Bernadette C. Holdener

Mesd Encodes an LRP5/6 Chaperone Essential for Specification of Mouse Embryonic Polarity

Specification of embryonic polarity and pattern formation in multicellular organisms requires inductive signals from neighboring cells. One approach toward understanding these interactions is to study mutations that disrupt development. Here, we demonstrate that mesd, a gene identified in the mesoderm development (mesd) deletion interval on mouse chromosome 7, is essential for specification of embryonic polarity and mesoderm induction. MESD functions in the endoplasmic reticulum as a specific chaperone for LRP5 and LRP6, which in conjunction with Frizzled, are coreceptors for canonical WNT signal transduction. Disruption of embryonic polarity and mesoderm differentiation in mesd-deficient embryos likely results from a primary defect in WNT signaling. However, phenotypic differences between mesd-deficient and wnt3(-)(/)(-) embryos suggest that MESD may function on related members of the low-density lipoprotein receptor (LDLR) family, whose members mediate diverse cellular processes ranging from cargo transport to signaling.

ARNT2 acts as the dimerization partner of SIM1 for the development of the hypothalamus.

One major function of the hypothalamus is to maintain homeostasis by modulating the secretion of pituitary hormones. The paraventricular (PVN) and supraoptic (SON) nuclei are major integration centers for the output of the hypothalamus to the pituitary. The bHLH-PAS transcription factor SIM1 is crucial for the development of several neuroendocrine lineages within the PVN and SON. bHLH-PAS proteins require heterodimerization for their function. ARNT, ARNT2, and BMAL1 are the three known general heterodimerization partners for bHLH-PAS proteins. Here, we provide evidence that Sim1 and Arnt2 form dimers in vitro, that they are co-expressed in the PVN and SON, and that their loss of function affects the development of the same sets of neuroendocrine cell types within the PVN and SON. Together, these results implicate ARNT2 as the in vivo dimerization partner of SIM1 in controlling the development of these neuroendocrine lineages.

O-fucosylation of thrombospondin type 1 repeats restricts epithelial to mesenchymal transition (EMT) and maintains epiblast pluripotency during mouse gastrulation

Thrombospondin type 1 repeat (TSR) superfamily members regulate diverse biological activities ranging from cell motility to inhibition of angiogenesis. In this study, we verified that mouse protein O-fucosyltransferase-2 (POFUT2) specifically adds O-fucose to TSRs. Using two Pofut2 gene-trap lines, we demonstrated that O-fucosylation of TSRs was essential for restricting epithelial to mesenchymal transition in the primitive streak, correct patterning of mesoderm, and localization of the definitive endoderm. Although Pofut2 mutant embryos established anterior/posterior polarity, they underwent extensive mesoderm differentiation at the expense of maintaining epiblast pluripotency. Moreover, mesoderm differentiation was biased towards the vascular endothelial cell lineage. Localization of Foxa2 and Cer1 expressing cells within the interior of Pofut2 mutant embryos suggested that POFUT2 activity was also required for the displacement of the primitive endoderm by definitive endoderm. Notably, Nodal, BMP4, Fgf8, and Wnt3 expression were markedly elevated and expanded in Pofut2 mutants, providing evidence that O-fucose modification of TSRs was essential for modulation of growth factor signaling during gastrulation. The ability of Pofut2 mutant embryos to form teratomas comprised of tissues from all three germ layer origins suggested that defects in Pofut2 mutant embryos resulted from abnormalities in the extracellular environment. This prediction is consistent with the observation that POFUT2 targets are constitutive components of the extracellular matrix (ECM) or associate with the ECM. For this reason, the Pofut2 mutants represent a valuable tool for studying the role of O-fucosylation in ECM synthesis and remodeling, and will be a valuable model to study how post-translational modification of ECM components regulates the formation of tissue boundaries, cell movements, and signaling.

MESD is essential for apical localization of megalin/LRP2 in the visceral endoderm

Deletion of the Mesd gene region blocks gastrulation and mesoderm differentiation in mice. MESD is a chaperone for the Wnt co‐receptors: low‐density lipoprotein receptor‐related protein (LRP) 5 and 6 (LRP5/6). We hypothesized that loss of Wnt signaling is responsible for the polarity defects observed in Mesd‐deficient embryos. However, because the Mesd‐deficient embryo is considerably smaller than Lrp5/6 or Wnt3 mutants, we predicted that MESD function extends more broadly to the LRP family of receptors. Consistent with this prediction, we demonstrated that MESD function in vitro was essential for maturation of the β‐propeller/EGF domain common to LRPs. To begin to understand the role of MESD in LRP maturation in vivo, we generated a targeted Mesd knockout and verified that loss of Mesd blocks WNT signaling in vivo. Mesd mutants continue to express the pluripotency markers Oct4, Nanog, and Sox2, suggesting that Wnt signaling is essential for differentiation of the epiblast. Moreover, we demonstrated that MESD was essential for the apical localization of the related LRP2 (Megalin/MEG) in the visceral endoderm, resulting in impaired endocytic function. Combined, our results provide evidence that MESD functions as a general LRP chaperone and suggest that the Mesd phenotype results from both signaling and endocytic defects resulting from misfolding of multiple LRP receptors. Developmental Dynamics 240:577–588, 2011.

