Terence D. Capellini

Human-specific loss of regulatory DNA and the evolution of human-specific traits

Humans differ from other animals in many aspects of anatomy, physiology, and behaviour; however, the genotypic basis of most human-specific traits remains unknown. Recent whole-genome comparisons have made it possible to identify genes with elevated rates of amino acid change or divergent expression in humans, and non-coding sequences with accelerated base pair changes. Regulatory alterations may be particularly likely to produce phenotypic effects while preserving viability, and are known to underlie interesting evolutionary differences in other species. Here we identify molecular events particularly likely to produce significant regulatory changes in humans: complete deletion of sequences otherwise highly conserved between chimpanzees and other mammals. We confirm 510 such deletions in humans, which fall almost exclusively in non-coding regions and are enriched near genes involved in steroid hormone signalling and neural function. One deletion removes a sensory vibrissae and penile spine enhancer from the human androgen receptor (AR) gene, a molecular change correlated with anatomical loss of androgen-dependent sensory vibrissae and penile spines in the human lineage. Another deletion removes a forebrain subventricular zone enhancer near the tumour suppressor gene growth arrest and DNA-damage-inducible, gamma (GADD45G), a loss correlated with expansion of specific brain regions in humans. Deletions of tissue-specific enhancers may thus accompany both loss and gain traits in the human lineage, and provide specific examples of the kinds of regulatory alterations and inactivation events long proposed to have an important role in human evolutionary divergence.

The TALE homeodomain protein Pbx2 is not essential for development and long-term survival.

ABSTRACT Pbx2 is one of four mammalian genes that encode closely related TALE homeodomain proteins, which serve as DNA binding partners for a subset of Hox transcription factors. The expression and contributions of Pbx2 to mammalian development remain undefined, in contrast to the essential roles recently established for family members Pbx1 and Pbx3. Here we report that Pbx2 is widely expressed during embryonic development, particularly in neural and epithelial tissues during late gestation. Despite wide Pbx2 expression, mice homozygous mutant for Pbx2 are born at the expected Mendelian frequencies and exhibit no detectable abnormalities in development and organogenesis or reduction of long-term survival. The lack of an apparent phenotype in Pbx2− /− mice likely reflects functional redundancy, since the Pbx2 protein is present at considerably lower levels than comparable isoforms of Pbx1 and/or Pbx3 in embryonic tissues. In postnatal bone marrow and thymus, however, Pbx2 is the predominant high-molecular-weight (MW)-isoform Pbx protein detectable by immunoblotting. Nevertheless, the absence of Pbx2 has no measurable effect on steady-state hematopoiesis or immune function in adult mice, suggesting possible compensation by low-MW-isoform Pbx proteins present in these tissues. We conclude that the roles of Pbx2 in murine embryonic development, organogenesis, hematopoiesis, immune responses, and long-term survival are not essential.

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.

Scapula development is governed by genetic interactions of Pbx1 with its family members and with Emx2 via their cooperative control of Alx1

The genetic pathways underlying shoulder blade development are largely unknown, as gene networks controlling limb morphogenesis have limited influence on scapula formation. Analysis of mouse mutants for Pbx and Emx2 genes has suggested their potential roles in girdle development. In this study, by generating compound mutant mice, we examined the genetic control of scapula development by Pbx genes and their functional relationship with Emx2. Analyses of Pbx and Pbx1;Emx2 compound mutants revealed that Pbx genes share overlapping functions in shoulder development and that Pbx1 genetically interacts with Emx2 in this process. Here, we provide a biochemical basis for Pbx1;Emx2 genetic interaction by showing that Pbx1 and Emx2 can bind specific DNA sequences as heterodimers. Moreover, the expression of genes crucial for scapula development is altered in these mutants, indicating that Pbx genes act upstream of essential pathways for scapula formation. In particular, expression of Alx1, an effector of scapula blade patterning, is absent in all compound mutants. We demonstrate that Pbx1 and Emx2 bind in vivo to a conserved sequence upstream of Alx1 and cooperatively activate its transcription via this potential regulatory element. Our results establish an essential role for Pbx1 in genetic interactions with its family members and with Emx2 and delineate novel regulatory networks in shoulder girdle development.

