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Region-specific expression of the helix–loop–helix gene BETA3 in developing and adult brains
Mechanisms of Development 114 (2002) 125–128 www.elsevier.com/locate/modo
Gene expression pattern
Region-specific expression of the helix–loop–helix gene BETA3 in developing and adult brains Mary H. Kim, Jenny Gunnersen, Cheryl Augustine, Seong-Seng Tan* Brain Development Laboratory, Howard Florey Institute, The University of Melbourne, Parkville 3010, Victoria, Australia Received 14 December 2001; received in revised form 29 January 2002; accepted 29 January 2002
Abstract Members of the basic helix–loop–helix (bHLH) transcription factor family are crucial regulators of neuronal cell generation and cell fate. A number of bHLH genes are expressed in the developing cerebral cortex, including MASH-1, neurogenin2 and NeuroD implying the existence of a regulatory and possibly redundant network of family members. BETA3 is a novel member originally cloned from pancreatic cells but we report here highly restricted expression patterns in developing forebrain structures that are highly stage-specific. We show that BETA3 mRNA is found in both neocortex and archicortex, mainly in cells that have reached their migratory destinations but is largely absent from proliferative zones. These expression data would suggest that BETA3 function is linked to the establishment rather than the initiation of neuronal fates. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Helix–loop–helix; BETA2; BETA3; NeuroD; Neocortex; Serial analysis of gene expression
1. Results and discussion Basic helix–loop–helix (bHLH) transcription factors play important roles in neuronal determination and differentiation by regulating gene expression via binding to E box sequences (Lee, 1997). This binding occurs through the formation of heterodimers between tissue-specific class B bHLH family members and ubiquitous class A bHLH factors (Murre et al., 1989). BETA2 (b-cell E-box transactivator (2), also known as NeuroD1, is a class B bHLH factor cloned from a hamster insulinoma cell line and shown to be important for both pancreatic and brain development (Lee et al., 1995; Naya et al., 1995). Mice lacking BETA2 suffer arrested development of insulin-producing b cells in the pancreas (Naya et al., 1997), and severe reduction of neurons in the cochlear-vestibular ganglia of the inner ear (Liu et al., 2000). Another class B member, BETA3, was also cloned from pancreatic b cells but despite a high degree of homology with BETA2 (Peyton et al., 1996), it is unable to bind DNA even though it can dimerize with class A proteins. Indeed, competition studies demonstrate that BETA3 is a negative regulator of BETA2 transcriptional activity. It is noteworthy that BETA2 and BETA3 genes were both cloned from pancreatic and embryonic stem cells but their * Corresponding author. Tel.: 161-3-8344-7336; fax: 61-3-9348-1707. E-mail address: [email protected] (S.-S. Tan).
expression extends to other organs, including the brain. BETA2 mRNA expression is strongly detected in developing and adult brain including the cortex (Lee et al., 1995; Naya et al., 1995). The importance of this gene for neuronal development is demonstrated in the null mutant where granule cells fail to differentiate properly in the cerebellum and hippocampal dentate gyrus (Miyata et al., 1999; Liu et al., 2000). BETA3 mRNA is also expressed in the adult lung, kidney and brain (Peyton et al., 1996), but there is no information on whether or not it has a role in neuronal differentiation. Given its putative role in regulating BETA2 transcriptional activity, the expression pattern of BETA3 in the developing brain is of particular interest. In this report, we describe highly restricted patterns of BETA3 mRNA expression in the developing neocortex, hippocampus and cerebellum. Data from Northern blots and in situ hybridization patterns confirm that BETA3 expression in the brain is associated with developmental structures, and is absent from the adult brain with the exception of trace levels in the adult hippocampus. BETA3 was identified in a differential gene expression screen using serial analysis of gene expression (SAGE) (Velculescu et al., 1995). The SAGE technique permits identification of differentially expressed genes by comparing the abundance of 10 bp sequence tags (downstream of the most 3 0 CATG restriction enzyme site) taken from every sampled transcript. A comparison of 20,000 SAGE tags
0925-4773/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(02)00036-9
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Fig. 1. Northern blot analysis of BETA3 mRNA expression in embryonic, postnatal and adult mouse neocortex. BETA3 expression is detectable from E13.5 to P7, with strongest expression at E15.5 and E17.5, with an approximate 3.75 kb transcript size. BETA3 expression is barely detectable in the adult neocortex.
