Pbx1/Pbx2 requirement for distal limb patterning is mediated by the hierarchical control of Hox gene spatial distribution and Shh expression

Vertebrate limb development occurs along three cardinal axes-proximodistal, anteroposterior and dorsoventral-that are established via the organization of signaling centers, such as the zone of polarizing activity (ZPA). Distal limb development, in turn, requires a molecular feedback loop between the ZPA expression of sonic hedgehog (Shh) and the apical ectodermal ridge. The TALE homeoprotein Pbx1 has been shown to be essential for proximal limb development. In this study, we first uncover that Pbx1 and Pbx2 are co-expressed in the lateral plate and early limb field mesoderm. Later, Pbx2 is expressed throughout the limb, unlike Pbx1, which is expressed only in the proximal bud. By exploiting a Pbx1/Pbx2 loss-of-function mouse model, we demonstrate that, despite the lack of limb abnormalities in Pbx2-deficient (Pbx2(-/-)) embryos, compound Pbx1(-/-); Pbx2(+/-) mutants, in addition to their exacerbated proximal limb defects, exhibit novel and severe distal abnormalities. Additionally, we reveal that Pbx1(-/-); Pbx2(-/-) embryos lack limbs altogether. Furthermore, we establish that, unlike in flies, where the leg develops independently of Hox and where the Pbx ortholog Exd is required for specification of proximal (but not distal) limbs, in vertebrates, distal limb patterning is Pbx1/Pbx2 dependent. Indeed, we demonstrate that Pbx genetic requirement is mediated, at least in part, through their hierarchical control of Hox spatial distribution and Shh expression. Overall, we establish that, by controlling the spatial expression of Hox genes in the posterior limb and regulating ZPA function, Pbx1/Pbx2 exert a primary hierarchical function on Hox genes, rather than behaving merely as Hox ancillary factors.     

Hox11 acts cell autonomously in spleen development and its absence results in altered cell fate of mesenchymal spleen precursors

The genetic steps governing development of the spleen are largely unknown. Absence of Hox11 in mice results in asplenia, but it is unclear how Hox11 exerts its effect on spleen development. To more precisely define Hox11's role in spleen morphogenesis, we have examined the fate of the developing spleen in Hox11(-/-) mice. Perturbation of spleen development begins between dE13 and dE13.5. Cells of the spleen anlage persist past this developmental stage as an unorganized rudiment between the stomach and the pancreas. They fail to proliferate, and haematopoietic cells do not colonize the rudiment. At later stages of embryonic development, the cells can be observed in the mesenchyme of the pancreas, also an expression site of Hox11. In Hox11-/-<-->+/+ chimaeras, spleens were devoid of Hox11(-/-) cells, indicating that the genetic defect is cell autonomous and not due to failure of the organ anlage to attract and retain haematopoietic cells. In -/-<-->+/+ chimaeric embryos, Hox11(-/-) cells were initially present in the spleen anlage. However, at dE13, a reorganization of the spleen occurred in the chimaeras and Hox11(-/-) cells were subsequently excluded from the spleen, suggesting that a change in the affinity for one of the spleen cells had occurred. These observations demonstrate that spleen development consists of genetically separable steps and that absence of Hox11 arrests spleen development at an early stage. The formation of the spleen primordium before the entry of haematopoietic cells does not require the activity of Hox11. However, subsequent differentiation of spleen precursor cells is dependent on the Hox11 gene.     

Congenital asplenia in mice and humans with mutations in a Pbx/Nkx2-5/p15 module

The molecular determinants of spleen organogenesis and the etiology of isolated congenital asplenia (ICA), a life-threatening human condition, are unknown. We previously reported that Pbx1 deficiency causes organ growth defects including asplenia. Here, we show that mice with splenic mesenchyme-specific Pbx1 inactivation exhibit hyposplenia. Moreover, the loss of Pbx causes downregulation of Nkx2-5 and derepression of p15Ink4b in spleen mesenchymal progenitors, perturbing the cell cycle. Removal of p15Ink4b in Pbx1 spleen-specific mutants partially rescues spleen growth. By whole-exome sequencing of a multiplex kindred with ICA, we identify a heterozygous missense mutation (P236H) in NKX2-5 showing reduced transactivation in vitro. This study establishes that a Pbx/Nkx2-5/p15 regulatory module is essential for spleen development.     

Abnormalities of caudal pharyngeal pouch development in Pbx1 knockout mice mimic loss of Hox3 paralogs

Pbx1 is a TALE-class homeodomain protein that functions in part as a cofactor for Hox class homeodomain proteins. Previous analysis of the in vivo functions of Pbx1 by targeted mutagenesis in mice has revealed roles for this gene in skeletal patterning and development and in the organogenesis of multiple systems. Both RNA expression and protein localization studies have suggested a possible role for Pbx1 in pharyngeal region development. As several Hox mutants have distinct phenotypes in this region, we investigated the potential requirement for Pbx1 in the development of the pharyngeal arches and pouches and their organ derivatives. Pbx1 homozygous mutants exhibited delayed or absent formation of the caudal pharyngeal pouches, and disorganized patterning of the third pharyngeal pouch. Formation of the third pouch-derived thymus/parathyroid primordia was also affected, with absent or hypoplastic primordia, delayed expression of organ-specific differentiation markers, and reduced proliferation of thymic epithelium. The fourth pouch and the fourth pouch-derived ultimobranchial bodies were usually absent. These phenotypes are similar to those previously reported in Hoxa3(-/-) single mutants and Hoxa1(-/-);Hoxb1(-/-) or Hoxa3(+/-);Hoxb3(-/-);Hoxd3(-/-) compound mutants, suggesting that Pbx1 acts together with multiple Hox proteins in the development of the caudal pharyngeal region. However, some aspects of the Pbx1 mutant phenotype included specific defects that were less severe than those found in known Hox mutant mice, suggesting that some functions of Hox proteins in this region are Pbx1-independent.     

The pentapeptide motif of Hox proteins is required for cooperative DNA binding with Pbx1, physically contacts Pbx1, and enhances DNA binding by Pbx1.

The vertebrate Hox genes, which represent a subset of all homeobox genes, encode proteins that regulate anterior-posterior positional identity during embryogenesis and are cognates of the Drosophila homeodomain proteins encoded by genes composing the homeotic complex (HOM-C). Recently, we demonstrated that multiple Hox proteins bind DNA cooperatively with both Pbx1 and its oncogenic derivative, E2A-Pbx1. Here, we show that the highly conserved pentapeptide motif F/Y-P-W-M-R/K, which occurs in numerous Hox proteins and is positioned 8 to 50 amino acids N terminal to the homeodomain, is essential for cooperative DNA binding with Pbx1 and E2A-Pbx1. Point mutational analysis demonstrated that the tryptophan and methionine residues within the core of this motif were critical for cooperative DNA binding. A peptide containing the wild-type pentapeptide sequence, but not one in which phenylalanine was substituted for tryptophan, blocked the ability of Hox proteins to bind cooperatively with Pbx1 or E2A-Pbx1, suggesting that the pentapeptide itself provides at least one surface through which Hox proteins bind Pbx1. Furthermore, the same peptide, but not the mutant peptide, stimulated DNA binding by Pbx1, suggesting that interaction of Hox proteins with Pbx1 through the pentapeptide motif raises the DNA-binding ability of Pbx1.