Cascades of transcription factor function combined with elaborate
feedback and feedforward mechanisms are fundamental to
generating nervous system circuitry. These processes ensure the
generation of the appropriate balance of specific neuronal subtypes.
In the development of the dorsal spinal cord, a transcription factor
network is beginning to be defined that regulates the balance of
inhibitory and excitatory neurons generated from progenitor cells.
Central to this network are transcription factors from the basic helix-
loop-helix (bHLH) and homeodomain (HD) class of proteins
(Cheng et al., 2005; Cheng et al., 2004; Glasgow et al., 2005; Gowan
et al., 2001; Gross et al., 2002; Helms et al., 2005; Kriks et al., 2005;
Mizuguchi et al., 2006; Müller et al., 2002; Wildner et al., 2006).
The bHLH factor Ptf1a is particularly important for promoting
GABAergic inhibitory neurons while suppressing glutamatergic
excitatory neurons in both the dorsal horn of the spinal cord and the
cerebellum (Glasgow et al., 2005; Hori et al., 2008; Hoshino et al.,
2005; Pascual et al., 2007). Whereas it has been shown that Ptf1a is
required for expression of the HD factors Pax2, Lhx1 and Lhx5, and
suppression of Tlx3 and Lmx1b, no direct downstream targets of
Ptf1a have been identified in the nervous system. We demonstrate
that the gene encoding the bHLH factor Neurog2 is a direct target of
Ptf1a is a component of a unique transcription complex called
PTF1-J that includes two bHLH factors, Ptf1a and an E-protein such
as Tcfe2a-E12, and Rbpj (Beres et al., 2006; Hori et al., 2008; Masui
et al., 2007). Ptf1a is the tissue-specific component of the PTF1-J
complex, and thus its pattern of expression defines domains of
PTF1-J function. In mouse embryos between embryonic days (E)
10.5 and 13.5, Ptf1a is largely restricted to the dorsal neural tube
from the hindbrain to the tail, and to the pancreatic anlage (Glasgow
et al., 2005; Obata et al., 2001). Other domains of expression include
the embryonic retina in progenitors to amacrine and horizontal
neurons and a subset of cells in the developing hypothalamus
(Fujitani et al., 2006; Glasgow et al., 2005; Nakhai et al., 2007).
Within the caudal neural tube, Ptf1a is restricted to the progenitor
domain that gives rise to dI4 and dILAinterneurons, which
contribute to the GABAergic inhibitory neural network in the dorsal
horn (Glasgow et al., 2005) (Fig. 1B). The requirement for Ptf1a in
the generation of many of these cell types has been demonstrated in
mice and humans mutant for the gene (Dullin et al., 2007; Fujitani
et al., 2006; Glasgow et al., 2005; Hoshino et al., 2005; Kawaguchi
et al., 2002; Nakhai et al., 2007; Pascual et al., 2007; Sellick et al.,
2004; Yamada et al., 2007).
The PTF1-binding site is unique in that it contains both an E-box
for bHLH heterodimer binding, and a TC-box for Rbpj binding
(Beres et al., 2006; Masui et al., 2007). This bipartite consensus
sequence was defined for a form of PTF1 (PTF1-L) that contains the
Rbpj homolog Rbpj-l. PTF1-L controls pancreas specific targets in
the adult pancreas (Beres et al., 2006). We identify a sequence
similar to the PTF1 consensus site in a 3? regulatory enhancer for
Neurog2. The bHLH factor Neurog2 (Ngn2, Math4A) functions in
neuronal differentiation and is expressed in a precise spatial and
temporal pattern during the development of the vertebrate sensory
ganglia, spinal cord and brain (Fode et al., 1998; Gradwohl et al.,
1996; Kele et al., 2006; Ma et al., 1999; Sommer et al., 1996). We
provide evidence that the PTF1-J complex controls Neurog2
transcription in the dorsal neural tube and cerebellum through its 3?
enhancer. Furthermore, we demonstrate that regulation of Neurog2
by Ptf1a in the PTF1-J complex is direct. The identification of direct
targets of PTF1-J is the first step in providing mechanistic insight
into the transcriptional control generating the balance of inhibitory
and excitatory neuronal circuitry.
