1. Volume 104, Issue 6, Pages 861-873 (March 2001)
Groucho-Mediated Transcriptional Repression Establishes Progenitor Cell Pattern and Neuronal Fate in the Ventral Neural Tube 
Jonas Muhr, Elisabet Andersson, Madelen Persson, Thomas M. Jessell, Johan Ericson 
Cell 
Volume 104, Issue 6, Pages (March 2001)
DOI: /S (01)

2. Figure 1
Class II Nkx Proteins Act as TN Domain-Dependent Repressors and Interact with Gro/TLE Proteins In Vitro
(A) Position of the TN domain (white) and HD (dark grey) in Nkx proteins and comparison of the TN domain to the core sequence of the eh1 domain present in Engrailed (En). Sequences cover the TN domains of mouse (m) Nkx2.2 and Nkx2.9, rat Nkx6.1 (r), chick (c) Nkx6.2, Drosophila Vnd, and the eh1 domain of En. A Nkx TN domain consensus sequence was derived from the deduced amino acid sequence of 15 Nkx proteins (see Supplemental Figure S2 at Conserved amino acid positions are indicated by filled circles (>90%) or diamonds (70%–90%).
(B) Interaction between Drosophila Groucho (Gro) and Nkx proteins in vitro. A Gst-Gro fusion protein interacts with 35S-labeled Nkx2.2, Nkx2.9, Nkx6.1, and Nkx6.2. In (B) and (C), Input represents 10% of labeled protein used in binding assays.
(C) 35S-labeled mouse Grg4 interacts with Nkx2.2 and Nkx6.1 immobilized on antibody protein A-sepharose. Deletion of the TN domain in Nkx2.2 and Nkx6.1 reduced the interaction with Grg4 9-fold for Nkx2.2ΔTN and 5-fold for Nkx6.1ΔTN.
(D) The HDs of Nkx6.1 and Nkx6.2, but not Nkx2.2 or Nkx2.9, bind to a Nkx6.1 binding site (Jorgensen et al., 1999) in EMSA (Jorgensen et al., 1999). Nkx2.2HD and Nkx2.9HD, but not Nkx6.1HD or Nkx6.2HD, interact with a Nkx2.2 binding site (Watada et al., 2000). The Nkx2.2ΔTN and Nkx6.1ΔTN proteins did not differ from corresponding wild-type proteins in binding to their respective target DNA sequences (data not shown).
(E) Nkx proteins act as TN domain-dependent repressors in vitro.
Gal-Nkx2.2, Gal-Nkx6.1, and Gal-Nkx6.2 reduced luciferase reporter activity ∼15-fold and Gal-Nkx2.9 reduced activity ∼6-fold compared to the control (Gal-only). Deletion of the TN domain abolished the repressor activity of Nkx2.2 (Gal-Nkx2.2ΔTN) and Nkx2.9 (Gal-Nkx2.9ΔTN) and greatly reduced the repressor activity of Nkx6.1 (Gal-Nkx6.1ΔTN) and Nkx6.2 (Gal-Nkx6.2ΔTN). The repressor activity of Gal-Nkx2.2, Gal-Nkx2.9, Gal-Nkx6.1, and Gal-Nkx6.2 was enhanced up to ∼10-fold by introducing increasing amounts of Grg4. Increasing amounts of Grg4 had no corepressor activity in experiments with Gal-Nkx2.2ΔTN or Gal-Nkx2.9ΔTN, but weakly enhanced the repressor activity of Gal-Nkx6.1ΔTN or Gal-Nkx6.2ΔTN (∼2-fold). Grg4 alone did not reduce luciferase reporter activity. Error bars indicate standard deviation (SD) of 3–6 independent transfections
Cell  , DOI: ( /S (01) )

