July 1, 2002;
Distinct patterns of downstream target activation are specified by the helix-loop-helix domain of proneural basic helix-loop-helix transcription factors.
Both gain- and loss-of-function analyses indicate that proneural basic/helix-loop-helix (bHLH) proteins direct not only general aspects of neuronal differentiation but also specific aspects of neuronal identity within neural progenitors. In order to better understand the function of this family of transcription factors, we have used hormone-inducible fusion constructs to assay temporal patterns of downstream target regulation in response to proneural bHLH overexpression. In these studies, we have compared two distantly related Xenopus proneural bHLH genes, Xash1
. Our findings indicate that both Xash1
induce expression of the general neuronal differentiation marker, N-tubulin
, with a similar time course in animal cap
progenitor populations. In contrast, these genes each induce distinct patterns of early downstream target expression. Both genes induce expression of the HLH-containing gene, Xcoe2
, at early time points, but only XNgnr1
induces early expression of the bHLH genes, Xath3
. Structure:function analyses indicate that the distinct pattern of XNgnr1
-induced downstream target activation is linked to the XNgnr1
HLH domain, demonstrating a novel role for this domain in mediating the differential function of individual members of the proneural bHLH gene family.
[+] show captions
FIG. 1. Inducible Xash1 and XNgnr1 function. (A) (i–iii) Xash1 and XNgnr1 expression and function in Xenopus embryos. Xash1 (i),
XNgnr1 (ii), and N-tubulin (iii) expression in Xenopus embryos at stage 15. (i) Asterisks indicate three bilaterally symmetric domains of
Xash1 expression. Arrowhead indicates medial expression domain. (ii and iii) Asterisks indicate domains of primary neurogenesis in the
embryo. (iv–vi) Hormone-inducible Xash1 and XNgnr1 function in the embryo. Embryos injected with GRXash1 (iv and v) or GRXNgnr1
(vi) and incubated either in the absence (iv) or presence (v and vi) of Dex to stage 15/16. N-tubulin expression marks domains of ectopic
neurogenesis (dark blue/purple staining) and -gal (blue) expression serves as a lineage marker. (B) PCR analysis of GRXash1 and GRXNgnr1
function in Xenopus animal caps. Sequence-specific primers were used to assay N-tubulin (N-tub) and Neurofilament-M (NF-M) expression
in GRXash1- and GRXNgnr1-injected animal cap samples incubated for 20 h with (D) or without ( ) Dex. Embryo (E) and uninjected animal
caps exposed to Dex for 20 h (C) were used as controls in these experiments. Expression of muscle specific actin (MSA) was used to control
for mesodermal contamination, and expression of EF1 served as an internal quantitation control.
FIG. 2. Early target regulation in GRXash1-, GRXNgnr1-, GRXash3-,
and GRXath1-injected animal caps. PCR analysis with sequencespecific
primers was used to assay gene expression in animal caps
isolated from GRbHLH injected embryos and incubated either in the
presence (D) or absence ( ) of Dex for 3 h. At this time, control
embryos had reached stage 11.5/12. Whole embryo (E) and uninjected
animal caps exposed to Dex for 3 h (C) were used as controls.
Expression of Brachyury (Bra) was used as a control for mesodermal
contamination, and EF1 served as an internal quantitation control.
FIG. 3. GRXash1 dose response. Gene expression was analyzed by RT-PCR at 3 (A) and 20 h (B) post-Dex addition in response to increasing
doses of GRXash1. Control animal caps exposed to Dex (C) and animal caps injected with 1 ng of GRXash1 and cultured in the absence of
Dex (no dex) were used as negative controls for these experiments. Whole embryos (E) were used as positive controls. Muscle specific actin
(MSA; A) and Brachyury (Bra; B) were used to control for mesoderm contamination, and EF1 was used as an internal quantitation control.
FIG. 4. Time course of GRXash1 and GRXNgnr1 function. PCR analyses were used to analyze gene expression at successive time points
following activation of GRXash1 and GRXNgnr1 function by hormone addition. Control embryos had reached stage 11.5 (3 h), stage 12.5
(5 h), stage 19 (10 h), stage 22 (13 h), stage 24 (16 h), and stage 27 (20 h) at time of animal cap harvest. Injected animal caps incubated without
Dex ( ) as well as uninjected animal caps incubated in the presence of Dex (C/D) were assayed at 3 and 20 h to serve as controls for these
experiments. Whole embryo (E) cDNA was also assayed. In these experiments, muscle specific actin (MSA) was used as a control for
mesoderm contamination, and EF1 was used for internal quantitation.
FIG. 5. The role of the HLH domain in patterns of downstream target activation. (A) Diagram of chimeric molecules generated from Xash1
and XNgnr1 protein coding sequences. In NHX, the HLH domain of XNgnr1 specifically replaces Xash1 HLH domain. In NBX, the basic
domain of XNgnr1 specifically replaces the Xash1 basic domain. In NH1X, sequences encoding the first 17 N-terminal amino acids of the
XNgnr1 HLH domain replace corresponding Xash1 sequences. In this diagram, lines between Xash1 and XNgnr1 sequences indicate amino
acid identity, while dashed lines indicate conservative substitutions. All chimeric molecules were subcloned in-frame with glucocorticoid
receptor ligand-binding domain sequences to yield GR fusion constructs (GRNBX, GRNHX, GRNH1X). (B, C) The chimeric constructs (B:
GRNBX, GRNHX; C: GRNH1X) as well as the wild type Xash1 and XNgnr1 genes were analyzed in animal cap assays as described in the
text (Results). Dex was added to animal cap samples at time of isolation (stage 8–9), animal caps were harvested at stage 11.5, and gene
expression was assayed by RT-PCR. Normal embryos (E) and uninjected animal caps (C) served as controls for these experiments.