Neurog2 is a direct downstream target of the Ptf1a-Rbpj
transcription complex in dorsal spinal cord
R. Michael Henke1,*, Trisha K. Savage1,*, David M. Meredith1, Stacey M. Glasgow1, Kei Hori1, Judy Dumas1,
Raymond J. MacDonald2and Jane E. Johnson1,†
PTF1-J is a trimeric transcription factor complex essential for generating the correct balance of GABAergic and glutamatergic
interneurons in multiple regions of the nervous system, including the dorsal horn of the spinal cord and the cerebellum. Although
the components of PTF1-J have been identified as the basic helix-loop-helix (bHLH) factor Ptf1a, its heterodimeric E-protein partner,
and Rbpj, no neural targets are known for this transcription factor complex. Here we identify the neuronal differentiation gene
Neurog2 (Ngn2, Math4A, neurogenin 2) as a direct target of PTF1-J. A Neurog2 dorsal neural tube enhancer localized 3? of the
Neurog2 coding sequence was identified that requires a PTF1-J binding site for dorsal activity in mouse and chick neural tube. Gain
and loss of Ptf1a function in vivo demonstrate its role in Neurog2 enhancer activity. Furthermore, chromatin immunoprecipitation
from neural tube tissue demonstrates that Ptf1a is bound to the Neurog2 enhancer. Thus, Neurog2 expression is directly regulated
by the PTF1-J complex, identifying Neurog2 as the first neural target of Ptf1a and revealing a bHLH transcription factor cascade
functioning in the specification of GABAergic neurons in the dorsal spinal cord and cerebellum.
KEY WORDS: bHLH transcription factor, Cerebellum development, Dorsal neural tube development, Gene regulation, Spinal cord
Development 136, 2945-2954 (2009) doi:10.1242/dev.035352
1Department of Neuroscience and 2Department of Molecular Biology, University of
Texas Southwestern Medical Center, Dallas, TX 75390, USA.
*These authors contributed equally to this work
†Author for correspondence (email@example.com)
Accepted 23 June 2009
MATERIALS AND METHODS
Mutant mouse strains have been previously described, including Ascl1null
(Mash1null) (Guillemot et al., 1993), Ptf1aCre(p48Cre) (Kawaguchi et al.,
2002) used here as the Ptf1a null, and Ptf1aW298A(Masui et al., 2007).
Transgenic mice were generated by standard procedures (Brinster et al.,
1985) using fertilized eggs from B6D2F1 (C57BL/6?DBA) or B6SJLF1
(C57BL/6J?SJL) crosses. The TgN2-4 transgenes and variants were
isolated from vector sequences using standard procedures and injected into
the pronucleus of fertilized mouse eggs at 1-3 ng/μl in 10 mM Tris (pH 7.5),
0.1 mM EDTA. Transgenic embryos were identified by PCR for the lacZ
gene using yolk sac DNA. Embryos were staged based on assumed
copulation halfway through the dark cycle, designated E0. All procedures
on animals follow NIH Guidelines and were approved by the UT
Southwestern Institutional Animal Care and Use Committee.
TgN2-4 transgene was previously published and contains a 1047 bp
enhancer located 226 bp 3?of the Neurog2coding sequence stop codon, and
when paired with a β-globin heterologous basal promoter [in BGZA (Yee
and Rigby, 1993)] it directs expression of a lacZ reporter gene to the dorsal
neural tube as well as a small ventral neural tube domain at E11.5
in transgenic mice (Simmons et al., 2001). Mutant variations of this
transgene were generated by PCR; E-boxes (CANNTG) were mutated to
(atNNTG) and TC-boxes (WTTCCCA) were mutated to (WTagaCA). The
4?PTF1 transgene has four copies of the sequence AATGG CT -
GGCATCTGCTCTATTCCCATTGCTGTCT, which contains a PTF1
consensus sequence (underlined) plus some flanking nucleotides cloned in
the 5? polylinker of the BGZA reporter. The Ptf1a expression construct for
the chick electroporations was in the expression vector pMiWIII, as
previously described (Hori et al., 2008). All plasmids were sequence
Immunofluorescence and β β-gal staining
For immunofluorescence, E11.5 mouse embryos were dissected in ice-cold
0.1 M sodium phosphate buffer pH 7.4, fixed in 4% formaldehyde for 2
hours at 4°C and washed three times in 0.1 M sodium phosphate buffer pH
7.4 for 2 hours. Embryos were sunk overnight in 30% sucrose in 0.1 M
sodium phosphate buffer pH 7.4, embedded in OCT and cryosectioned at 30
μm. All sections of neural tubes were from the upper limb level.
