Microfibril-associated glycoprotein 4 (Mfap4) regulates haematopoiesis in zebrafish.

Microfibril-associated glycoprotein 4 (MFAP4) is an extracellular matrix protein belonging to the fibrinogen-related protein superfamily. MFAP4 is produced by vascular smooth muscle cells and is highly enriched in the blood vessels of the heart and lung, where it is thought to contribute to the structure and function of elastic fibers. Genetic studies in humans have implicated MFAP4 in the pathogenesis of Smith-Magenis syndrome, in which patients present with multiple congenital abnormalities and mental retardation, as well as in the severe cardiac malformation left-sided congenital heart disease. Comprehensive genetic analysis of the role of MFAP4 orthologues in model organisms during development and tissue homeostasis is however lacking. Here, we demonstrate that zebrafish mfap4 transcripts are detected embryonically, resolving to the macrophage lineage by 24 h post fertilization. mfap4 null mutant zebrafish are unexpectedly viable and fertile, without ostensible phenotypes. However, tail fin amputation assays reveal that mfap4 mutants have reduced numbers of macrophages, with a concomitant increase in neutrophilic granulocytes, although recruitment of both cell types to the site of injury was unaffected. Molecular analyses suggest that loss of Mfap4 alters the balance between myeloid and lymphoid lineages during both primitive and definitive haematopoiesis, which could significantly impact the downstream function of the immune system.

The PDZ domain protein Mcc is a novel effector of non-canonical Wnt signaling during convergence and extension in zebrafish.

During vertebrate gastrulation, a complex set of mass cellular rearrangements shapes the embryonic body plan and appropriately positions the organ primordia. In zebrafish and Xenopus, convergence and extension (CE) movements simultaneously narrow the body axis mediolaterally and elongate it from head to tail. This process is governed by polarized cell behaviors that are coordinated by components of the non-canonical, ß-catenin-independent Wnt signaling pathway, including Wnt5b and the transmembrane planar cell polarity (PCP) protein Vangl2. However, the intracellular events downstream of Wnt/PCP signals are not fully understood. Here, we show that zebrafish mutated in colorectal cancer (mcc), which encodes an evolutionarily conserved PDZ domain-containing putative tumor suppressor, is required for Wnt5b/Vangl2 signaling during gastrulation. Knockdown of mcc results in CE phenotypes similar to loss of vangl2 and wnt5b, whereas overexpression of mcc robustly rescues the depletion of wnt5b, vangl2 and the Wnt5b tyrosine kinase receptor ror2. Biochemical experiments establish a direct physical interaction between Mcc and the Vangl2 cytoplasmic tail. Lastly, CE defects in mcc morphants are suppressed by downstream activation of RhoA and JNK. Taken together, our results identify Mcc as a novel intracellular effector of non-canonical Wnt5b/Vangl2/Ror2 signaling during vertebrate gastrulation.

The SMAD2/3 corepressor SNON maintains pluripotency through selective repression of mesendodermal genes in human ES cells.

Activin/Nodal signaling via SMAD2/3 maintains human embryonic stem cell (hESC) pluripotency by direct transcriptional regulation of NANOG or, alternatively, induces mesoderm and definitive endoderm (DE) formation. In search of an explanation for these contrasting effects, we focused on SNON (SKIL), a potent SMAD2/3 corepressor that is expressed in hESCs but rapidly down-regulated upon differentiation. We show that SNON predominantly associates with SMAD2 at the promoters of primitive streak (PS) and early DE marker genes. Knockdown of SNON results in premature activation of PS and DE genes and loss of hESC morphology. In contrast, enforced SNON expression inhibits DE formation and diverts hESCs toward an extraembryonic fate. Thus, our findings provide novel mechanistic insight into how a single signaling pathway both regulates pluripotency and directs lineage commitment.

Activin and BMP4 synergistically promote formation of definitive endoderm in human embryonic stem cells.

