type and Id4-/- animals. However, the expression of markers identifying early differentiating neurons, NeuroD(Fig. 3C,C′) and Tbr2 (arrow in Fig. 2D,D′), was consistently increased in the mantle zone of the dorsomedial cortex in Id4-/- animals. Furthermore,scattered Tbr2-expressing cells were found ectopically in the VZ(dorsal to its normal expression domain in the VP and LP(Bulfone et al., 1999; Yun et al., 2001)) of the dorsal and medial pallium (arrowhead in Fig. 2D,D′). Each of these findings is indicative of precocious differentiation of early cortical progenitors.

Fig. 3.

E11.5 (A,A′), E12.5 (B-C′), E15.5 (D,D′) and E18.5(E-F′) coronal (A-D′) and saggital (E-F′) sections of wild-type (A-F) and Id4-/- (A′-F′)telencephalon are compared. Immunofluorescence was detected using the antibodies listed above each panel (A,A′,D-E′) and in situ hybridization was performed using the S-35-labeled riboprobes listed above each panel (B-C′,F,F′). A pan-neuronal marker, MAP2 (A,A′),shows reproducibly increased expression in the mutant dorsomedial cortex(arrowhead). Markers of differentiating neurons, NeuroD (C,C′)and Tbr2 (see Fig. 2D,D′), also show increased expression in the dorsomedial mantle (arrowheads in C,C′). TBR1 expression marks differentiating cortical neurons that will form deep cortical layers (D-E′). Arrowheads in F,F′ indicate disorganized cortical layers and areas. hp,hippocampus; ctx, neocortex; LV, lateral ventricle; VP, ventral pallium; st,striatum.

Fig. 3.

E11.5 (A,A′), E12.5 (B-C′), E15.5 (D,D′) and E18.5(E-F′) coronal (A-D′) and saggital (E-F′) sections of wild-type (A-F) and Id4-/- (A′-F′)telencephalon are compared. Immunofluorescence was detected using the antibodies listed above each panel (A,A′,D-E′) and in situ hybridization was performed using the S-35-labeled riboprobes listed above each panel (B-C′,F,F′). A pan-neuronal marker, MAP2 (A,A′),shows reproducibly increased expression in the mutant dorsomedial cortex(arrowhead). Markers of differentiating neurons, NeuroD (C,C′)and Tbr2 (see Fig. 2D,D′), also show increased expression in the dorsomedial mantle (arrowheads in C,C′). TBR1 expression marks differentiating cortical neurons that will form deep cortical layers (D-E′). Arrowheads in F,F′ indicate disorganized cortical layers and areas. hp,hippocampus; ctx, neocortex; LV, lateral ventricle; VP, ventral pallium; st,striatum.

Early-born (E11.5-E13.5) neurons of the cortex form deep layer neurons(layers 5 and 6) (Takahashi et al.,1999). At E15.5 and E18.5, TBR1 expression in postmitotic cortical neurons (Hevner et al., 2001)identified an increased number of deep layer neurons in Id4-/- animals (Fig. 3D-E′). In addition, TBR1-expressing neurons were found in more dispersed positions in Id4-/- animals at E15.5(Fig. 3D′). Consistently, Id2 expression at E18.5(Rubenstein et al., 1999)revealed disorganized layering/differentiation within the cortical layers(Fig. 3F,F′). These observations provide additional evidence that early cortical progenitors are precociously differentiating in the absence of the functional Id4gene.