from embryonic day (E) 15.5 mouse neocortex with a comparable pool of transcripts from postnatal day (P) 1 revealed the tag GCGATCTGCC to be expressed more highly in the E15.5 library (seven tags) than at P1 (two tags). Using the Mus musculus Unigene ‘reliable’ tag-togene mapping dataset (www.ncbi.nlm.nih.gov/Unigene/), the tag was mapped to Unigene cluster Mm. 143811 and identified as BETA3. A cDNA clone (GenBank ID BE650599) was used to generate probes for Northern blot and in situ hybridization analyses. Northern blot analysis was performed using mRNA obtained from developing and adult neocortical tissues from E11.5 onwards (Fig. 1). The results showed a single BETA3 transcript corresponding to the 3.75 kb band first reported by Xu et al. (1999). In the developing cortex, BETA3 was barely detectable at E11.5, the stage when the
Fig. 2. Expression of BETA3 in the mouse embryonic forebrain. BETA3 expression is observed in the preplate at E11.5 (A, arrow) with no expression in the VZ. At E13.5, BETA3 is found in the cortical plate of the neocortex and the piriform cortex (B), and the thalamic eminences (C). There is no expression in the ganglionic eminence of the developing striatum or in the septum (B). (D) Higher magnification of the boxed area in (C) shows the localization of BETA3 expression to the cortical plate of the neocortex and limbic cortex. (E) A posterior coronal section at E13.5 displays expression in a cluster of cells lateral to the ventricular layer of the third ventricle (higher magnification in F). (G) At E15.5, BETA3 expression is at the greatest intensity, involving both cortical plate and the underlying subventricular zone. Expression is slightly reduced at the piriform cortex region. (H) BETA3 expression terminates abruptly at the cortical hem marking the site of hippocampal development. (I) At E17.5, BETA3 expression remains intense in the cortical plate and subventricular zone. Abbreviations: A; amygdala; CP, cortical plate; DT, dorsal thalamus; GE, ganglionic eminence; H, hippocampus; Hyp, hypothalamus; LV, lateral ventricle; Ncx, neocortex; Px, piriform cortex; Pp, preplate; r, reservoir; S, septum; SVZ, subventricular zone; TE, thalamic eminence; V3, third ventricle; VZ, ventricular zone. Scale bar: (A, D, F), 270mm; (B, E, G–I), 690 mm.
M.H. Kim et al. / Mechanisms of Development 114 (2002) 125–128
preplate is established and before any cortical neurons are generated. Expression of BETA3 was clearly demonstrated at E13.5, with stronger signal intensities at E15.5 and E17.5 and still highly expressed until P7. In the adult cortex, hardly any BETA3 expression was seen. The expression levels suggested by Northern blot analysis are supported by spatial localization of mRNA patterns using in situ hybridization. At E11.5, the cerebral wall is composed of a pseudostratified neuroepithelium with an emerging row of preplate cells at the pial surface. BETA3 is expressed in the preplate (PP) but not in the ventricular zone (VZ) (Fig. 2A). From E13.5 to E17.5, the VZ is the most prominent layer as intense cell proliferation is occurring during this period while large numbers of postmitotic neuroblasts are known to migrate towards the cortical plate (CP). However, there is no BETA3 expression in the VZ of either neocortex or underlying ganglionic eminences but