Pbx1/Pbx2 govern axial skeletal development by controlling Polycomb and Hox in mesoderm and Pax1/Pax9 in sclerotome

The post-cranial axial skeleton consists of a metameric series of vertebral bodies and intervertebral discs, as well as adjoining ribs and sternum. Patterning of individual vertebrae and distinct regions of the vertebral column is accomplished by Polycomb and Hox proteins in the paraxial mesoderm, while their subsequent morphogenesis depends partially on Pax1/Pax9 in the sclerotome. In this study, we uncover that Pbx1/Pbx2 are co-expressed during successive stages of vertebral and rib development. Next, by exploiting a Pbx1/Pbx2 loss-of-function mouse, we show that decreasing Pbx2 dosage in the absence of Pbx1 affects axial development more severely than single loss of Pbx1. Pbx1/Pbx2 mutants exhibit a homogeneous vertebral column, with loss of vertebral identity, rudimentary ribs, and rostral hindlimb shifts. Of note, these axial defects do not arise from perturbed notochord function, as cellular proliferation, apoptosis, and expression of regulators of notochord signaling are normal in Pbx1/Pbx2 mutants. While the observed defects are consistent with loss of Pbx activity as a Hox-cofactor in the mesoderm, we additionally establish that axial skeletal patterning and hindlimb positioning are governed by Pbx1/Pbx2 through their genetic control of Polycomb and Hox expression and spatial distribution in the mesoderm, as well as of Pax1/Pax9 in the sclerotome.

Pbx homeodomain proteins: TALEnted regulators of limb patterning and outgrowth

Limb development has long provided an excellent model for understanding the genetic principles driving embryogenesis. Studies utilizing chick and mouse have led to new insights into limb patterning and morphogenesis. Recent research has centered on the regulatory networks underlying limb development. Here, we discuss the hierarchical, overlapping, and iterative roles of Pbx family members in appendicular development that have emerged from genetic analyses in the mouse. Pbx genes are essential in determining limb bud positioning, early bud formation, limb axes establishment and coordination, and patterning and morphogenesis of most elements of the limb and girdle. Pbx proteins directly regulate critical effectors of limb and girdle development, including morphogen‐encoding genes like Shh in limb posterior mesoderm, and transcription factor‐encoding genes like Alx1 in pre‐scapular domains. Interestingly, at least in limb buds, Pbx appear to act not only as Hox cofactors, but also in the upstream control of 5′ HoxA/D gene expression. Developmental Dynamics 240:1063–1086, 2011.

Conservation of Notochord Gene Expression Across Chordates: Insights From the Leprecan Gene Family

The notochord is a defining character of the chordates, and the T‐box transcription factor Brachyury has been shown to be required for notochord development in all chordates examined. In the ascidian Ciona intestinalis, at least 44 notochord genes have been identified as bona fide transcriptional targets of Brachyury. We examined the embryonic expression of a subset of murine orthologs of Ciona Brachyury target genes in the notochord to assess its conservation throughout chordate evolution. We focused on analyzing the Leprecan gene family, which in mouse is composed of three genes, as opposed to the single‐copy Ciona gene. We found that all three mouse Leprecan genes are expressed in the notochord. Additionally, while Leprecan expression in C. intestinalis is confined to the notochord, expression of its mouse orthologs includes dorsal root ganglia, limb buds, branchial arches, and developing kidneys. These results have interesting implications for the evolution and development of chordates. genesis 46:683–696, 2008.

