HMGB factors are required for posterior digit development through integrating signaling pathway activities

Abstract

The chromatin factors Hmgb1 and Hmgb2 have critical roles in cellular processes, including transcription and DNA modification. To identify the function of Hmgb genes in embryonic development, we generated double mutants of Hmgb1;Hmgb2 in mice. While double null embryos arrest at E9.5, Hmgb1(-/-) ; Hmgb2(+/-) embryos exhibit a loss of digit5, the most posterior digit, in the forelimb. We show that Hmgb1(-/-) ; Hmgb2(+/-) forelimbs have a reduced level of Shh signaling, as well as a clear downregulation of Wnt and BMP target genes in the posterior region. Moreover, we demonstrate that hmgb1 and hmgb2 in zebrafish embryos enhance Wnt signaling in a variety of tissues, and that double knockdown embryos have reduced Wnt signaling and shh expression in pectoral fin buds. Our data show that Hmgb1 and Hmgb2 function redundantly to enhance Wnt signaling in embryos, and further suggest that integrating Wnt, Shh, and BMP signaling regulates the development of digit5 in forelimbs.

Fig. 1. Skeletal phenotype of the Hmgb1^-/-; Hmgb2^+/- limb

Alcian Blue and Alizarin red-stained E16.5 forelimbs (A-H) and hindlimbs (I-P) of Hmgb1; Hmgb2 mutants are shown. Genotypes of Hmgb1; Hmgb2 are indicated on the top: (A, E, I, M) +/+;+/+, (B, F, J, N) +/–;+/–, (C, G, K, O) +/–;–/–, (D, H, L, P) –/–;+/–. AD and I-L show lateral views of entire forelimb and hindlimb skeletons, respectively. E-H and M-P show dorsal views of the autopod. In (A) and (I) the stylopod, zeugopod and autopod are indicated as s, z and a, and in (E) and (H) digits are indicated with 1-5. The loss of digit 5 is indicated by red arrows in (D) and (H).

Fig. 2. Specific defects in the posterior development in the Hmgb1^-/-; Hmgb2^+/- limb

Control (A, C, E, G) and Hmgb1^-/-; Hmgb2^+/- (B, D, F, H) forelimb buds at E13.5 (A, B) and E11.5 (C-H). Alcian Blue staining (A, B) and Sox9 staining (C, D) visualized the loss of digit 5 in Hmgb1^-/-; Hmgb2^+/- forelimb bud (red arrows). Digit tips are marked by asterisks. Pax9 expression is normal in both control (E) and mutant (F) forelimb bud (black arrows). Hoxd13 expression in the autopod region is detected in both control (G) and mutant (H) forelimb bud. The posterior defect is visible in the mutant limb bud at E11.5 (red arrows). The Hoxd13-non-expressiong domain showed a slight expansion (marked by dotted line). All panels are dorsal views with the anterior to the top.

Fig. 3. Downregulation of Shh signaling in the Hmgb1^-/-; Hmgb2^+/- forelimb bud

Control (A, C, E, G, I, K) and Hmgb1^-/-; Hmgb2^+/- (B, D, F, H, J, L) forelimb buds at E10.0 (A-D) and at E10.5 (E-L). Shh (A, B, E, F), Gli1 (C, D), Ptc1 (G, H), Gre1 (I, J) and CycD1 (K, L) expression are shown. Compared to the expression in the control forelimb bud (black arrows), expression in the mutant forelimb bud is not initiated (red arrowhead in B) or downregulated (red arrows). All panels are dorsal views with the anterior to the top.

Fig. 4. Normal signaling by AER-FGF and Wnt7a from the dorsal ectoderm

Control (A, B, E, G, I, K, L) and Hmgb1^-/-; Hmgb2^+/- (C, D, F, H, J, L, M, N) forelimb buds at E10.5. Fgf8 in the AER and Shh in the posterior mesenchyme are co-stained (A-D). Fgf10 (E, F) and Mkp3 (G, H) expression in response to AER-FGF are shown. Despite the downregulation of Shh (A-D), Fgf8 expression in the AER (A-D) and expression of Fgf10 (E, F) and Mkp3 (G, H) are normal. (I, J) pERK1/2 staining on sections is also normal. Dotted lines show the boundary of the pERK1/2 signal. Patchy signals are derived from blood cells and were positive in all fluorescent channels. Lmx1b expression in the control (K, L) and mutant (M, N) forelimb bud shows normal Wnt7a signaling into dorsal mesenchyme. All panels except for B, D, I, J, L and N are dorsal views, and B, D, L and N are distal views with the anterior to the top, and I and J are section images with the AER to the bottom.