Prolonged G1-S transition in the Id4-/- cortex

To determine whether defects in progenitor proliferation also contributed to the Id4-/- mutant phenotype, we examined markers of cell cycle phases and found that G1-S transition of the Id4-/-neuroepithelial cells is compromised at E12.5. We compared progenitor cells in wild-type and Id4-/- cortex by analyzing the expression of markers that identify all cells actively proliferating (PCNA, proliferating cell nuclear antigen), or cells in M-phase (PH3: Phosphorylated Histone 3), or cells in S-phase, by a short pulse of BrdU (Bromodeoxyuridine)-labeling. At E12.5, the density of proliferating cells in the VZ is indistinguishable in wild-type and Id4-/- animals (PCNA+ cells in Fig. 4A,A′,D′), but the VZ is approximately 15% thicker in the Id4-/- mutant(see Fig. 1C,C′, Fig. 4A,A′). To determine whether this increased thickness could be attributed to cells at a particular phase of the cell cycle, we first examined cells in M phase. We found that the actual number of cells in M-phase was indistinguishable in the wild-type and Id4-/- animals at this age(Fig. 4C,C′, and not shown). BrdU labeling at E12.5 identified cells that were in S phase(Fig. 4B,B′). Although the thickness of the S-phase zone varied within the cortical VZ (thinner dorsomedially) in the mutant animal, the most striking difference between the control and Id4-/- animals we found was in the thickness of the zone corresponding to the G-phases of the cell cycle. The G-phase zone is sandwiched between the BrdU-positive S-phase cells(Fig. 4B,B′) and the PH3-positive M-phase cells (Fig. 4C,C′). The thickening of this zone (distance between the arrowheads in Fig. 4B,B′)clearly accounts for the overall increased thickness of the VZ in mutant animals. The presence of cycling cells in this region, as identified by PCNA expression (Fig. 4A,A′,D′), suggested that progenitor cells in the Id4-/- animal might make a prolonged transition between G1-S and/or G2-M phases of the cell cycle. We, therefore, evaluated the cell cycle distribution of cortical cells isolated from E12.5 dorsal telencephalon by FACS analysis. In multiple experiments (n=7) examining cells from E12.5 or E13.5 littermate embryos, we invariably observed a reduction in the percentage of cells in S-phase and a corresponding increase in the percentage of cells in G0/G1, as indicated in representative experiments from E12.5 shown in Table 1. In wild-type animals at E12.5, virtually all VZ cells are dividing and no G0 cells are found among them (Takahashi et al.,1995a). Our finding that the thicker mutant VZ is composed of PCNA+ proliferating cells (Fig. 4A′,D′) indicates that the increased percentage of cells in G0/G1 detected by FACS analysis(Table 1) represents a prolonged G1 in the mutant cells at this age. Although the increased number of prematurely differentiating cells observed at this age may also contribute to the higher percentage of G0/G1 cells in the ID4 mutants, the fact that most cells in the G-phase zone are PCNA positive(Fig. 4A′,D′)indicates that a contribution of prematurely differentiating cells to the thickening of the VZ is unlikely. Together, these two independent lines of investigation indicate that Id4 is required for normal G1-S transition in early neural stem cells. Thus, the reduced brain size in the Id4-/- cortex results from both premature differentiation and compromised cell cycle transition of early neural stem cells.

Fig. 4.

E12.5 (A-C′,D′) and E15.5 (E-H′) coronal sections through control (A-H) and Id4-/- (A′-H′) mutant cortex. Markers of cell cycle used are: proliferating cells, PCNA(A,A′,D′); S-phase, BrdU pulse (B,B′,H,H′); and M-phase, PH3 (C,C′,G,G′). (B,B′) The G-phase zone is indicated by the distance between the arrowheads; yellow dashes indicate the ventricular surface. (D′) High magnification image of PCNA staining(red), double labeled with DAPI (blue) to visualize all nuclei. At E15.5,ectopically positioned Ngn2- and NeuroD-expressing cells were detected outside of the VZ and SVZ (arrowheads in E′,F′). Immunofluorescence analysis was performed using antibodies against PH3 and BrdU (following a 45-minute labelling pulse) in littermate control and Id4 null animals (arrowheads in G′,H′ indicate ectopically proliferating cells). BrdU is shown in green and DAPI in blue(H,H′). Arrow in H′ indicates increased BrdU labeling in the Id4-/- SVZ at E15.5. LV, lateral ventricle; hp,hippocampus; ctx, neocortex; LGE, lateral ganglionic eminence; CGE, caudal ganglionic eminence; VZ, ventricular zone; SVZ, subventricular zone.

Fig. 4.