Human embryonic stem cells (hESCs) herald tremendous promise for the production of clinically useful cell types for the treatment of injury and disease. Numerous reports demonstrate their differentiation into definitive endoderm (DE) cells, the germ layer from which pancreatic ß cells and hepatocytes arise, solely from exposure to a high dose of recombinant Activin/Nodal. We show that combining a second related ligand, BMP4, in combination with Activin A yields 15%-20% more DE as compared with Activin A alone. The addition of recombinant BMP4 accelerates the downregulation of pluripotency genes, particularly SOX2, and results in upregulation of endogenous BMP2 and BMP4, which in turn leads to elevated levels of phospho-SMAD1/5/8. Combined Activin A and BMP4 treatment also leads to an increase in the expression of DE genes CXCR4, SOX17, and FOXA2 when compared with Activin A addition alone. Comparative microarray studies between DE cells harvested on day 3 of differentiation further reveal a novel set of genes upregulated in response to initial BMP4 exposure. Several of these, including APLNR, LRIG3, MCC, LEPREL1, ROR2, and LZTS1, are expressed in the mouse primitive streak, the site of DE formation. Thus, this synergism between Activin A and BMP4 during the in vitro differentiation of hESC into DE suggests a complex interplay between BMP and Activin/Nodal signaling during the in vivo allocation and expansion of the endoderm lineage.

Mutated in colorectal cancer (Mcc), a candidate tumor suppressor, is dynamically expressed during mouse embryogenesis.

Mutated in Colorectal Cancer (MCC) encodes a multiple PSD-95/Dlg/ZO-1 (PDZ) domain-containing protein implicated, as its name suggests, in the pathogenesis of human colon cancer. To date, however, what role, if any, MCC plays in normal tissue homeostasis and development remains unclear. In an effort to expand our understanding of MCC function and distribution, we examined the expression of the evolutionarily conserved mouse Mcc homolog between embryonic days (E) 6.5 and 12.5 using conventional whole-mount in situ hybridization and two independent Mcc reporter alleles. Mcc is expressed in the posterior primitive streak during gastrulation and in diverse tissues of both mesodermal and endodermal origin. In addition, Mcc transcripts localize to the posterior neural tube and identify discrete neuronal subtypes and ganglia within the developing central nervous system. Genetically, however, Mcc is entirely dispensable, as mice homozygous for the Mcc(Gt(D062B07)) gene trap allele, which generates a loss-of-function mutation, are viable and fertile, with no ostensible phenotype.

Localization and regulation of aromatase liver receptor homologue-1 in the developing rat testis.

The enzyme aromatase converts androgens to estrogens, which have recently been postulated to be essential for testicular development and fertility. Understanding the mechanisms that regulate aromatase activity in the testis may therefore have implications for treatment of male infertility. Aromatase is encoded by the CYP19 gene, which uses multiple tissue-specific alternative promoters. In the testis, the proximal promoter PII drives aromatase expression. PII activity requires a nuclear receptor half-site, CAAGGTCA, to which two orphan receptors; SF-1 and LRH-1, have been shown to bind in vitro. The aim of this study was to investigate expression of aromatase and LRH-1 in the developing rat testis and define the ability of LRH-1 to induce aromatase expression in the testicular cells where both are expressed. We show that aromatase and LRH-1 are present throughout all stages of development of the rat testis, although the sites and levels of expression vary. The pattern of LRH-1 expression was broadly similar to that of aromatase. In adult animals higher levels of expression were observed in Leydig and germ cells. Over-expression of LRH-1 in primary rat Leydig and germ cells by adenoviral infection strongly increased endogenous aromatase mRNA levels, demonstrating the ability of LRH-1 to stimulate aromatase expression in vivo. We also observed binding of endogenous LRH-1 to the aromatase promoter II by chromatin immunoprecipitation. These data provide evidence that LRH-1 plays an important role in the regulation of testicular aromatase expression, and implicate LRH-1 as a regulator of rat spermatogenesis, in which estrogens are emerging as important mediators.