Fossil hominin shoulders support an African ape-like last common ancestor of humans and chimpanzees

Significance Knowing the direction and pace of evolutionary change is critical to understanding what selective forces shaped our ancestors. Unfortunately, the human fossil record is sparse, and little is known about the earliest members of our lineage. This unresolved ancestor complicates reconstructions of what behavioral shifts drove major speciation events. Using 3D shape measurements of the shoulder, we tested competing evolutionary models of the last common ancestor against the fossil record. We found that a sustained shift in scapular shape occurred during hominin evolution from an African ape-like ancestor to a modern human-like form, first present in our genus, Homo. These data suggest a long, gradual shift out of the trees and increased reliance on tools as our ancestors became more terrestrial. Reconstructing the behavioral shifts that drove hominin evolution requires knowledge of the timing, magnitude, and direction of anatomical changes over the past ∼6–7 million years. These reconstructions depend on assumptions regarding the morphotype of the Homo–Pan last common ancestor (LCA). However, there is little consensus for the LCA, with proposed models ranging from African ape to orangutan or generalized Miocene ape-like. The ancestral state of the shoulder is of particular interest because it is functionally associated with important behavioral shifts in hominins, such as reduced arboreality, high-speed throwing, and tool use. However, previous morphometric analyses of both living and fossil taxa have yielded contradictory results. Here, we generated a 3D morphospace of ape and human scapular shape to plot evolutionary trajectories, predict ancestral morphologies, and directly test alternative evolutionary hypotheses using the hominin fossil evidence. We show that the most parsimonious model for the evolution of hominin shoulder shape starts with an African ape-like ancestral state. We propose that the shoulder evolved gradually along a single morphocline, achieving modern human-like configuration and function within the genus Homo. These data are consistent with a slow, progressive loss of arboreality and increased tool use throughout human evolution.

Cooperation between p27 and p107 during endochondral ossification suggests a genetic pathway controlled by p27 and p130

ABSTRACT Pocket proteins and cyclin-dependent kinase (CDK) inhibitors negatively regulate cell proliferation and can promote differentiation. However, which members of these gene families, which cell type they interact in, and what they do to promote differentiation in that cell type during mouse development are largely unknown. To identify the cell types in which p107 and p27 interact, we generated compound mutant mice. These mice were null for p107 and had a deletion in p27 that prevented its binding to cyclin-CDK complexes. Although a fraction of these animals survived into adulthood and looked similar to single p27 mutant mice, a larger number of animals died at birth or within a few weeks thereafter. These animals displayed defects in chondrocyte maturation and endochondral bone formation. Proliferation of chondrocytes was increased, and ectopic ossification was observed. Uncommitted mouse embryo fibroblasts could be induced into the chondrocytic lineage ex vivo, but these cells failed to mature normally. These results demonstrate that p27 carries out overlapping functions with p107 in controlling cell cycle exit during chondrocyte maturation. The phenotypic similarities between p107−/−p27D51/D51 and p107−/−p130−/− mice and the cells derived from them suggest that p27 and p130 act in an analogous pathway during chondrocyte maturation.

DNA methylation Landscape of body size variation in sheep

Sub-populations of Chinese Mongolian sheep exhibit significant variance in body mass. In the present study, we sequenced the whole genome DNA methylation in these breeds to detect whether DNA methylation plays a role in determining the body mass of sheep by Methylated DNA immunoprecipitation – sequencing method. A high quality methylation map of Chinese Mongolian sheep was obtained in this study. We identified 399 different methylated regions located in 93 human orthologs, which were previously reported as body size related genes in human genome-wide association studies. We tested three regions in LTBP1, and DNA methylation of two CpG sites showed significant correlation with its RNA expression. Additionally, a particular set of differentially methylated windows enriched in the “development process” (GO: 0032502) was identified as potential candidates for association with body mass variation. Next, we validated small part of these windows in 5 genes; DNA methylation of SMAD1, TSC1 and AKT1 showed significant difference across breeds, and six CpG were significantly correlated with RNA expression. Interestingly, two CpG sites showed significant correlation with TSC1 protein expression. This study provides a thorough understanding of body size variation in sheep from an epigenetic perspective.