Genetic and biochemical evidence that gastrulation defects in Pofut2 mutants result from defects in ADAMTS9 secretion.

Protein O-fucosyltransferase 2 (POFUT2) adds O-linked fucose to Thrombospondin Type 1 Repeats (TSR) in 49 potential target proteins. Nearly half the POFUT2 targets belong to the A Disintegrin and Metalloprotease with ThromboSpondin type-1 motifs (ADAMTS) or ADAMTS-like family of proteins. Both the mouse Pofut2 RST434 gene trap allele and the Adamts9 knockout were reported to result in early embryonic lethality, suggesting that defects in Pofut2 mutant embryos could result from loss of O-fucosylation on ADAMTS9. To address this question, we compared the Pofut2 and Adamts9 knockout phenotypes and used Cre-mediated deletion of Pofut2 and Adamts9 to dissect the tissue-specific role of O-fucosylated ADAMTS9 during gastrulation. Disruption of Pofut2 using the knockout (LoxP) or gene trap (RST434) allele, as well as deletion of Adamts9, resulted in disorganized epithelia (epiblast, extraembryonic ectoderm, and visceral endoderm) and blocked mesoderm formation during gastrulation. The similarity between Pofut2 and Adamts9 mutants suggested that disruption of ADAMTS9 function could be responsible for the gastrulation defects observed in Pofut2 mutants. Consistent with this prediction, CRISPR/Cas9 knockout of POFUT2 in HEK293T cells blocked secretion of ADAMTS9. We determined that Adamts9 was dynamically expressed during mouse gastrulation by trophoblast giant cells, parietal endoderm, the most proximal visceral endoderm adjacent to the ectoplacental cone, extraembryonic mesoderm, and anterior primitive streak. Conditional deletion of either Pofut2 or Adamts9 in the epiblast rescues the gastrulation defects, and identified a new role for O-fucosylated ADAMTS9 during morphogenesis of the amnion and axial mesendoderm. Combined, these results suggested that loss of ADAMTS9 function in the extra embryonic tissue is responsible for gastrulation defects in the Pofut2 knockout. We hypothesize that loss of ADAMTS9 function in the most proximal visceral endoderm leads to slippage of the visceral endoderm and altered characteristics of the extraembryonic ectoderm. Consequently, loss of input from the extraembryonic ectoderm and/or compression of the epiblast by Reicherts membrane blocks gastrulation. In the future, the Pofut2 and Adamts9 knockouts will be valuable tools for understanding how local changes in the properties of the extracellular matrix influence the organization of tissues during mammalian development.

The Structure of MESD45–184 Brings Light into the Mechanism of LDLR Family Folding

Mesoderm development (MESD) is a 224 amino acid mouse protein that acts as a molecular chaperone for the low-density lipoprotein receptor (LDLR) family. Here, we provide evidence that the region 45-184 of MESD is essential and sufficient for this function and suggest a model for its mode of action. NMR studies reveal a β-α-β-β-α-β core domain with an α-helical N-terminal extension that interacts with the β sheet in a dynamic manner. As a result, the structural ensemble contains open (active) and closed (inactive) forms, allowing for regulation of chaperone activity through substrate binding. The mutant W61R, which is lethal in Drosophila, adopts only the open state. The receptor motif recognized by MESD was identified by in vitro-binding studies. Furthermore, in vivo functional evidence for the relevance of the identified contact sites in MESD is provided.

Mouse Fzd4 maps within a region of chromosome 7 important for thymus and cardiac development

Summary: The cardiac neural crest (CNC) plays a central role in development of the thymus gland and cardiovascular system. Through morphological and histological characterization of embryos homozygous for the Del(7)Tyrc‐112K and Del(7)Tyrc‐3H albino deletions, we identified abnormalities that are consistent with aberrant development of tissues requiring CNC contributions. The defects include incompletely penetrant heart and great vessel patterning defects and hypoplastic thymus glands. The CNC phenotype is complemented by the partially overlapping deletion Del(7)Tyrc‐23DVT. Combined, these results suggest that a functional region necessary for development of CNC derived tissues is located between the Del(7)Tyrc‐23DVT and Del(7)Tyrc‐112K distal deletion breakpoints. This interval encompasses a functional region previously identified as important for juvenile survival (juvenile development and fertility, jdf). Using deletion mapping, we localized the Frizzled4 (Fzd4) gene to the jdf/thymus and cardiac development intervals. genesis 27:64–75, 2000.