Fig. 5. Reduced Wnt/ß-catenin signaling and BMP signaling in the posterior Hmgb1^-/-; Hmgb2^+/- forelimb bud

Control (A, C, E, G, I) and Hmgb1^-/-; Hmgb2^+/- (B, D, F, H, J) forelimb buds at E10.5, stained with Msx2 (A-D), Msx1 (E, F), Axin2 (G, H) and Dkk1 (I, J). Msx2 expression in the posterior region is lost in Hmgb1^-/-; Hmgb2^+/- limbs (B, D, red arrows), compared to control limbs (A, C, black arrows). (E, F) Msx1 expression in the posterior region is also lost in Hmgb1^-/-; Hmgb2^+/- limbs (F, red arrow), compared to control limbs (E, black arrow). (G, H) The posterior boundary of Axin2 expression in the AER, and the posterior boundary of the forelimbs are indicated by arrowheads. The domain without Axin2 expression is longer in the Hmgb1^-/-; Hmgb2^+/- limb bud (H), compared to the control limb bud (G). (I, J) The posterior Dkk1 expression domain in the forelimb bud is indicated by arrowheads. The Dkk1-expressing domain is smaller in the Hmgb1^-/-; Hmgb2^+/- limb bud (J), compared to control limb bud (I). All panels except for C and D are dorsal views, and C and D are distal-posterior views, with the anterior to the top.

Fig. 6. hmgb1 and hmgb2 are required for enhancement of Wnt/ß-catenin signaling in zebrafish embryos

Lateral views of 18 somite stage embryos (A-E) and 36 hpf embryos (F-J) injected with control MO (A, F), hmgb1 MO (B, G), hmgb2 MO (C, H), both hmgb1 MO and hmgb2 MO (D, I) and hmgb1 MO, hmgb2 MO, Hmgb1 mRNA and Hmgb2 mRNA (E, J). (A-E) Top-GFP expression is visualized by in situ hybridization. Wnt/ß-catenin signaling in forebrain (arrows), midbrain (asterisks) and tail bud (arrowhead) is visualized in control morphants (A), but downregulated in hmgb-downregulated embryos (B-D). In mRNA co-injected double morphants, Top-GFP expression is partially restored (E). Normal body extension (F) is impaired in hmgb1-morphants (G). The phenotype is less severe in hmgb2-morphants (H), but becomes more severe in double morphants (I). This phenotype was partially restored by co-injection of Hmgb1 mRNA and Hmgb2 mRNA (J).

Fig. 7. Downregulation of Wnt signaling and shh signaling in hmgb-knockdown pectoral fin buds

Dorsal views (A-H) and lateral views (I, J) of 36 hpf embryos injected with control MO (A, C, E, G, I) or hmgb1 MO + hmgb2 MO (B, D, F, H, J). (A, B) Normal expression of prx1 in the pectoral fin bud of control (A, arrows) and hmgb-knockdown embryos (B, arrows). (C-H) Expression of Top-GFP (D), shh (F) and ptc1 (H) in the pectoral fin bud of hmgb-knockdown embryos (D, F, H, red arrows) is downregulated, compared to control embryos (C, E, G, black arrows). (I, J) High magnification images of the pectoral fin bud stained with shh shows smaller expression domain of shh in the pectoral fin bud of hmgb-knockdown embryos (J, red arrow), compared to the control fin bud (I, black arrow).

Fig. 8. A model of function of Hmgb1 and Hmgb2 during mouse forelimb development

Combined function of Hmgb1 and Hmgb2 integrates the Hox-Shh pathway, Wnt/ß-catenin signaling and BMP signaling for proper development of digit 5 in the forelimb bud. See detail in the text.