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

Abstract

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.

Using Xenopus to understand human disease and developmental disorders

“…Xenopus also has greatly enhanced our understanding of DNA replication and repair (Kalb, Mallery, Larkin, Huang, & Hiom, ; Long, Joukov, Budzowska, & Walter, ; Räschle et al, ; Shintomi, Takahashi, & Hirano, ; Zhang et al, ), and the mechanisms of genome organization, transcriptional regulation, and epigenetics (Belikov, Berg, & Wrange, ; Bogdanović et al, ; Buisine et al, ; Gazdag, Jacobi, van Kruijsbergen, Weeks, & Veenstra, ; Gao et al, ; Hontelez et al, ; Owens et al, ; Tamaoki et al, ; Wang et al, ; Wen, Fu, Guo, Chen, & Shi, ). Numerous studies in Xenopus elucidate mechanisms of morphogenesis and organogenesis ( Agricola et al, ; Bestman, Huang, Lee‐Osbourne, Cheung, & Cline, ; Metikala, Neuhaus, & Hollemann, ; Mimoto, Kwon, Green, Goldman, & Christian, ; Nie & Bronner, ; Okada, Wen, Miller, Su, & Shi, ; Ossipova et al, ; Tanizaki, Ishida‐Iwata, Obuchi‐Shimoji, & Kato, ). Many of the components of the well‐known signaling pathways were discovered in Xenopus and fly, and new discoveries in this field continue to rely on Xenopus (e.g., Lee et al, ; Lee, Shi, & Zheng, ; Park et al, ; Zhang et al, ; Zhao, Shi, Winey, & Klymkowsky, ).…”

An in vivo brain–bacteria interface: the developing brain as a key regulator of innate immunity

“…Spib-is a highly conserved ETS transcription factor that marks the primitive myeloid-cell lineage and is required for its development. 3,10 spib expression in myeloid precursors has been characterized in Xenopus, 10 mouse, 46 and humans. 47 To specifically probe the role of the brain in establishing key cell types involved in innate immunity, we studied the spiba-positive 10 cells in UTI and NI early-staged embryos (st. 28), subjected to the different surgical interventions (Ctrl, BR -, SC -, Tail -; Supplementary Fig.…”

“…3,10 mmp7 is a secreted metalloproteinase involved in extracellular matrix remodeling and extensively used as macrophage differentiation and migration markers. 3,10,11,48,82 mmp7 probe was used to label migrating embryonic macrophages on st. 35-36 embryos, as relative to other myeloid markers, mmp7 is expressed considerably later. 10 Probes were generated in vitro from linearized templates using a digoxigenin (DIG)-labeling mix (Sigma-Aldrich, #11277065910 Roche).…”

“…Innate immunity provides a first line of defense against pathogens 1 and plays a crucial role in initiating adaptive immune responses. 2 Most of the cell types of hematopoietic origin and genes known to be involved in mammalian innate and adaptive immunity have been identified in X. laevis, 3,4 making the frog model an important emerging tool for understanding the cell and molecular biology of immunity. 1,5,6 The immune response in the Xenopus embryo is provided uniquely by innate immunity during the first 12 days (d) post-fertilization, 7 allowing studies of response to infection by wide range of pathogens without the confounding influences of adaptive immune component.…”

Two-Color Fluorescence in Situ Hybridization using Chromogenic Substrates in Xenopus

“…While spib.a and mpo are known to be expressed in primitive myeloid cells (7), it is unclear whether these genes are expressed uniformly in the myeloid population or if there are subpopulations with different expression profiles. The more strongly expressed mpo probe was used with FL and the Vector Red AP substrate (Vector Laboratories, Burlingame, CA) to compensate for the lower intensity of the Vector Red fluorescence signal compared with the other substrate used in this study, NBT/BCIP (Roche Diagnostics).…”