Immunofluorescence was performed using the following primary
antibodies: chick anti-β-gal (1:250; Abcam), rabbit anti-Pax2 (1:1,000;
Zymed), guinea pig anti-Brn3a (Pou4f1 – Mouse Genome Informatics)
(1:10,000; generated for this study using GST-Brn3a from E. Turner,
UCSD), guinea pig anti-Ptf1a (1:10,000) (Hori et al., 2008) and mouse anti-
Neurog2 (1:10; D. Anderson, Caltech). Fluorescence conjugated species-
specific secondary antibodies were used from Molecular Probes.
Fluorescence imaging was carried out on a Bio-Rad MRC 1024 confocal
microscope. For each experiment multiple sections from at least three
different animals were analyzed.
For β-gal staining, E10.5 embryos or E17.5 brains were dissected in ice-
cold 0.1 M sodium phosphate buffer pH 7.4, fixed in 4% formaldehyde and
processed in whole mount for β-gal staining as described (Simmons et al.,
2001). After imaging, embryos and brains were agarose embedded and
vibratome sectioned at 100 μm and mounted on slides for imaging.
Chicken in ovo electroporation
Fertilized White Leghorn eggs were obtained from the Texas A&M Poultry
Department (College Station, TX) and incubated at 39°C for 5 days.
Solutions of supercoiled plasmid DNA (2 μg/μl) in PBS/0.02% Trypan Blue
were injected into the lumen of the closed neural tube at stage HH14-16, and
embryos were electroporated as previously described (Timmer et al., 2001).
Embryos were harvested 48 hours later, fixed with 4% formaldehyde for 1
hour and processed as above for β-gal staining or for immunofluorescence.
Embryos were embedded in OCT, cryosectioned at 30 μm and mounted on
slides. Images shown in Fig. 7 are representative of expression seen in more
than eight embryos per condition.
In vitro translated mouse Tcfe2a-E12, mouse Ptf1a, the mutant Ptf1aW298A
and human Rbpj proteins were synthesized in vitro using SP6 and T7 TNT
Quick Coupled lysate systems (Promega, Madison, WI), and were quantified
using 35S-Met according to the manufacturer’s directions. TNT lysates were
incubated in binding buffer (10 mM Hepes pH 7.9, 4 mM Tris-HCl pH 8.0,
Development 136 (17)
Fig. 1. A dorsal-neural-tube-specific enhancer
for Neurog2. (A,D,E) Diagram of the Neurog2
gene and transgenic constructs (A) showing the
location on mouse chromosome 3 of the 3?
enhancer (TgN2-4) that directs expression of a lacZ
reporter (BgZA) to both a dorsal (DNT) and a
ventral (VNT) region in the E11.5 neural tube of
transgenic mice (β-gal stained neural tube section
shown in D) (Simmons et al., 2001), and a deletion
that specifically disrupts the dorsal pattern (TgN2-
4ΔDNT; β-gal-stained neural tube section shown in
E). # exp is the number of independently derived
transgenic embryos analyzed that expressed the
transgene. (B)Ptf1a immunofluorescence on a
transverse section of the neural tube illustrates
Ptf1a is restricted to and defines the dorsal
progenitor domain 4 (dP4) that will give rise to
dorsal interneuron population 4 (dI4) (Glasgow et
al., 2005). (C)The complex pattern of endogenous
Neurog2 in the neural tube is shown by mRNA in
situ hybridization. The dashed boxes in C-E indicate
the Neurog2 expression domain that is the focus of
this study. (F)Mouse sequence (133 bp) from the
Neurog2 3? enhancer that contains the dorsal-
specific regulatory region deleted in TgN2-4ΔDNT.
The nucleotides conserved between mouse and
human are shown in uppercase letters, and non-
conserved nucleotides are in lowercase. E-boxes
(red), TC-boxes (blue) and the PTF1-binding site
(shaded) that were tested in transgenic mouse
assays (see Fig. 6) are indicated.
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