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expression is intense in the CP (Fig. 2B–D), suggesting that at E13.5, BETA3 is activated after the cessation of cell migration and acquisition of neuronal fates. In the thalamus, most neurons become postmitotic between E10.5 and E14.5 (Angevine, 1970). In the ventral thalamus, BETA3 is expressed in the thalamic eminences (Fig. 2C), and in the dorsal thalamus, faint staining is seen in a ribbon of cells lateral to the VZ of the third ventricle (Fig. 2E,F). Peak neurogenesis in the neocortex occurs at E15.5 when the number of departed neurons is matched by the number of remaining proliferating cells in the VZ (Takahashi et al., 1996). At this time point, BETA3 expression in the CP rises to its greatest intensity and other cells (possibly still migrating) present in the intermediate and subventricular zones (SVZ) are also positive (Fig. 2G). At more caudal regions, a section across the E15.5 hippocampus reveals BETA3 staining in the cortical hem from which the hippo-
Fig. 3. Expression of BETA3 in the mouse postnatal forebrain and cerebellum. From P1 onwards, BETA3 is strongly expressed in the superficial layers of the neocortex (A,B), with intense expression in the hippocampus until P7. Arrows in (A), (B), and (G) indicate an abrupt break in expression intensities at the border between the cingulate and neocortex. (C) BETA3 expression persists at a lower level in the upper layer of the piriform cortex. (D) BETA3 is also expressed in the developing cerebellum. (E) Higher power view of boxed area shows expression in the Purkinje cell layer (arrow) and the internal granule cell layer (arrowhead). (F–H) At older stages, BETA3 expression in the neocortex begins to wane and until in the adult, no expression can be seen in the neocortex. On the other hand, expression in the hippocampus remains strong at P3 and P7, but in the adult, BETA3 expression is only confined to CA3 field and dentate gyrus. No staining may be observed in CA1. Abbreviations: Cer, Cerebellum; Cc, cingulate cortex; CA1, cornu ammonis 1; CA3, cornu ammonis 3; DG, dentate gyrus; GE, ganglionic eminence; LV, lateral ventricle; Ncx, neocortex; Px, piriform cortex; SVZ, subventricular zone (striatum). Scale bar: (A–D, F– H), 690 mm; (E), 270 mm.
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campal pyramidal neurons will later emerge (Fig. 2H). No staining can be observed in the subpallial ganglionic eminence. Similarly, no expression was detected at E15.5 in the midbrain or the trunk neural tube (data not shown). Staining at E17.5 is prominent in the CP and SVZ of the neocortex and hippocampus but appears to break abruptly at the junction of the neo- and piriform cortices (Fig. 2I). Staining in the postnatal period is dominated by highly restricted expression patterns in the CP and the hippocampus. At P1, BETA3 expression is strong in the upper CP but decreases abruptly at the medial limbic cortex (Fig. 3A,B arrows). The most superficial aspects of the cingulate cortex continue to express the gene (Fig. 3B). The developing hippocampus at P1 shows strong BETA3 staining in all CA fields and in the dentate gyrus (Fig. 3B). In the lateral limbic cortex, BETA3 expression is also diminished in the piriform region (Fig. 3C). More posteriorly, the developing cerebellum is also stained for BETA3 in the Purkinje cell layer and in the granule cells (Fig. 3D,E). Staining in the neocortex begins to wane by P3 and only weak staining is P7 (Fig. 3F,G). The hippocampus continues to demonstrate staining at these stages. However, in the adult brain, the neocortex is no longer stained for BETA3 (Fig. 3H). Interestingly, staining in the adult hippocampus persists in the dentate gyrus and CA3 but not in the CA1 field suggesting BETA3 to be a marker for the adult CA3 field.
