Stephanie Grainger

Cdx2 regulates patterning of the intestinal epithelium.

Cdx1, Cdx2 and Cdx4 encode homeodomain transcription factors that are involved in vertebral anterior-posterior (AP) patterning. Cdx1 and Cdx2 are also expressed in the intestinal epithelium during development, suggesting a role in this tissue. Intestinal defects have not been reported in Cdx1 null mutants, while Cdx2 null mutants die at embryonic day 3.5 (E3.5), thus precluding assessment of the null phenotype at later stages. To circumvent this latter shortcoming, we have used a conditional Cre-lox strategy to inactivate Cdx2 in the intestinal epithelium. Using this approach, we found that ablation of Cdx2 at E13.5 led to a transformation of the small intestine to a pyloric stomach-like identity, although the molecular nature of the underlying mesenchyme remained unchanged. Further analysis of Cdx1-Cdx2 double mutants suggests that Cdx1 does not play a critical role in the development of the small intestine, at least after E13.5.

Cdx function is required for maintenance of intestinal identity in the adult

The homeodomain transcription factors Cdx1 and Cdx2 are expressed in the intestinal epithelium from early development, with expression persisting throughout the life of the animal. While our understanding of the function of Cdx members in intestinal development has advanced significantly, their roles in the adult intestine is relatively poorly understood. In the present study, we found that ablation of Cdx2 in the adult small intestine severely impacted villus morphology, proliferation and intestinal gene expression patterns, resulting in the demise of the animal. Long-term loss of Cdx2 in a chimeric model resulted in loss of all differentiated intestinal cell types and partial conversion of the mucosa to a gastric-like epithelium. Concomitant loss of Cdx1 did not exacerbate any of these phenotypes. Loss of Cdx2 in the colon was associated with a shift to a cecum-like epithelial morphology and gain of cecum-associated genes which was more pronounced with subsequent loss of Cdx1. These findings suggest that Cdx2 is essential for differentiation of the small intestinal epithelium, and that both Cdx1 and Cdx2 contribute to homeostasis of the colon.

Cdx1 and Cdx2 Function as Tumor Suppressors

Background: Cdx function is essential for intestinal homeostasis and may impact tumorigenesis. Results: Cdx loss increases tumor incidence and alters tumor phenotype in an APC mutant background. Conclusion: Cdx transcription factors impact intestinal tumorigenesis. Significance: Cdx members modulate both the frequency and phenotype of intestinal tumors through previously unrecognized mechanisms. In humans, colorectal cancer is often initiated through APC loss of function, which leads to crypt hyperplasia and polyposis driven by unrestricted canonical Wnt signaling. Such polyps typically arise in the colorectal region and are at risk of transforming to invasive adenocarcinomas. Although colorectal cancer is the third most common cause of cancer-related death worldwide, the processes impacting initiation, transformation, and invasion are incompletely understood. Murine APCMin/+ mutants are often used to model colorectal cancers; however, they develop nonmetastatic tumors confined largely to the small intestine and are thus not entirely representative of the human disease. APCMin/+ alleles can collaborate with mutations impacting other pathways to recapitulate some aspects of human colorectal cancer. To this end, we assessed APCMin/+-induced polyposis following somatic loss of the homeodomain transcription factor Cdx2, alone or with a Cdx1 null allele, in the adult gastrointestinal tract. APCMin/+-Cdx2 mutants recapitulated several aspects of human colorectal cancer, including an invasive phenotype. Notably, the concomitant loss of Cdx1 led to a significant increase in the incidence of tumors in the distal colon, relative to APCMin/+-Cdx2 offspring, demonstrating a previously unrecognized role for this transcription factor in colorectal tumorigenesis. These findings underscore previously unrecognized roles for Cdx members in intestinal tumorigenesis.