E12.5 (A-C′,D′) and E15.5 (E-H′) coronal sections through control (A-H) and Id4-/- (A′-H′) mutant cortex. Markers of cell cycle used are: proliferating cells, PCNA(A,A′,D′); S-phase, BrdU pulse (B,B′,H,H′); and M-phase, PH3 (C,C′,G,G′). (B,B′) The G-phase zone is indicated by the distance between the arrowheads; yellow dashes indicate the ventricular surface. (D′) High magnification image of PCNA staining(red), double labeled with DAPI (blue) to visualize all nuclei. At E15.5,ectopically positioned Ngn2- and NeuroD-expressing cells were detected outside of the VZ and SVZ (arrowheads in E′,F′). Immunofluorescence analysis was performed using antibodies against PH3 and BrdU (following a 45-minute labelling pulse) in littermate control and Id4 null animals (arrowheads in G′,H′ indicate ectopically proliferating cells). BrdU is shown in green and DAPI in blue(H,H′). Arrow in H′ indicates increased BrdU labeling in the Id4-/- SVZ at E15.5. LV, lateral ventricle; hp,hippocampus; ctx, neocortex; LGE, lateral ganglionic eminence; CGE, caudal ganglionic eminence; VZ, ventricular zone; SVZ, subventricular zone.

Table 1.

Cell cycle analysis of E12.5 Id4+/- and Id4-/- dorsal telencephalon

ExperimentGenotypeSample size, nPercentage in G0/G1*Percentage in S*Percentage in G2/M*
+/- 60.70±1.85 27.16±0.03 12.14±1.83 
 -/- 63.03±2.87 25.31±4.65 11.67±1.77 
+/- 52.41 41.31 6.28 
 -/- 55.94±1.29 35.96±1.14 8.10±0.16 
+/- 58.04±1.51 38.80±2.42 3.17±1.74 
 -/- 60.84 37.65 1.51 
+/- 61.03±1.01 31.82±2.63 7.15±2.92 
 -/- 65.36±0.50 27.45±0.28 7.19±0.71 
ExperimentGenotypeSample size, nPercentage in G0/G1*Percentage in S*Percentage in G2/M*
+/- 60.70±1.85 27.16±0.03 12.14±1.83 
 -/- 63.03±2.87 25.31±4.65 11.67±1.77 
+/- 52.41 41.31 6.28 
 -/- 55.94±1.29 35.96±1.14 8.10±0.16 
+/- 58.04±1.51 38.80±2.42 3.17±1.74 
 -/- 60.84 37.65 1.51 
+/- 61.03±1.01 31.82±2.63 7.15±2.92 
 -/- 65.36±0.50 27.45±0.28 7.19±0.71 

Shown here are typical experiments from PI labeling and FACS scanning of Id4+/- and Id4-/- dorsal telencephalon cells at E12.5. n identifies the numbers of Id4-/- or control littermates in each experiment. Quantitation using ModFit software revealed the increased percentage of G1-phase cells and the decreased percentage of S-phase cells in Id4-/- samples. Consistent shifts in cell cycle profile were observed in every litter examined, a total of seven experiments representing seven independent litters (four litters from E12.5 and three litters from E13.5, not shown). The total number of embryos we examined was nine Id4-/- and 12 Id4+/- at E12.5, and seven Id4-/- and eight Id4+/- at E13.5. The data from Experiment 4 was evaluated by t-test, a representative experiment with a large enough sample size for a t-test, resulting in a P value of 0.0026. If there were no association, the probability of always observing an increased percentage of cells in G0/G1 in Id4-/- animals in seven total experiments would be 0.00781.

*

Values are ±s.e.m. where applicable

Ectopically positioned proliferating cells

To determine whether the proliferation defect in Id4-/-animals at E12.5 was also detectable at later stages of development, we examined markers of cortical progenitors and cellular proliferation at E15.5. Surprisingly, we found a greater number of proliferating cells outside of the VZ in the Id4-/- animals. In addition to progenitors in the SVZ, many cells in the mantle (postmitotic) region ectopically expressed markers of VZ and SVZ, such as Ngn2, Ngn1, NeuroD, Emx1 and cadherin 8 (Fig. 4E-F′, and not shown). We interpret this expression pattern to reflect the presence of proliferating cells in the postmitotic region. Therefore, we sought evidence for ectopic proliferation at E15.5 by evaluating cortical tissue following a 45-minute BrdU pulse to identify S-phase cells and PH3+immunostaining to identify M phase cells. As indicated in Fig. 4G-H′, we observed many BrdU-positive, as well as PH3-positive, cells in the mantle zone. This observation indicates that, in contrast to the compromised proliferation observed at E12.5, cells in the postmitotic region are aberrantly proliferating in the Id4 mutant at E15.5.