2. Experimental procedures C57/BL6 mice were used throughout the study. The morning of the vaginal plug was defined as embryonic day 0.5. For Northern blot analysis, total RNA was isolated from neocortical tissues dissected at various time points using standard protocols. In situ hybridization was carried out on 20 mm sections with antisense probes transcribed with T3 RNA polymerase and the digoxigenin label detected using anti-digoxigenin Fabs coupled to alkaline
phosphatase and NBT/BCIP (Roche). Signals were not detected in the sense controls. References Angevine Jr, J.B., 1970. Time of neuron origin in the diencephalon of the mouse. An autoradiography study. J. Comp. Neurol. 139, 129–187. Lee, J.E., 1997. Basic helix–loop–helix genes in neural development. Curr. Opin. Neurobiol. 7, 13–30. Lee, J.E., Hollenberg, S.M., Snider, L., Turner, D.L., Lipnick, N., Weintraub, H., 1995. Conversion of Xenopus ectoderm into neurons by NeuroD, a basic helix–loop–helix protein. Science 268, 836–844. Liu, M., Pereira, F.A., Price, S.D., Chu, M.J., Shope, C., Himes, D., Eatock, R.A., Brownell, W.E., Lysakowski, A., Tsai, M.J., 2000. Essential role of BETA2/NeuroD1 in development of the vestibular and auditory systems. Genes Dev. 14, 2839–2854. Miyata, T., Maeda, T., Lee, J.E., 1999. NeuroD is required for differentiation of the granule cells in the cerebellum and hippocampus. Genes Dev. 13, 1647–1652. Murre, C., McCaw, P.S., Vaessin, H., Caudy, M., Jan, L.Y., Jan, Y.N., Cabrera, C.V., Buskin, J.N., Hauschka, S.D., Lassar, A.B., et al., 1989. Interactions between heterologous helix–loop–helix proteins generate complexes that bind specifically to a common DNA sequence. Cell 58, 537–544. Naya, F.J., Stellrecht, C.M., Tsai, M.J., 1995. Tissue-specific regulation of the insulin gene by a novel basic helix–loop–helix transcription factor. Genes Dev. 9, 1009–1019. Naya, F.J., Huang, H.P., Qui, Y., Mutoh, H., DeMayo, F.J., Leiter, A.B., Tsai, M.J., 1997. Diabetes, defective pancreatic morphogenesis, and abnormal enteroendocrine differentiation in BETA2/neuroD-deficient mice. Genes Dev. 11, 2323–2334. Peyton, M., Stellrecht, C.M., Naya, F.J., Samora, P.J., Tsai, M.J., 1996. BETA3, a novel helix–loop–helix protein, can act as a negative regulator of BETA2 and MyoD-responsive genes. Mol. Cell. Biol. 16, 626– 633. Takahaski, T., Nowakowski, R.S., Caviness, V.C., 1996. The leaving or Q fraction of the murine cerebral proliferative epithelium: a general model of neocortical neuronogenesis. J. Neurosci. 16, 6183–6196. Velculescu, V.E., Zhang, L., Vogelstein, B., Kinzler, K.W., 1995. Serial analysis of gene expression. Science 270, 484–487. Xu, Z., Dutra, A., Piatigorsky, J., 1999. Functional and structural dissection of a novel human BETA3 gene, a bHLH transcription factor. 49th Annual meeting of the American Society of Human Genetics, San Fransisco, CA.
Gene expression pattern
Region-specific expression of the helix–loop–helix gene BETA3 in developing and adult brains Mary H. Kim, Jenny Gunnersen, Cheryl Augustine, Seong-Seng Tan* Brain Development Laboratory, Howard Florey Institute, The University of Melbourne, Parkville 3010, Victoria, Australia Received 14 December 2001; received in revised form 29 January 2002; accepted 29 January 2002
Abstract Members of the basic helix–loop–helix (bHLH) transcription factor family are crucial regulators of neuronal cell generation and cell fate. A number of bHLH genes are expressed in the developing cerebral cortex, including MASH-1, neurogenin2 and NeuroD implying the existence of a regulatory and possibly redundant network of family members. BETA3 is a novel member originally cloned from pancreatic cells but we report here highly restricted expression patterns in developing forebrain structures that are highly stage-specific. We show that BETA3 mRNA is found in both neocortex and archicortex, mainly in cells that have reached their migratory destinations but is largely absent from proliferative zones. These expression data would suggest that BETA3 function is linked to the establishment rather than the initiation of neuronal fates. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Helix–loop–helix; BETA2; BETA3; NeuroD; Neocortex; Serial analysis of gene expression
1. Results and discussion Basic helix–loop–helix (bHLH) transcription factors play important roles in neuronal determination and differentiation by regulating gene expression via binding to E box sequences (Lee, 1997). This binding occurs through the formation of heterodimers between tissue-specific class B bHLH family members and ubiquitous class A bHLH factors (Murre et al., 1989). BETA2 (b-cell E-box transactivator (2), also known as NeuroD1, is a class B bHLH factor cloned from a hamster insulinoma cell line and shown to be important for both pancreatic and brain development (Lee et al., 1995; Naya et al., 1995). Mice lacking BETA2 suffer arrested development of insulin-producing b cells in the pancreas (Naya et al., 1997), and severe reduction of neurons in the cochlear-vestibular ganglia of the inner ear (Liu et al., 2000). Another class B member, BETA3, was also cloned from pancreatic b cells but despite a high degree of homology with BETA2 (Peyton et al., 1996), it is unable to bind DNA even though it can dimerize with class A proteins. Indeed, competition studies demonstrate that BETA3 is a negative regulator of BETA2 transcriptional activity. It is noteworthy that BETA2 and BETA3 genes were both cloned from pancreatic and embryonic stem cells but their * Corresponding author. Tel.: 161-3-8344-7336; fax: 61-3-9348-1707. E-mail address: [email protected] (S.-S. Tan).