Biallelic mutations in the 3′ exonuclease TOE1 cause pontocerebellar hypoplasia and uncover a role in snRNA processing

Deadenylases are best known for degrading the poly(A) tail during mRNA decay. The deadenylase family has expanded throughout evolution and, in mammals, consists of 12 Mg2+-dependent 3′-end RNases with substrate specificity that is mostly unknown. Pontocerebellar hypoplasia type 7 (PCH7) is a unique recessive syndrome characterized by neurodegeneration and ambiguous genitalia. We studied 12 human families with PCH7, uncovering biallelic, loss-of-function mutations in TOE1, which encodes an unconventional deadenylase. toe1-morphant zebrafish displayed midbrain and hindbrain degeneration, modeling PCH-like structural defects in vivo. Surprisingly, we found that TOE1 associated with small nuclear RNAs (snRNAs) incompletely processed spliceosomal. These pre-snRNAs contained 3′ genome-encoded tails often followed by post-transcriptionally added adenosines. Human cells with reduced levels of TOE1 accumulated 3′-end-extended pre-snRNAs, and the immunoisolated TOE1 complex was sufficient for 3′-end maturation of snRNAs. Our findings identify the cause of a neurodegenerative syndrome linked to snRNA maturation and uncover a key factor involved in the processing of snRNA 3′ ends.

Cdx regulates Dll1 in multiple lineages.

Vertebrate Cdx genes encode homeodomain transcription factors related to caudal in Drosophila. The murine Cdx homologues Cdx1, Cdx2 and Cdx4 play important roles in anterior-posterior patterning of the embryonic axis and the intestine, as well as axial elongation. While our understanding of the ontogenic programs requiring Cdx function has advanced considerably, the molecular bases underlying these functions are less well understood. In this regard, Cdx1-Cdx2 conditional mutants exhibit abnormal somite formation, while loss of Cdx1-Cdx2 in the intestinal epithelium results in a shift in differentiation toward the Goblet cell lineage. The aim of the present study was to identify the Cdx-dependent mechanisms impacting on these events. Consistent with prior work implicating Notch signaling in these pathways, we found that expression of the Notch ligand Dll1 was reduced in Cdx mutants in both the intestinal epithelium and paraxial mesoderm. Cdx members occupied the Dll1 promoter both in vivo and in vitro, while genetic analysis indicated interaction between Cdx and Dll1 pathways in both somitogenesis and Goblet cell differentiation. These findings suggest that Cdx members operate upstream of Dll1 to convey different functions in two distinct lineages.

Wnt9a Is Required for the Aortic Amplification of Nascent Hematopoietic Stem Cells

Summary All mature blood cell types in the adult animal arise from hematopoietic stem and progenitor cells (HSPCs). However, the developmental cues regulating HSPC ontogeny are incompletely understood. In particular, the details surrounding a requirement for Wnt/β-catenin signaling in the development of mature HSPCs are controversial and difficult to consolidate. Using zebrafish, we demonstrate that Wnt signaling is required to direct an amplification of HSPCs in the aorta. Wnt9a is specifically required for this process and cannot be replaced by Wnt9b or Wnt3a. This proliferative event occurs independently of initial HSPC fate specification, and the Wnt9a input is required prior to aorta formation. HSPC arterial amplification occurs prior to seeding of secondary hematopoietic tissues and proceeds, in part, through the cell cycle regulator myca (c-myc). Our results support a general paradigm, in which early signaling events, including Wnt, direct later HSPC developmental processes.

Cdx1 and Cdx2 exhibit transcriptional specificity in the intestine.

The caudal-related homeodomain transcription factors Cdx1 and Cdx2 are expressed in the developing endoderm with expression persisting into adulthood. Cdx1−/− mutants are viable and fertile and display no overt intestinal phenotype. Cdx2 null mutants are peri-implantation lethal; however, conditional mutation approaches have revealed that Cdx2 is required for patterning the intestinal epithelium and specification of the colon. Cdx2 is also necessary for homeostasis of the intestinal tract in the adult, where Cdx1 and Cdx2 appear to functionally overlap in the distal colon, but not during intestinal development. Cdx1 and Cdx2 exhibit complete overlap of expression in the intestine, although they differ in their relative levels, with Cdx1 maximal in the distal colon and Cdx2 peaking in the proximal cecum. Moreover, Cdx1 protein is graded along the crypt-villus axis, being abundant in the crypts and diminishing towards the villi. Cdx2 is expressed uniformly along this axis, but is differentially phosphorylated; the functional relevance of these expression domains and phosphorylation is currently unknown. Cdx1 and Cdx2 have been suggested to exhibit functional specificity in the intestinal tract. In the present study, using cell-based models, we found that relative to Cdx1, Cdx2 was significantly less potent at effecting a transcriptional response from the Cdx1 promoter, a known Cdx target gene. We subsequently assessed this relationship in vivo using a “gene swap” approach and found that Cdx2 cannot substitute for Cdx1 in this autoregulatory loop. This is in marked contrast with the ability of Cdx2 to support Cdx1 expression and function in paraxial mesoderm and vertebral patterning, thus providing novel in vivo evidence of context-dependent transcriptional specificity between these transcription factors.