Discussion

Unlike other mouse lines in which inactivation of a single ID gene family member does not show an observable CNS phenotype(Lyden et al., 1999; Yokota, 2001), the Id4-/- mouse has an obviously smaller brain. For example, Id1 and Id3 single mutants do not show an obvious CNS phenotype, although a phenotype in the telencephalon does emerge when Id1 and Id3 are simultaneously inactivated(Lyden et al., 1999). Here, we provide multiple lines of evidence indicating that the Id4 mutant phenotype arises from the requirement, in vivo, of ID4 function in cortical stem cells for their proliferation and differentiation. Importantly, we show different effects of Id4 loss of function on cortical cell proliferation at different times during development.

Requirement for Id4 during early stem cell proliferation

Neural epithelium of the developing cortex expands laterally through the rapid division of early stem cells. Cortical neurogenesis commences at around E11.5 and the onset of an observable Id4-/- phenotype coincides with this major milestone in cortical development. At E12.5, the mutant cortical VZ lateral expansion is greatly compromised, and a progressively reduced cortical VZ area is found from this time forward. For example, at E15.5, the cortical VZ surface area is less than 50% of the control littermate brains (∼60% mediolaterally×∼70% rostrocaudally). At E12.5, the Id4 mutant VZ is thicker and this thickening can be attributed to a prolonged G1 phase of the cell cycle (Fig. 4 and Table 1). This phenotype is consistent with in vitro studies indicating that ID genes enhance proliferation by facilitating the G1-S transition. For example, ID2 has been shown to bind to the RB (Retinoblastoma) protein thereby enhancing the G1-S transition (Iavarone et al.,1994), and other ID genes have been shown to regulate the expression of the G1 cyclin-dependent kinase inhibitors p16 and p21 at the transcriptional level (Alani et al.,2001; Lasorella et al.,1996; Prabhu et al.,1997). ID genes are in turn regulated during the cell cycle where their peak level of expression is observed early in G1, after serum stimulation (Christy et al.,1991; Hara et al.,1994). However, our analysis of these G1-S regulators in the Id4-/- telencephalon did not reveal significant changes in their expression levels (data not shown), suggesting that Id4 does not regulate their expression during cortical development. We are currently examining the possibility that ID4 interacts with these molecules at the protein:protein level to affect their function.

Our findings provide evidence supporting an integrated proliferative regulatory mechanism that coordinates the timing, the number and the neuronal specificity of cortical neurons, as proposed by Caviness and his colleagues in their theoretical model (Caviness et al.,2003). Their model predicts that increasing the proportion of daughter cells exiting the cell cycle early will lead to reduced lateral expansion of the neuroepithelium, as a result of the reduced proportion of daughter cells available to expand the proliferating population. It also predicts that the early cortical plate will be transiently thicker (because of an increased output of postmitotic cells), but significantly thinner at the end of the neurogenic period because of the loss of stem cells early on. In addition, an increase in the proportion of daughter cells exiting the VZ is associated with a lengthening of the cell cycle time, particularly G1, during normal development. With the exception of a thicker, rather than thinner,cortical plate in the Id4 mutant at E18.5, the Id4 mutant phenotype is consistent with this model. Hence, our findings generally support the existence of an integrated regulatory mechanism, and identify Id4as a key regulator that controls the time domain, the output domain, and neuronal specification during cortical development.