expression extends to other organs, including the brain. BETA2 mRNA expression is strongly detected in developing and adult brain including the cortex (Lee et al., 1995; Naya et al., 1995). The importance of this gene for neuronal development is demonstrated in the null mutant where granule cells fail to differentiate properly in the cerebellum and hippocampal dentate gyrus (Miyata et al., 1999; Liu et al., 2000). BETA3 mRNA is also expressed in the adult lung, kidney and brain (Peyton et al., 1996), but there is no information on whether or not it has a role in neuronal differentiation. Given its putative role in regulating BETA2 transcriptional activity, the expression pattern of BETA3 in the developing brain is of particular interest. In this report, we describe highly restricted patterns of BETA3 mRNA expression in the developing neocortex, hippocampus and cerebellum. Data from Northern blots and in situ hybridization patterns confirm that BETA3 expression in the brain is associated with developmental structures, and is absent from the adult brain with the exception of trace levels in the adult hippocampus. BETA3 was identified in a differential gene expression screen using serial analysis of gene expression (SAGE) (Velculescu et al., 1995). The SAGE technique permits identification of differentially expressed genes by comparing the abundance of 10 bp sequence tags (downstream of the most 3 0 CATG restriction enzyme site) taken from every sampled transcript. A comparison of 20,000 SAGE tags
0925-4773/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(02)00036-9
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Fig. 1. Northern blot analysis of BETA3 mRNA expression in embryonic, postnatal and adult mouse neocortex. BETA3 expression is detectable from E13.5 to P7, with strongest expression at E15.5 and E17.5, with an approximate 3.75 kb transcript size. BETA3 expression is barely detectable in the adult neocortex.
from embryonic day (E) 15.5 mouse neocortex with a comparable pool of transcripts from postnatal day (P) 1 revealed the tag GCGATCTGCC to be expressed more highly in the E15.5 library (seven tags) than at P1 (two tags). Using the Mus musculus Unigene ‘reliable’ tag-togene mapping dataset (www.ncbi.nlm.nih.gov/Unigene/), the tag was mapped to Unigene cluster Mm. 143811 and identified as BETA3. A cDNA clone (GenBank ID BE650599) was used to generate probes for Northern blot and in situ hybridization analyses. Northern blot analysis was performed using mRNA obtained from developing and adult neocortical tissues from E11.5 onwards (Fig. 1). The results showed a single BETA3 transcript corresponding to the 3.75 kb band first reported by Xu et al. (1999). In the developing cortex, BETA3 was barely detectable at E11.5, the stage when the
Fig. 2. Expression of BETA3 in the mouse embryonic forebrain. BETA3 expression is observed in the preplate at E11.5 (A, arrow) with no expression in the VZ. At E13.5, BETA3 is found in the cortical plate of the neocortex and the piriform cortex (B), and the thalamic eminences (C). There is no expression in the ganglionic eminence of the developing striatum or in the septum (B). (D) Higher magnification of the boxed area in (C) shows the localization of BETA3 expression to the cortical plate of the neocortex and limbic cortex. (E) A posterior coronal section at E13.5 displays expression in a cluster of cells lateral to the ventricular layer of the third ventricle (higher magnification in F). (G) At E15.5, BETA3 expression is at the greatest intensity, involving both cortical plate and the underlying subventricular zone. Expression is slightly reduced at the piriform cortex region. (H) BETA3 expression terminates abruptly at the cortical hem marking the site of hippocampal development. (I) At E17.5, BETA3 expression remains intense in the cortical plate and subventricular zone. Abbreviations: A; amygdala; CP, cortical plate; DT, dorsal thalamus; GE, ganglionic eminence; H, hippocampus; Hyp, hypothalamus; LV, lateral ventricle; Ncx, neocortex; Px, piriform cortex; Pp, preplate; r, reservoir; S, septum; SVZ, subventricular zone; TE, thalamic eminence; V3, third ventricle; VZ, ventricular zone. Scale bar: (A, D, F), 270mm; (B, E, G–I), 690 mm.