Mechanisms of Wnt signaling and control

The Wnt signaling pathway is a highly conserved system that regulates complex biological processes across all metazoan species. At the cellular level, secreted Wnt proteins serve to break symmetry and provide cells with positional information that is critical to the patterning of the entire body plan. At the organismal level, Wnt signals are employed to orchestrate fundamental developmental processes, including the specification of the anterior–posterior body axis, induction of the primitive streak and ensuing gastrulation movements, and the generation of cell and tissue diversity. Wnt functions extend into adulthood where they regulate stem cell behavior, tissue homeostasis, and damage repair. Disruption of Wnt signaling activity during embryonic development or in adults results in a spectrum of abnormalities and diseases, including cancer. The molecular mechanisms that underlie the myriad of Wnt‐regulated biological effects have been the subject of intense research for over three decades. This review is intended to summarize our current understanding of how Wnt signals are generated and interpreted.

Cdx1 Interacts Physically with a Subset of Hox Proteins

Cdx and Hox gene families encode homeodomain-containing transcription factors involved in anterior-posterior vertebral patterning. Although Cdx proteins are direct transcriptional regulators of Hox gene expression, both Hox and Cdx proteins are known to interact with other homeodomain transcription factors, leading us to speculate that Cdx and Hox proteins may also interact physically. In testing this, we found that that Cdx1 is indeed capable of associating with a subset of Hox proteins. This interaction is localized to the homeodomain region of both classes of proteins, is reliant on specific arginine residues in helix I of the Hox homeodomain, and is further modulated by N-terminal Hox sequences. More promiscuous interactions were seen with Hox proteins expressed in vivo, suggestive of bridging factors or post-translational modifications. Finally, we demonstrate that this interaction modulates Cdx-Hox transcriptional activity on a Hox-responsive element. This study is the first example of Cdx-Hox protein interactions and suggests that such complexes may modulate Hox and/or Cdx function.

EGFR confers exquisite specificity of Wnt9a-Fzd9b signaling in hematopoietic stem cell development

The mechanisms of Wnt-Frizzled (Fzd) signaling selectivity and their biological implications remain unclear. We demonstrate for the first time that the epidermal growth factor receptor (EGFR) is required as a co-factor for Wnt signaling. Using genetic studies in zebrafish, paired with in vitro cell biology and biochemistry, we have determined that Fzd9b signals specifically with Wnt9a in vivo and in vitro to elicit β-catenin dependent Wnt signals that regulate hematopoietic stem and progenitor cell (HSPC) development in the dorsal aorta. This requirement is conserved in the derivation of HSPCs from human embryonic stem cells. Wnt9a-Fzd9b specificity requires two intracellular domains in Fzd9b, which interact with EGFR as a required co-factor to promote signal transduction. EGFR phosphorylates one tyrosine residue on Fzd9b, a requirement for the Wnt signal. These findings indicate that Wnt signaling interactions can be exquisitely specific and inform protocols for derivation of HSPCs in vitro. Highlights An in vitro signaling screen identifies Fzd9b as a Wnt9a-specific receptor. Fzd9b and Wnt9a regulate hematopoietic stem cell development as a cognate pair. WNT9A and FZD9 are required for HSPC derivation from human pluripotent cells in vitro. EGFR confers specificity to Wnt9a-Fzd9b signaling in zebrafish and human cells.