Formation and characterization of BMP2/GDF5 and BMP4/GDF5 heterodimers.

BACKGROUND: Proteins of the TGFß family, which are largely studied as homodimers, are also known to form heterodimers with biological activity distinct from their component homodimers. For instance, heterodimers of bone morphogenetic proteins, including BMP2/BMP7, BMP2/BMP6, and BMP9/BMP10, among others, have illustrated the importance of these heterodimeric proteins within the context of TGFß signaling. RESULTS: In this study, we have determined that mature GDF5 can be combined with mature BMP2 or BMP4 to form BMP2/GDF5 and BMP4/GDF5 heterodimer. Intriguingly, this combination of a BMP2 or BMP4 monomer, which exhibit high affinity to heparan sulfate characteristic to the BMP class, with a GDF5 monomer with low heparan sulfate affinity produces a heterodimer with an intermediate affinity. Using heparin affinity chromatography to purify the heterodimeric proteins, we then determined that both the BMP2/GDF5 and BMP4/GDF5 heterodimers consistently signaled potently across an array of cellular and in vivo systems, while the activities of their homodimeric counterparts were more context dependent. These differences were likely driven by an increase in the combined affinities for the type 1 receptors, Alk3 and Alk6. Furthermore, the X-ray crystal structure of BMP2/GDF5 heterodimer was determined, highlighting the formation of two asymmetric type 1 receptor binding sites that are both unique relative to the homodimers. CONCLUSIONS: Ultimately, this method of heterodimer production yielded a signaling molecule with unique properties relative to the homodimeric ligands, including high affinity to multiple type 1 and moderate heparan binding affinity.

Identification of genes expressed in the migrating primitive myeloid lineage of Xenopus laevis.

BACKGROUND: During primitive hematopoiesis in Xenopus, cebpa and spib expressing myeloid cells emerge from the anterior ventral blood island. Primitive myeloid cells migrate throughout the embryo and are critical for immunity, healing, and development. Although definitive hematopoiesis has been studied extensively, molecular mechanisms leading to the migration of primitive myelocytes remain poorly understood. We hypothesized these cells have specific extracellular matrix modifying and cell motility gene expression. RESULTS: In situ hybridization screens of transcripts expressed in Xenopus foregut mesendoderm at stage 23 identified seven genes with restricted expression in primitive myeloid cells: destrin; coronin actin binding protein, 1a; formin-like 1; ADAM metallopeptidase domain 28; cathepsin S; tissue inhibitor of metalloproteinase-1; and protein tyrosine phosphatase nonreceptor 6. A detailed in situ hybridization analysis revealed these genes are initially expressed in the aVBI but become dispersed throughout the embryo as the primitive myeloid cells become migratory, similar to known myeloid markers. Morpholino-mediated loss-of-function and mRNA-mediated gain-of-function studies revealed the identified genes are downstream of Spib.a and Cebpa, key transcriptional regulators of the myeloid lineage. CONCLUSIONS: We have identified genes specifically expressed in migratory primitive myeloid progenitors, providing tools to study how different gene networks operate in these primitive myelocytes during development and immunity.

Disrupted endosomal trafficking of the Vangl-Celsr polarity complex underlies congenital anomalies in trachea-esophageal morphogenesis.

Disruptions in foregut morphogenesis can result in life-threatening conditions where the trachea and esophagus fail to separate properly, such as esophageal atresia (EA) and tracheoesophageal fistulas (TEF). The developmental basis of these congenital anomalies is poorly understood, but recent genome sequencing reveals that de novo variants in intracellular trafficking genes are enriched in EA/TEF patients. Here we show that mutation of orthologous genes in Xenopus disrupts trachea-esophageal separation similar to EA/TEF patients. We show that the Rab11a recycling endosome pathway is required to localize Vangl-Celsr polarity complexes at the cell surface where opposite sides of the common foregut tube fuse. Partial loss of endosome trafficking or the Vangl/Celsr complex disrupts epithelial polarity and cell division orientation. Mutant cells accumulate at the fusion point, fail to downregulate cadherin, and do not separate into distinct trachea and esophagus. These data provide new insights into the mechanisms of congenital anomalies and general paradigms of tissue fusion during organogenesis.

Endosome-Mediated Epithelial Remodeling Downstream of Hedgehog-Gli Is Required for Tracheoesophageal Separation.

The trachea and esophagus arise from the separation of a common foregut tube during early fetal development. Mutations in key signaling pathways such as Hedgehog (HH)/Gli can disrupt tracheoesophageal (TE) morphogenesis and cause life-threatening birth defects (TEDs); however, the underlying cellular mechanisms are unknown. Here, we use mouse and Xenopus to define the HH/Gli-dependent processes orchestrating TE morphogenesis. We show that downstream of Gli the Foxf1+ splanchnic mesenchyme promotes medial constriction of the foregut at the boundary between the presumptive Sox2+ esophageal and Nkx2-1+ tracheal epithelium. We identify a unique boundary epithelium co-expressing Sox2 and Nkx2-1 that fuses to form a transient septum. Septum formation and resolution into distinct trachea and esophagus requires endosome-mediated epithelial remodeling involving the small GTPase Rab11 and localized extracellular matrix degradation. These are disrupted in Gli-deficient embryos. This work provides a new mechanistic framework for TE morphogenesis and informs the cellular basis of human TEDs.

Two-color fluorescence in situ hybridization using chromogenic substrates in Xenopus.

Xenopus embryo yolk proteins present a serious barrier to fluorescence microscopy. Previously, in situ assays of gene transcripts in whole embryos was limited to the use of chromogenic alkaline phosphatase substrates, which restricted researchers' ability to gauge coexpression of transcripts. Here, we describe a modified in situ hybridization (ISH) protocol that uses fluorescent substrates and a novel yolk-clearing technique for simultaneous visualization of the expression of two genes in whole Xenopus embryos with high resolution. This protocol employs two well-known fluorescent substrates, nitro blue tetrazolium/5-bromo- 4-chloro-3-indolyl-phosphate (NBT/BCIP) and Vector Red, in a sequential dual in situ hybridization procedure. Subsequent clearing of the samples with refractive-index-matching solution (RIMS) renders the samples amenable to confocal microscopy, allowing imaging at sufficiently high resolution to discern coexpression of transcripts within individual cells.