Role of ID family members in telencephalic development

We were intrigued by the region-specific requirement for ID4 function, and the relatively mild mutant phenotype at P0 despite the early loss of stem cells due to both premature differentiation and compromised cell cycle transition. As Id1, Id2 and Id3 are also expressed in the developing telencephalon, these ID genes may functionally compensate for Id4 in non-affected areas. For example, mutational studies of Id1 and Id3 have revealed that they are functionally redundant during telencephalic development. To examine this possibility, we monitored expression levels of other ID genes in Id4 mutant telencephalon. Indeed Id1, Id2 and Id3 expression levels were increased in the Id4 mutant, when compared with a control littermate (Fig. 2E′ and not shown). In particular, Id2 expression was altered both spatially(Fig. 2E,E′) and quantitatively (not shown). Hence, we examined possible functional redundancies between Id2 and Id4 by analyzing Id2/4double null embryos. We were unable to detect evidence of altered telencephalic development in Id2-/- animals (data not shown). Interestingly, the brain size of the Id2/4 double null animal was only slightly smaller than that of the Id4 single mutant (data not shown), and no obvious defects were found in other areas of the telencephalon. This observation indicates that Id2 is not necessary to compensate for Id4 in other areas, and supports the idea that ID4 function is unique and necessary for the development of the telencephalic midline tissue. Id2/4 double mutant analysis also suggests the existence of other compensatory mechanisms, either at the molecular level by Id1 or Id3, or at the cellular level (see below).

Role of Id4 in differentiating neurons

In contrast to the reduced proliferation observed in the early neuroepithelium, the number of proliferating cells outside of the VZ in the Id4-/- brain after E15.5 is dramatically increased(Fig. 4). These ectopically positioned cells proliferate, as indicated by both S-phase and M-phase markers, and differentiate, as indicated by NeuroD expression, in ectopic positions. These results indicate a potential compensation mechanism at the cellular level for the reduced proliferation of early VZ progenitors, and may account for the relatively mild Id4 mutant phenotype observed at birth.

Ectopically positioned proliferating cells found in the Id4-/- cortex are reminiscent of changes observed in animals lacking the Rb gene. In Rb-/- animals,ectopically positioned proliferating cells are found in the cortex, and this phenotype has been interpreted as indicating that differentiating mantle cells have aberrantly re-entered the cell cycle in the absence of functional RB(Clarke et al., 1992; Lee et al., 1992; Lipinski et al., 2001; MacPherson et al., 2003). We therefore examined Rb levels in Id4-/- animals,but we did not observe a significant difference in its expression, either at the RNA or protein level (not shown). ID2 has been shown to bind RB protein and to inhibit its function post-translationally(Iavarone et al., 1994). Hence, a possible explanation for the observed phenotype is that the increased level of Id2 expression in the Id4 mutant(Fig. 2E′) leads to inactivation of RB. However, direct examination of this hypothesis indicates that this phenotype is not the result of an epigenetic event (increased Id2 levels). The Id2/4 double mutant cortex also contained many ectopically proliferating cells in the mantle region (not shown). These observations suggest that if the Id4 phenotype were mediated through the RB pathway, ID4 most likely acts to enhance RB-mediated inhibition of proliferation, in contrast to ID2, which negatively regulates RB. Among the many possibilities, direct interactions can be envisioned in which ID4 competes with ID2 for RB binding in differentiating neurons. Or, ID4 may directly interact with other molecules in the cell cycle machinery, and depending on the interacting partner and cellular context, either enhance or inhibit cell cycle transition.

Id4 interaction with bHLH factors

ID genes have been shown to inhibit differentiation in many different cell types in vitro and in vivo (Norton,2000). The main mechanism of this potent inhibitory effect is attributed to its ability to form dominant-negative dimers with bHLH proteins. In the developing cortical progenitor zone, at least three proneural bHLH genes, Ngn1, Ngn2 and Mash1, are important for progenitor proliferation, cell fate specification, and differentiation(Fode et al., 2000; Nieto et al., 2001; Parras et al., 2002; Ross et al., 2003). As ID proteins are thought to act as negative regulators of bHLH proteins by sequestering available E-proteins, we have postulated that proneural bHLH proteins are hyper-active in the Id4 mutant cortex. Consistent with this model, the Id4 mutant phenotype correlates well with both loss-of-function and gain-of-function studies of these proneural genes(Fode et al., 2000; Parras et al., 2002; Sun et al., 2001). For example, ectopic expression of Ngn1 clearly promotes neuronal differentiation (Sun et al.,2001), and the simultaneous loss of Ngn2 and Mash1 function results in reduced neurogenesis (and NeuroDexpression), particularly in the dorsomedial cortex(Fode et al., 2000). In Id4-/- animals, where proneural bHLH proteins are presumably free from ID4 inhibition, neural stem cells prematurely differentiate and this is most obvious in the dorsomedial region(Fig. 2D,D′, Fig. 3A,A′,C,C′). It is unclear, at present, why the dorsomedial region of the telencephalon is particularly sensitive to both positive and negative HLH protein activity, but these in vivo studies reveal an important requirement for bHLH transcription factor function in this area, and emphasize the role of Id4 in regulating its development.