M.H. Kim et al. / Mechanisms of Development 114 (2002) 125–128
preplate is established and before any cortical neurons are generated. Expression of BETA3 was clearly demonstrated at E13.5, with stronger signal intensities at E15.5 and E17.5 and still highly expressed until P7. In the adult cortex, hardly any BETA3 expression was seen. The expression levels suggested by Northern blot analysis are supported by spatial localization of mRNA patterns using in situ hybridization. At E11.5, the cerebral wall is composed of a pseudostratified neuroepithelium with an emerging row of preplate cells at the pial surface. BETA3 is expressed in the preplate (PP) but not in the ventricular zone (VZ) (Fig. 2A). From E13.5 to E17.5, the VZ is the most prominent layer as intense cell proliferation is occurring during this period while large numbers of postmitotic neuroblasts are known to migrate towards the cortical plate (CP). However, there is no BETA3 expression in the VZ of either neocortex or underlying ganglionic eminences but
127
expression is intense in the CP (Fig. 2B–D), suggesting that at E13.5, BETA3 is activated after the cessation of cell migration and acquisition of neuronal fates. In the thalamus, most neurons become postmitotic between E10.5 and E14.5 (Angevine, 1970). In the ventral thalamus, BETA3 is expressed in the thalamic eminences (Fig. 2C), and in the dorsal thalamus, faint staining is seen in a ribbon of cells lateral to the VZ of the third ventricle (Fig. 2E,F). Peak neurogenesis in the neocortex occurs at E15.5 when the number of departed neurons is matched by the number of remaining proliferating cells in the VZ (Takahashi et al., 1996). At this time point, BETA3 expression in the CP rises to its greatest intensity and other cells (possibly still migrating) present in the intermediate and subventricular zones (SVZ) are also positive (Fig. 2G). At more caudal regions, a section across the E15.5 hippocampus reveals BETA3 staining in the cortical hem from which the hippo-
Fig. 3. Expression of BETA3 in the mouse postnatal forebrain and cerebellum. From P1 onwards, BETA3 is strongly expressed in the superficial layers of the neocortex (A,B), with intense expression in the hippocampus until P7. Arrows in (A), (B), and (G) indicate an abrupt break in expression intensities at the border between the cingulate and neocortex. (C) BETA3 expression persists at a lower level in the upper layer of the piriform cortex. (D) BETA3 is also expressed in the developing cerebellum. (E) Higher power view of boxed area shows expression in the Purkinje cell layer (arrow) and the internal granule cell layer (arrowhead). (F–H) At older stages, BETA3 expression in the neocortex begins to wane and until in the adult, no expression can be seen in the neocortex. On the other hand, expression in the hippocampus remains strong at P3 and P7, but in the adult, BETA3 expression is only confined to CA3 field and dentate gyrus. No staining may be observed in CA1. Abbreviations: Cer, Cerebellum; Cc, cingulate cortex; CA1, cornu ammonis 1; CA3, cornu ammonis 3; DG, dentate gyrus; GE, ganglionic eminence; LV, lateral ventricle; Ncx, neocortex; Px, piriform cortex; SVZ, subventricular zone (striatum). Scale bar: (A–D, F– H), 690 mm; (E), 270 mm.