3. Figure 2
Nkx Proteins Act as TN Domain-Dependent Repressors in Neural Patterning In Vivo
(A) Constructs used: full-length Nkx2.2 and Nkx6.1; Nkx2.2ΔTN and Nkx6.1ΔTN with the TN domain (yellow) deleted; hybrid proteins consisting of the Nkx2.2 HD (2.2HD, red) or Nkx6.1 HD (6.1HD, orange) coupled to: (1) a myc-tagged EnR (InR, blue); (2) the transactivation domain of VP16 (VP16, green); (3) their respective isolated TN domains; (4) a myc-tag (dark gray).
(B) Constructs outlined in panel 2A were fused to Gal4 and examined for their activity in a transcription reporter assay in COS-7 cells. Error bars indicate standard deviation of three independent transfections.
(C) (i): Nkx2.2 and Pax6 are expressed in mutually exclusive domains in the spinal cord of normal HH stage 20 chick embryos. (ii) to (xi): Consequences of misexpression of Nkx2.2, Nkx2.2ΔTN, Nkx2.2HD-EnR, Nkx2.2HD-TN, and Nkx2.2-VP16 on progenitor HD protein expression.
(D) Nkx6.1 and Dbx2 are expressed in mutually exclusive domains in the ventral spinal cord of HH stage 20 chick embryos (i). (ii) to (xi): Consequences of misexpression of Nkx6.1, Nkx6.1ΔTN, Nkx6.1HD-EnR, Nkx6.1HD-TN, and Nkx6.1-VP16 on progenitor HD protein expression. Forced expression of Nkx6.1ΔTN resulted in a small reduction in numbers of Dbx2+ cells (12 ± 6% cells/section n = 5). The efficiency of repression was determined by comparing the numbers of Dbx2+ cells on the electroporated and control sides
Cell  , DOI: ( /S (01) )

4. Figure 3
The Repressor Activity of Class II Nkx Proteins Specifies Ventral Neuronal Cell Fate
(A) Consequences of misexpression of Nkx2.2, Nkx2.2ΔTN, Nkx2.2HD-EnR, Nkx2.2HD-TN, and Nkx2.2-VP16 on Sim1 expression. Expression of Nkx2.2HD-EnR resulted in the ectopic expression of endogenous Nkx2.2 protein in a few cells scattered within the ventral neural tube (see Supplemental Figure S1 at likely reflecting the ability of Nkx2.2HD-EnR to repress Pax6 (Ericson et al., 1997). However, few ectopic Nkx2.2+ cells were detected, and it is unlikely that the activation of endogenous Nkx2.2 expression accounts for the broad activation of Sim1 expression observed.
(B) Consequences of misexpression of Nkx2.2, Nkx2.2ΔTN, Nkx2.2HD-EnR, Nkx2.2HD-TN, and Nkx2.2-VP16 on MN marker expression.
(C) Consequences of misexpression of Nkx6.1, Nkx6.1ΔTN, Nkx6.1HD-EnR, Nkx6.1HD-TN, and Nkx6.1-VP16 on MN (MNR2 and Hb9) and interneuron (Lim3, Chx10, and En1) marker expression
Cell  , DOI: ( /S (01) )

5. Figure 4
Class I Proteins that Interact with Gro/TLE Proteins Act as eh1 Domain-Dependent Repressors In Vivo
(A) The class I proteins Dbx1, Dbx2, Pax3, and Pax7 contain eh1-like domains (white). HDs (dark gray) and paired domains (light gray) are also indicated. The Nkx TN domain consensus sequence and the eh1 domain of En are shown. Conserved amino acids are indicated by filled circles (>90%) or diamonds (70%–90%).
(B–F) Interaction between Gst-Gro fusion protein with 35S-labeled class I proteins.
(G–J) Consequences of ectopic expression of Dbx2, Dbx2ΔEh1, Dbx2HD-EnR, and Dbx2HD-VP16 on Nkx6.1 expression.
(K–N) Consequences of misexpression of Pax6, Pax6PD-VP16, and Pax6PD-EnR on Nkx2.2 expression in p3 domain progenitors
Cell  , DOI: ( /S (01) )

6. Figure 5 Patterns of Grg Gene Expression in the Neural Tube
(A–D) Localization of Grg1, Grg3b, Grg4, and Grg5 expression in E10 mouse spinal cord. At E9.5, little or no Grg5 expression was detected in neural progenitor cells (data not shown). Most post-mitotic spinal neurons express Grg5.
(E–G) Expression of Grg4 in developing chick neural tube
Cell  , DOI: ( /S (01) )