CNS development requires complex molecular interactions that control progenitor proliferation and differentiation, and Id4 can now be recognized to be important for these processes. Our finding that Id4inhibits neural progenitor differentiation is consistent with the assignment of this function to other ID family members. In addition, we provide in vivo evidence for a novel function of Id4 in preventing aberrant cell cycle entry in differentiating neurons. These observations underscore the importance of cell context for ID gene function, and in particular for Id4, which either enhances or blocks cell cycle transition in the same developing tissue at different times. This study reveals a crucial role for Id4 in cortical development, and identifies the molecular pathways over which its effects are mediated, while suggesting important new areas for future study.

Acknowledgements

We thank Sharon Liu, Jian Yang and Eric DuFour for their excellent help with animal care. We also thank John Hill for sharing the ID gene RT-PCR primers we used, and Nathan Watson for his assistance with western analysis. We thank Dr Bernard F. Cole for his advice on statistical analysis. This study was supported by NIH grant #CA84280 (M.A.I.), the Betz Foundation (M.A.I.),and the Jordan and Kyra Memorial Fund (M.A.I.).

References

Alani, R. M., Young, A. Z. and Shifflett, C. B.(
2001
). Id1 regulation of cellular senescence through transcriptional repression of p16/Ink4a.
PNAS
98
,
7812
-7816.
Andres-Barquin, P. J., Hernandez, M. C. and Israel, M. A.(
2000
). Id genes in nervous system development.
Histol. Histopath.
15
,
603
-618.
Benezra, R., Davis, R. L., Lockshon, D., Turner, D. L. and Weintraub, H. (
1990
). The protein Id: a negative regulator of helix-loop-helix DNA binding proteins.
Cell
61
,
49
-59.
Bulfone, A., Martinez, S., Marigo, V., Campanella, M., Basile,A., Quaderi, N., Gattuso, C., Rubenstein, J. L. and Ballabio, A.(
1999
). Expression pattern of the Tbr2 (Eomesodermin) gene during mouse and chick brain development.
Mech. Dev.
84
,
133
-138.
Caviness, V. S., Jr, Goto, T., Tarui, T., Takahashi, T., Bhide,P. G. and Nowakowski, R. S. (
2003
). Cell output, cell cycle duration and neuronal specification: a model of integrated mechanisms of the neocortical proliferative process.
Cereb. Cortex
13
,
592
-598.
Christy, B. A., Snaders, L. K., Lau, L. F., Copeland, N. G.,Jenkins, N. A. and Nathans, D. (
1991
).
Proc. Natl. Acad. Sci. USA
88
,
1815
-1819.
Clarke, A. R., Maandag, E. R., van Roon, M., van der Lugt, N. M. T., van der Valk, M., Hooper, M. L., Berns, A. and Riele, H.(
1992
). Requirement for a functional Rb1 gene in murine development.
Nature
359
,
328
-330.
Fode, C., Ma, Q., Casarosa, S., Ang, S. L., Anderson, D. J. and Guillemot, F. (
2000
). A role for neural determination genes in specifying the dorsoventral identity of telencephalic neurons.
Genes Dev.
14
,
67
-80.
Gradwohl, G., Fode, C. and Guillemot, F.(
1996
). Restricted expression of a novel murine atonal-related bHLH protein in undifferentiated neural precursors.
Dev. Biol.
180
,
227
-241.
Guillemot, F. (
1999
). Vertebrate bHLH genes and the determination of neuronal fates.
Exp. Cell Res.
