Border formation in a Bmp gradient reduced to single dissociated cells - PubMed (original) (raw)
Border formation in a Bmp gradient reduced to single dissociated cells
Jia Sheng Hu et al. Proc Natl Acad Sci U S A. 2008.
Abstract
Conversions of signaling gradients into sharp "all-or-none" borders are fundamental to tissue and organismal development. However, whether such conversions can be meaningfully reduced to dissociated cells in culture has been uncertain. Here we describe ultrasensitivity, the phenomenon equivalent to an all-or-none response, in dissociated neural precursor cells (NPCs) exposed to bone morphogenetic protein 4 (Bmp4). NPC ultrasensitivity is evident at the population and single-cell levels based on Msx1 induction, a well known Bmp target response, and occurs in the context of gene expression changes consistent with Bmp4 activity as a morphogen. Dissociated NPCs also display immediate early kinetics and irreversibility for Msx1 induction after brief Bmp4 exposure, which are attractive features for initial border formation. Relevance to border formation in vivo is provided by Bmp4 gain-of-function studies in explants and evidence for single-cell ultrasensitivity in normal and mutant Bmp gradient contexts in the developing forebrain. Together, these studies demonstrate relatively simple, robust, and reducible cell-intrinsic properties that contribute to developmental border formation within a signaling gradient.
Conflict of interest statement
The authors declare no conflict of interest.
Figures
Fig. 1.
Confirmations of graded Bmp activity and NPC threshold conversion in dorsal forebrain explants. (A) Coronal schematics of the E10.5 telencephalon (light blue) and Msx1-expressing dorsal midline (dark blue). (B) Graded Bmp activity (pSmad1/5/8) and three thresholded Msx1 readouts at the E10.5 dorsal midline. Msx2 is more broadly expressed than Msx1 in this region (50), accounting for the larger expression domain detected with the Msx1/2 antibody. Right highlights expression in neural tissue for clarity. (Scale bar: 0.1 mm.) (C) Models of Msx1 threshold conversion with and without exogenous Bmp4 point sources (beads), which predict dose-, distance-, and orientation-dependent ectopic Msx1 induction. The endogenous Bmp gradient profile is based on previous measurements (10). (D) Explant and bead schematic. (E and F) Ectopic Msx1 mRNA and nlacZ inductions (arrowheads) around Bmp4-soaked, but not BSA-soaked, blue Affigel (E) or clear heparin acrylic beads (F) in E10.5 explants cultured for 2 days. Findings confirm predictions described in C and establish Bmp4 and cortical NPC sufficiencies for threshold conversion in explants. [Scale bars: 1 mm (low power) and 0.2 mm (high power).] c, cortex; d, diencephalon; dm, dorsal midline; ee, epidermal ectoderm; l, lateral ganglionic eminence; m, medial ganglionic eminence; me, mesenchyme.
Fig. 2.
NPC ultrasensitivity and irreversibility in dissociated cultures at the population level. (A and B) Schematic Michaelis–Menten, ultrasensitive, bistable, and irreversible response curves. (C–F) Hill plots of real-time qRT-PCR data from E10.5 Msx1-nlacZ (D), E12.5 Msx1-nlacZ (E), and E12.5 wild-type (F) cortical NPC cultures. Native and nlacZ-containing Msx1 transcript inductions display significant ultrasensitivity in response to Bmp4 (nH = 2.4–3.8), whereas Tgif induction is non-ultrasensitive (nH = 0.3 using data points up to 128 or 256 ng/ml Bmp4). Tgif up-regulations at the two highest Bmp4 doses were significant by t test (P < 0.05). (G) Real-time qRT-PCR data from E10.5 Msx1-nlacZ cells for other midline genes. Unlike endogenous Msx1 transcripts, Ttr is up-regulated at intermediate Bmp4 concentrations, whereas both Ttr and Lmx1a display suppression at higher Bmp4 concentrations associated with maximal Msx1 induction, consistent with a morphogen effect. Ttr up-regulations at 8 and 16 ng/ml Bmp4 were significant by t test (P < 0.05). (H–J) Induction kinetics, wild-type cortical NPCs, 50 ng/ml Bmp4. Msx1 up-regulation is detected by 15 min, is statistically significant by 2 h, and is stable for at least 5 days in the continuous presence of Bmp4. (K and L) Washout studies, wild-type cortical NPCs, 50 ng/ml Bmp4. Maximal Msx1 induction requires only brief Bmp4 exposure (no more than 15 min) and does not require Bmp4 thereafter, thus displaying irreversibility. Error bars show standard errors. t tests: *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Fig. 3.
NPC ultrasensitivity in dissociated cultures at the single-cell level. (A) Schematics of graded and ultrasensitive responses at the single-cell level. (B) Culture schematic. (C) Double label immunocytochemistry of E12.5 wild-type cortical NPCs. Msx1/2 induction (red) is ultrasensitive compared with pSmad (green), which is graded and more homogeneous. Similar graded pSmad findings were seen in four independent cultures. (Scale bar: 0.05 mm.) (D) Percentage of X-Gal-positive cells in E12.5 Msx1-nlacZ cortical NPCs (
SI Fig. 6
). Msx1-nlacZ induction occurs equally well in cells that are completely isolated compared with those in contact with other cells. Errors bars show standard errors from two independent cultures.
Fig. 4.
Evidence for single-cell ultrasensitivity in intact tissues. (A) Msx1/2-positive NPCs (arrows) surrounded by negative cells near the E10.5 cortex–midline border. [Scale bars: 0.1 mm (low power) and 0.05 mm (high power).] (B) Scattered Msx1-nlacZ-positive NPCs (arrows) amid negative cells surrounding a Bmp4-soaked heparin acrylic bead in E10.5 explants cultured for 2 days. B Right Inset is inverted and thresholded for clarity. Dashed lines, beads; arrowhead, endogenous Msx1-nlacZ-expressing dorsal midline domain. [Scale bars: 0.2 mm (low power) and 0.05 mm (high power).] (C) Scattered Msx1 transcript-positive NPCs (arrowheads) amid negative cells in the E10.5 dorsal midline region after roof plate ablation. The pSmad1/5/8 gradient, which is reduced and flattened compared with normal (10), remains continuous across the cortex–midline border. C Right highlights neural expression for clarity. [Scale bars: 0.1 mm (low power) and 0.05 mm (high power).] (D) Model using NPC-intrinsic ultrasensitivity and irreversibility to initially convert the Bmp gradient into a crude Msx1 border, which is then refined by additional mechanisms into a sharp cortex–midline boundary. cx, cortex; dm, dorsal midline.
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