7. Figure 6
In Vivo Expression of Grg5 Deregulates Class I and Class II Protein Expression
(A) Grg5, but not Grg5ΔQ, interacts with Drosophila Gro.
(B) Grg5 inhibits Nkx-mediated repression in vitro. Grg5, but not Grg5ΔQ, reduced the repressor activities of Gal-Nkx2.2 and Gal-Nkx6.1 in COS-7 cell reporter assays. Grg5, but not Grg5ΔQ, also inhibits the ability of Grg4 to enhance the repressor activity of Gal-Nkx2.2 or Gal-Nkx6.1. Grg5 also reduces the repressor activity of Gal-Nkx2.9 and Gal-Nkx6.2 (data not shown).
(C) Consequences of ectopic expression of Grg5 (i–vii) and Grg5ΔQ (viii–xiv) on marker gene expression in chick neural tube.
(D) Consequences of ectopic expression of Nkx6.1 and Grg5 or Grg5ΔQ on Lim3 expression in chick neural tube. Coelectroporation of Nkx6.1 and myc-tagged Grg5ΔQ (i) results in induction of Lim3 (29 ± 8 cells, n = 3 cells/section) (ii) and Hb9 expression (data not shown). Few ectopic dorsal Lim3 cells (4 ± 3 cells/section, n = 5) (iv) and HB9+ cells (data not shown) were detected after Nkx6.1 was coelectroporated with myc-tagged Grg5 (iii). The endogenous ventral domains of Lim3 and MNR2 expression appeared unaffected in experiments in which Grg5 had been misexpressed (iv and data not shown). Expression of Grg5 or Grg5ΔQ alone did not result in ectopic induction of Lim3 or HB9 expression (data not shown). The neural dorsal boundary of Lim3 expression is indicated by dashed line
Cell  , DOI: ( /S (01) )

8. Figure 7
Gro/TLE-Mediated Repression and the Pattern of Neuronal Specification in the Ventral Neural Tube
Schematic model showing: (1) the spatial pattern of progenitor HD protein expression in the ventral neural tube, (2) the position of progenitor domains, and (3) selected interactions between progenitor HD proteins and neuronal subtype determinants. This model depicts repressive interactions as direct, but this remains to be shown. For simplicity, corresponding interactions within the p0 and p2 domains are not shown.
p3 domain : Nkx2.2-Gro/TLE-mediated repression prevents expression of Pax6. Nkx6.1 is also expressed within p3 progenitors (not shown), but the repressive action of Nkx2.2-Gro/TLE prevents expression of MNR2, thus permitting expression of a V3 determinant (det).
pMN domain : Pax6 activates expression of a Gro/TLE-dependent repressor (R), which represses Nkx2.2 and a V3 neuronal subtype determinant. Nkx6.1-Gro/TLE activity represses the expression of Dbx2 and other class I proteins (not shown), resulting in the derepression of MNR2. Nkx6.1-Gro/TLE activity also represses V1 (and other ventral interneuron) subtype determinants, thus ensuring that cells do not inappropriately initiate the generation of more dorsal neuronal subtypes.
p1 domain : Dbx2-Gro/TLE activity represses expression of Nkx6.1 and MNR2, permitting expression of a V1 subtype determinant.
pD domain : In the dorsal (D) neural tube, equivalent Gro/TLE-dependent repressors normally prevent expression of ventral progenitor HD proteins such as Nkx6.1 (and Nkx2.2, not shown), and prevent expression of neuronal subtype determinants, such as MNR2 (and a V3 determinant, not shown). These dorsal Gro/TLE-dependent repressors are subject to repression by Nkx6.1 and Nkx2.2. The distinct ventral neuronal subtypes that emerge after ectopic dorsal expression of Nkx6.1 (MNs) and Nkx2.2 (V3 neurons) can be explained by the ability of Nkx2.2 but not Nkx6.1 to repress Pax6. Ectopic Nkx6.1 permits expression of Pax6, the activation of (R), and the repression of the V3 neuronal subtype determinant
Cell  , DOI: ( /S (01) )