253
,
357
-364.
Hara, E., Yamaguchi, T., Nojima, H., Ide, T., Campisi, J.,Okayama, H. and Oda, K. (
1994
). Id-related genes encoding helix-loop-helix proteins are required for G1 progression and are repressed in senescent human fibroblasts.
J. Biol. Chem.
269
,
2139
-2145.
Hevner, R. F., Shi, L., Justice, N., Hsueh, Y., Sheng, M.,Smiga, S., Bulfone, A., Goffinet, A. M., Campagnoni, A. T. and Rubenstein, J. L. (
2001
). Tbr1 regulates differentiation of the preplate and layer 6.
Neuron
29
,
353
-366.
Iavarone, A., Garg, P., Lasorelle, A., Hsu, J. and Israel, M. A. (
1994
). The helix-loop-helix protein Id2 enhances cell proliferation and binds to the retinoblastoma protein.
Genes Dev.
8
,
1270
-1284.
Jen, Y., Manova, K. and Benezra, R. (
1997
). Each member of the Id gene family exhibits a unique expression pattern in mouse gastrulation and neurogenesis.
Dev. Dyn.
208
,
92
-106.
Kondo, T. and Raff, M. (
2000
). The Id4 HLH protein and the timing of oligodendrocyte differentiation.
EMBO J.
19
,
1998
-2007.
Lasorella, A., Iavarone, A. and Israel, M. A.(
1996
). Id2 specifically alters regulation of the cell cycle by tumor suppressor proteins.
Mol. Cell. Biol.
16
,
2570
-2578.
Lee, E. Y., Chang, C. Y., Hu, N., Wang, Y. J., Lai, C. C.,Herrup, K., Lee, W. H. and Bradley, A. (
1992
). Mice deficient for Rb are nonviable and show defects in neurogenesis and haematopoiesis.
Nature
359
,
288
-294.
Lee, S. M., Tole, S., Grove, E. and McMahon, A. P.(
2000
). A local Wnt-3a signal is required for development of the mammalian hippocampus.
Development
127
,
457
-467.
Lipinski, M. M., Macleod, K. F., Williams, B. O., Mullaney, T. L., Crowley, D. and Jacks, T. (
2001
). Cell-autunomous and non-cell-autonomous functions of the Rb tumor suppressor in developing central nervous system.
EMBO J.
20
,
3402
-3413.
Lyden, D., Young, A. Z., Zagzag, D., Yan, W., Gerald, W.,O'Reilly, R., Bader, B. L., Hynes, R. O., Zhuang, Y., Manova, K. et al.(
1999
). Id1 and Id3 are required for neurogenesis, angiogenesis,and vascularization of tumor xenografts.
Nature
401
,
670
-677.
MacPherson, D., Sage, J., Crowley, D., Trumpp, A., Bronson, R. T. and Jacks, T. (
2003
). Conditional mutation of Rb causes cell cycle defects without apoptosis in the central nervous system.
Mol. Cell. Biol.
23
,
1044
-1053.
Mori, S., Nishikawa, S. and Yokota, Y. (
2000
). Lactation defect in mice lacking the helix-loop-helix inhibitor Id2.
EMBO J.
19
,
5772
-5781.
Nieto, M., Schuurmans, C., Britz, O. and Guillemot, F.(
2001
). Neural bHLH genes control the neuronal versus glial fate decision in cortical progenitors.
Neuron
29
,
401
-413.
Norton, J. D. (
2000
). ID helix-loop-helix proteins in cell growth, differentiation and tumorigenesis.
J. Cell Sci.
113
,
3897
-3905.
Parras, C. M., Schuurmans, C., Scardigli, R., Kim, J., Anderson,D. J. and Guillemot, F. (
2002
). Divergent functions of the proneural genes Mash1 and Ngn2 in the specification of neuronal subtype identity.
Genes Dev.
16
,
324
-338.
Prabhu, S., Ignatova, A., Park, S. T. and Sun, X. H.(
1997
). Regulation of the expression of cyclin-dependent kinase inhibitor p21 by E2A and Id proteins.
Mol. Cell. Biol.
17
,
5888
-5896.