Phenotypic and physical analysis of a chemically induced mutation disrupting anterior axial development in the mouse

Phenotypic analysis of radiation-induced mutations has identified a region of mouse Chromosome (Chr) 7, embryonic ectoderm development (eed), that is required for axial development (Faust et al. 1995; Niswander et al. 1988). The eed mutant phenotype is first recognizable at the onset of gastrulation [embryonic day (E) 6.5)] (Faust et al. 1995). By E 8.5, mutant embryos are easily distinguished from their wild-type littermates by their unusual morphology. Characteristically, the extraembryonic structures, including amnion, chorion, and allantois, are well developed in eed mutants, although the allantois appears excessively large. In contrast, no morphologically distinct head process or notochord are observed, and there is no visible induction of the neural axis (Faust et al. 1995; Niswander et al. 1988). Despite the morphological absence of these structures, the midline distribution of Shh, Hnf3fS, and T transcripts in these mutants suggests that axial development occurs to some extent (Faust et al. 1995). Also intriguing is the observation that both T and evxl exhibit overlapping patterns of transcription at ectopic proximal anterior locations. Together, morphological characteristics and transcript distribution in mutant embryos suggest a role for eed in anterior primitive-streak function. eed is located approximately 1-2 cM distal to the albino (c) locus, within a maximum 150-kb interval located between the c 3H and c 3Deub distal breakpoints (Holdener et al. 1995; refer to Fig. 1). The proximal limit is defined by the genomic locus DTCwr3D, and the distal limit is located within the 30-kb interval proximal to DTRn5 (Fig. 1; Holdener et al. 1995; Potter et al. 1995). Because the eed region spans a relatively large interval, it is conceivable that the anterior primitive streak defect is due to loss of multiple genes from the interval. To address this question, we were able to

Sequence analysis of a radiation-induced deletion breakpoint fusion in mouse

C l lDSD is an X-ray-induced deletion generated at the Oak Ridge National Laboratory (Russell et al. 1979) that spans ~3 cM (Sharan et al. 1991) of mouse Chromosome (Chr) 7 including the c (albino) locus. The c11DSD-deletion breakpoint fusion fragment was cloned from a C3H/101 background (Sharan et al. 1991). Clones from a wild-type 129/SV phage genomic library (Stratagene) corresponding to the proximal and distal breakpoints of the deletion were isolated. The cHDSD-breakpoint fusion fragment and wildtype clones were sequenced and compared. The purpose was to determine whether any flanking rearrangements and/or sequence motifs were associated with the fusion site. Sequence flanking the cllDSD-breakpoint fusion was aligned with the wild-type proximal and distal sequences (Fig. 1). Homology between the C l lDSD and proximal wildtype sequence ends at the fusion site, after which homology between the distal wild-type and c 11DsD sequences begins (Fig. 1). The exact fusion site could not be determined because of a two-base overlap marked by the box in Fig. 1. A single base mismatch was present in both homologous proximal and distal sequences, and these are probably the result of polymorphism between strains. No significant sequence homology was found between the proximal and distal sides of the fusion. A BLASTN (Altschul et al. 1990) search showed the proximal side to be ~80% homologous to rat (Bilofsky and Burks 1988) and mouse (Shehee et al. 1987) long interspersed elements (LINEs), whereas the distal side showed no homology matches. This breakpoint fusion sequence in context with two other sequenced radiation-induced breakpoint fusions, pCp (Nakatsuet al. 1993) and Aa2 (Strobel et al. 1990), illustrates the precise nature with which induced double-strand breaks can be joined.

Development of a Conditional Mesd (Mesoderm Development) Allele for Functional Analysis of the Low-Density Lipoprotein Receptor-Related Family in Defined Tissues

The Low-density lipoprotein receptor-Related Protein (LRP) family members are essential for diverse processes ranging from the regulation of gastrulation to the modulation of lipid homeostasis. Receptors in this family bind and internalize a diverse array of ligands in the extracellular matrix (ECM). As a consequence, LRPs regulate a wide variety of cellular functions including, but not limited to lipid metabolism, membrane composition, cell motility, and cell signaling. Not surprisingly, mutations in single human LRPs are associated with defects in cholesterol metabolism and development of atherosclerosis, abnormalities in bone density, or aberrant eye vasculature, and may be a contributing factor in development of Alzheimer’s disease. Often, members of this diverse family of receptors perform overlapping roles in the same tissues, complicating the analysis of their function through conventional targeted mutagenesis. Here, we describe development of a mouse Mesd (Mesoderm Development) conditional knockout allele, and demonstrate that ubiquitous deletion of Mesd using Cre-recombinase blocks gastrulation, as observed in the traditional knockout and albino-deletion phenotypes. This conditional allele will serve as an excellent tool for future characterization of the cumulative contribution of LRP members in defined tissues.