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campal pyramidal neurons will later emerge (Fig. 2H). No staining can be observed in the subpallial ganglionic eminence. Similarly, no expression was detected at E15.5 in the midbrain or the trunk neural tube (data not shown). Staining at E17.5 is prominent in the CP and SVZ of the neocortex and hippocampus but appears to break abruptly at the junction of the neo- and piriform cortices (Fig. 2I). Staining in the postnatal period is dominated by highly restricted expression patterns in the CP and the hippocampus. At P1, BETA3 expression is strong in the upper CP but decreases abruptly at the medial limbic cortex (Fig. 3A,B arrows). The most superficial aspects of the cingulate cortex continue to express the gene (Fig. 3B). The developing hippocampus at P1 shows strong BETA3 staining in all CA fields and in the dentate gyrus (Fig. 3B). In the lateral limbic cortex, BETA3 expression is also diminished in the piriform region (Fig. 3C). More posteriorly, the developing cerebellum is also stained for BETA3 in the Purkinje cell layer and in the granule cells (Fig. 3D,E). Staining in the neocortex begins to wane by P3 and only weak staining is P7 (Fig. 3F,G). The hippocampus continues to demonstrate staining at these stages. However, in the adult brain, the neocortex is no longer stained for BETA3 (Fig. 3H). Interestingly, staining in the adult hippocampus persists in the dentate gyrus and CA3 but not in the CA1 field suggesting BETA3 to be a marker for the adult CA3 field.
2. Experimental procedures C57/BL6 mice were used throughout the study. The morning of the vaginal plug was defined as embryonic day 0.5. For Northern blot analysis, total RNA was isolated from neocortical tissues dissected at various time points using standard protocols. In situ hybridization was carried out on 20 mm sections with antisense probes transcribed with T3 RNA polymerase and the digoxigenin label detected using anti-digoxigenin Fabs coupled to alkaline
phosphatase and NBT/BCIP (Roche). Signals were not detected in the sense controls. References Angevine Jr, J.B., 1970. Time of neuron origin in the diencephalon of the mouse. An autoradiography study. J. Comp. Neurol. 139, 129–187. Lee, J.E., 1997. Basic helix–loop–helix genes in neural development. Curr. Opin. Neurobiol. 7, 13–30. Lee, J.E., Hollenberg, S.M., Snider, L., Turner, D.L., Lipnick, N., Weintraub, H., 1995. Conversion of Xenopus ectoderm into neurons by NeuroD, a basic helix–loop–helix protein. Science 268, 836–844. Liu, M., Pereira, F.A., Price, S.D., Chu, M.J., Shope, C., Himes, D., Eatock, R.A., Brownell, W.E., Lysakowski, A., Tsai, M.J., 2000. Essential role of BETA2/NeuroD1 in development of the vestibular and auditory systems. Genes Dev. 14, 2839–2854. Miyata, T., Maeda, T., Lee, J.E., 1999. NeuroD is required for differentiation of the granule cells in the cerebellum and hippocampus. Genes Dev. 13, 1647–1652. Murre, C., McCaw, P.S., Vaessin, H., Caudy, M., Jan, L.Y., Jan, Y.N., Cabrera, C.V., Buskin, J.N., Hauschka, S.D., Lassar, A.B., et al., 1989. Interactions between heterologous helix–loop–helix proteins generate complexes that bind specifically to a common DNA sequence. Cell 58, 537–544. Naya, F.J., Stellrecht, C.M., Tsai, M.J., 1995. Tissue-specific regulation of the insulin gene by a novel basic helix–loop–helix transcription factor. Genes Dev. 9, 1009–1019. Naya, F.J., Huang, H.P., Qui, Y., Mutoh, H., DeMayo, F.J., Leiter, A.B., Tsai, M.J., 1997. Diabetes, defective pancreatic morphogenesis, and abnormal enteroendocrine differentiation in BETA2/neuroD-deficient mice. Genes Dev. 11, 2323–2334. Peyton, M., Stellrecht, C.M., Naya, F.J., Samora, P.J., Tsai, M.J., 1996. BETA3, a novel helix–loop–helix protein, can act as a negative regulator of BETA2 and MyoD-responsive genes. Mol. Cell. Biol. 16, 626– 633. Takahaski, T., Nowakowski, R.S., Caviness, V.C., 1996. The leaving or Q fraction of the murine cerebral proliferative epithelium: a general model of neocortical neuronogenesis. J. Neurosci. 16, 6183–6196. Velculescu, V.E., Zhang, L., Vogelstein, B., Kinzler, K.W., 1995. Serial analysis of gene expression. Science 270, 484–487. Xu, Z., Dutra, A., Piatigorsky, J., 1999. Functional and structural dissection of a novel human BETA3 gene, a bHLH transcription factor. 49th Annual meeting of the American Society of Human Genetics, San Fransisco, CA.