Puelles, L., Kuwana, E., Puelles, E., Bulfone, A., Shimamura,K., Keleher, J., Smiga, S. and Rubenstein, J. L. (
2000
). Pallial and subpallial derivatives in the embryonic chick and mouse telencephalon, traced by the expression of the genes Dlx-2, Emx-1, Nkx-2.1,Pax-6, and Tbr-1.
J. Comp. Neurol.
424
,
409
-438.
Qiu, M., Bulfone, A., Martinez, S., Meneses, J. J., Shimamura,K., Pedersen, R. A. and Rubenstein, J. L. (
1995
). Null mutation of Dlx-2 results in abnormal morphogenesis of proximal first and second branchial arch derivatives and abnormal differentiation in the forebrain.
Genes Dev.
9
,
2523
-2538.
Ross, S. E., Greenberg, M. E. and Stiles, C. D.(
2003
). Basic helix-loop-helix factors in cortical development.
Neuron
39
,
13
-25.
Rubenstein, J. L., Anderson, S., Shi, L., Miyashita-Lin, E.,Bulfone, A. and Hevner, R. (
1999
). Genetic control of cortical regionalization and connectivity.
Cereb. Cortex
9
,
524
-532.
Sauvageot, C. M. and Stiles, C. D. (
2002
). Molecular mechanisms controlling cortical gliogenesis.
Curr. Opin. Neurobiol.
12
,
244
-249.
Sun, Y., Nadal-Vicens, M., Misono, S., Lin, M. Z., Zubiaga, A.,Hua, X., Fan, G. and Greenberg, M. E. (
2001
). Neurogenin promotes neurogenesis and inhibits glial differentiation by independent mechanisms.
Cell
104
,
365
-376.
Takahashi, T., Nowakowski, R. S. and Caviness, V. S., Jr(
1992
). BUdR as an S-phase marker for quantitative studies of cytokinetic behaviour in the murine cerebral ventricular zone.
J. Neurocytol.
21
,
185
-197.
Takahashi, T., Nowakowski, R. S. and Caviness, V. S., Jr(
1993
). Cell cycle parameters and patterns of nuclear movement in the neocortical proliferative zone of the fetal mouse.
J. Neurosci.
13
,
820
-833.
Takahashi, T., Nowakowski, R. S. and Caviness, V. S., Jr(
1995a
). The cell cycle of the pseudostratified ventricular epithelium of the embryonic murine cerebral wall.
J. Neurosci.
15
,
6046
-6057.
Takahashi, T., Nowakowski, R. S. and Caviness, V. S., Jr(
1995b
). Early ontogeny of the secondary proliferative population of the embryonic murine cerebral wall.
J. Neurosci.
15
,
6058
-6068.
Takahashi, T., Goto, T., Miyama, S., Nowakowski, R. S. and Caviness, V. S., Jr (
1999
). Sequence of neuron origin and neocortical laminar fate: relation to cell cycle of origin in the developing murine cerebral wall.
J. Neurosci.
19
,
10357
-10371.
Toma, J. G., El-Bizri, H., Barnabe-Heider, F., Aloyz, R. and Miller, F. D. (
2000
). Evidence that Helix-loop-helix proteins collaborate with Retinoblastoma tumor suppressor protein to regulate cortical neurogenesis.
J. Neurosci.
20
,
7648
-7656.
Yokota, Y. (
2001
). Id and development.
Oncogene
20
,
8290
-8298.
Yokota, Y., Mansouri, A., Mori, S., Sugawara, S., Adachi, S.,Nishikawa, S. and Gruss, P. (
1999
). Development of peripheral lymphoid organs and natural killer cells depends on the helix-loop-helix inhibitor Id2.
Nature
397
,
702
-706.
Yun, K., Potter, S. and Rubenstein, J. L.(
2001
). Gsh2 and Pax6 play complementary roles in dorsoventral patterning of the mammalian telencephalon.
Development
128
,
193
-205.
Zebedee, Z. and Hara, E. (
2001
). Id proteins in cell cycle control and cellular senescence.
Oncogene
20
,
8317
-8325.