Xpbx1b and Xmeis1b play a collaborative role in hindbrain and neural crest gene expression in Xenopus embryos

Abstract

Pbx1 is a homeodomain protein that functions in complexes with other homeodomain-containing proteins to regulate gene expression during embryogenesis and oncogenesis. Pbx proteins bind DNA cooperatively as heterodimers or higher order complexes with Meis family members and Hox proteins and are believed to specify cell identity during development. Here, we present evidence that Pbx1, in partnership with Meis1b, can regulate posterior neural markers and neural crest marker genes during Xenopus development. A Xenopus homolog of the Pbx1b homeodomain protein was isolated and shown to be expressed throughout embryogenesis. Xpbx1b expression overlaps with Xmeis1 in several areas, including the lateral neural folds, caudal branchial arch, hindbrain, and optic cup. When ectopically expressed, Xpbx1b can synergize with Xmeis1b to promote posterior neural and neural crest gene expression in ectodermal explants. Further, a physical interaction between these two homeodomain proteins is necessary for induction of these genes in embryonic tissue. In addition, coexpression of Xmeis1b and Xpbx1b leads to a prominent shift in the localization of Xmeis1b from the cytoplasm to the nucleus, suggesting that nuclear transport or retention of Xmeis1b may depend upon Xpbx1b. Finally, expression of a mutant construct in which Xpbx1b protein is fused to the repressor domain from Drosophila Engrailed inhibits posterior neural and neural crest gene expression. These data indicate that Xpbx1b and its partner, Xmeis1b, function in a transcriptional activation complex during hindbrain and neural crest development.

Figure 1

Xpbx1b encodes a TALE family homeodomain-containing protein. Amino acid comparison of the coding sequences of Xenopus Pbx1b, Mouse Pbx1b, Drosophila extradenticle, Zebrafish Pbx 4(Lazarus), and C. elegans Pbx (Ceh-20). Identical residues are shaded in black, conservative differences are shaded in gray, and white represents nonhomology. The bracketed area above the sequence denotes the homeodomain region.

Figure 2

Temporal and spatial distribution of XPbx1b and Xmeis1 RNA expression during embryogenesis. (A) Northern blot analysis of RNA extracted from embryos at the indicated stages with Xpbx1b and Xmeis1 specific probe. The 18S RNA probe was used as a loading control. Whole mount in situ hybridization analysis of the tissue distribution of XPbx1b (B–F) and Xmeis1 (G–I) transcripts in X. laevis embryos. (B) Stage 15, anterior view. Note expression in lateral neural folds. (C) Stage 21, dorso-anterior view. Note strong staining in the presumptive hindbrain and along neural folds. (D) Lateral view of cleared stage-26 embryo. (E) Stage 26 dorsal view. (F) Enlarged lateral view of enclosure from D. Note the distinct Xpbx1 staining in the forebrain, hindbrain, and caudal branchial arch. (G) Lateral view of cleared stage-25 embryo. (H) Stage 25 dorsal view. (I) Enlarged lateral view of enclosure from G. Note strong expression of Xmeis1 in the midbrain, hindbrain, caudal and rostral branchial arches, and somites. (J) Transverse section through the pronephros of a stage-28 embryo. Note the strong Xpbx1 staining in lateral edge of neural tube (white arrowheads); expression also is observed in the lateral mesoderm surrounding the pronephritic anlage. (B–J) Black arrowheads indicate caudal branchial arch. Black brackets denote the indicated tissues. Fb, forebrain; Hb, hindbrain; Mb, midbrain; NF, neural fold; Nt, neural tube; Pn, pronephros.

Figure 3

Interaction of XPbx1b and Xmeis1b induces posterior neural and neural crest markers in animal cap explants in the absence of mesoderm. (A) The animal pole region of two-cell stage embryos were injected with either XPbx1b RNA (1.0 ng per embryo), Xmeis1b RNA (0.5 ng per embryo), or both RNAs. Animal pole explants were excised at stage 9 and cultured until stage 26. RT-PCR gene analysis was performed for N-CAM (pan-neural), N-tubulin (pan-neuronal), Xtwist (neural crest), Otx-2 (forebrain), XAG-1 (cement gland), Krox-20 (hindbrain), HoxB9 (spinal cord), Xzic3 (proneural and early neural crest marker), muscle actin (dorsal mesoderm), and EF-1a (loading control). Stage-24 embryonic RNA with or without reverse transcriptase (RT+ or RT−) also was used as a positive and negative reaction control. Note that coinjection of XPbx1b and Xmeis1b RNA induced the expression of posterior neural and neural crest cell markers. (B) Schematic representation of the Xmeis1b mutants. Wild-type Xmeis1b consists of a Meis-Homothorax domain (MH, yellow box) and homeodomain (HD, green box). MH domain possesses two subdomains: M1 box (amino acids 71–96) and M2 box (amino acids 148–161) that are important for Pbx binding. Deletions of one or more subdomains are indicated. (C) XPbx1b and Xmeis1b physically interact. The animal pole region of two-cell stage embryos were injected with either XPbx1b (2.5 ng per embryo) or Xmeis1b (2.5 ng per embryo) RNA alone or were coinjected with XPbx1b and Xmeis1b wild-type and mutant RNAs. Embryos were cultured until stage 9, and embryonic extracts were prepared and either directly immunoblotted (Lower) or immunoprecipitated with an anti-Xmeis1 antibody before immunoblotting (Upper). Anti-Meis1 antibody or anti-Pbx1b antibody was used to detect the indicated protein. Note: the Xpbx1b protein was coimmunoprecipitated with the wild-type Xmeis1b but not with any of the Xmeis1b mutants. (D) Nuclear localization of the Xmeis1b protein depends upon the XPbx1b protein in frog embryonic cells. The animal pole region of two-cell stage embryos was injected with either Flag tagged XPbx1b (2.5 ng per embryo) or Flag-tagged Xmeis1b RNA (2.5 ng per embryo) alone or in combination with Xmeis1b or XPbx1b RNA (2.5 ng per embryo). Injected embryos were fixed at stage 9, embedded, sectioned, and immunostained with anti-Flag antibody. Note that Xmeis1b protein was in the cytoplasm when expressed alone but was localized in the nuclei in the presence of XPbx1b. In contrast, XPbx1b was localized in the nuclei regardless of whether exogenous Xmeis1b was present.

Figure 4

XPbx1b/EnR suppresses posterior neural and neural crest markers during embryonic development. (A) Schematic representation of XPbx1b protein fused with an Engrailed repressor domain. The three major conserved domains with vertebrate Pbx1 and C. elegans Ceh-20; PBC-A, PBC-B, and the homeodomain (HD) also are indicated. (B) One blastomere of two-cell stage embryos was either injected with XPbx1b/EnR RNA (0.75 ng per embryo) alone or coinjected with wild-type XPbx1b RNA (2.5 ng per embryo), as indicated. Engrailed control embryos were injected with EnR RNA (2.5 ng per embryo) as noted. Embryos were cultured until stage 16 (Xslug images), stage 18 (Krox-20, Krox-20/Otx-2, and Xzic3 images), or stage 24 (Xap-2 images). Embryos were fixed, and in situ hybridization was performed by using the indicated probe. Arrow indicates injected side of embryo. Note that XPbx1b/EnR suppressed Krox-20 (hindbrain), Xzic3 (proneural and neural crest), Xslug (neural crest), and Xap-2 (neural crest) expression on the injected side, but Otx-2 (anterior neural) expression remained. Also note that coexpression of wild-type Xpbx1b rescued Xslug and Krox-20 expression.