Epidermal growth factor suppresses intestinal epithelial cell shedding through a MAPK-dependent pathway.

Cell shedding from the intestinal villus is a key element of tissue turnover that is essential to maintain health and homeostasis. However, the signals regulating this process are not well understood. We asked whether shedding is controlled by epidermal growth factor receptor (EGFR), an important driver of intestinal growth and differentiation. In 3D ileal enteroid culture and cell culture models (MDCK, IEC-6 and IPEC-J2 cells), extrusion events were suppressed by EGF, as determined by direct counting of released cells or rhodamine-phalloidin labeling of condensed actin rings. Blockade of the MEK-ERK pathway, but not other downstream pathways such as phosphoinositide 3-kinase (PI3K) or protein kinase C (PKC), reversed EGF inhibition of shedding. These effects were not due to a change in cell viability. Furthermore, EGF-driven MAPK signaling inhibited both caspase-independent and -dependent shedding pathways. Similar results were found in vivo, in a novel zebrafish model for intestinal epithelial shedding. Taken together, the data show that EGF suppresses cell shedding in the intestinal epithelium through a selective MAPK-dependent pathway affecting multiple extrusion mechanisms. EGFR signaling might be a therapeutic target for disorders featuring excessive cell turnover, such as inflammatory bowel diseases.

tbx6l and tbx16 are redundantly required for posterior paraxial mesoderm formation during zebrafish embryogenesis.

BACKGROUND: T-box genes encode a large transcription factor family implicated in many aspects of development. We are focusing on two related zebrafish T-box genes, tbx6l and tbx16, that are expressed in highly overlapping patterns in embryonic paraxial mesoderm. tbx16 mutants are deficient in trunk, but not tail, somites; we explored whether presence of tail somites in tbx16 mutants was due to compensatory function provided by the tbx6l gene. RESULTS: We generated two zebrafish tbx6l mutant alleles. Loss of tbx6l has no apparent effect on embryonic development, nor does tbx6l loss enhance the phenotype of two other T-box gene mutants, ta and tbx6, or of the mesp family gene mutant msgn1. In contrast, loss of tbx6l function dramatically enhances the paraxial mesoderm deficiency of tbx16 mutants. CONCLUSIONS: These data demonstrate that tbx6l and tbx16 genes function redundantly to direct tail somite development. tbx6l single mutants develop normally because tbx16 fully compensates for loss of tbx6l function. However, tbx6l only partially compensates for loss of tbx16 function. These results resolve the question of why loss of function of tbx16 gene, which is expressed throughout the ventral and paraxial mesoderm, profoundly affects somite development in the trunk but not the tail. Developmental Dynamics 246:759-769, 2017. © 2017 Wiley Periodicals, Inc.

The differentiation and movement of presomitic mesoderm progenitor cells are controlled by Mesogenin 1.

Somites are formed from the presomitic mesoderm (PSM) and give rise to the axial skeleton and skeletal muscles. The PSM is dynamic; somites are generated at the anterior end, while the posterior end is continually renewed with new cells entering from the tailbud progenitor region. Which genes control the conversion of tailbud progenitors into PSM and how is this process coordinated with cell movement? Using loss- and gain-of-function experiments and heat-shock transgenics we show in zebrafish that the transcription factor Mesogenin 1 (Msgn1), acting with Spadetail (Spt), has a central role. Msgn1 allows progression of the PSM differentiation program by switching off the progenitor maintenance genes ntl, wnt3a, wnt8 and fgf8 in the future PSM cells as they exit from the tailbud, and subsequently induces expression of PSM markers such as tbx24. msgn1 is itself positively regulated by Ntl/Wnt/Fgf, creating a negative-feedback loop that might be crucial to regulate homeostasis of the progenitor population until somitogenesis ends. Msgn1 drives not only the changes in gene expression in the nascent PSM cells but also the movements by which they stream out of the tailbud into the PSM. Loss of Msgn1 reduces the flux of cells out of the tailbud, producing smaller somites and an enlarged tailbud, and, by delaying exhaustion of the progenitor population, results in supernumerary tail somites. Through its combined effects on gene expression and cell movement, Msgn1 (with Spt) plays a key role both in genesis of the paraxial mesoderm and in maintenance of the progenitor population from which it derives.

Discovery and characterization of an amidinotransferase involved in the modification of archaeal tRNA.

The presence of the 7-deazaguanosine derivative archaeosine (G(+)) at position 15 in tRNA is one of the diagnostic molecular characteristics of the Archaea. The biosynthesis of this modified nucleoside is especially complex, involving the initial production of 7-cyano-7-deazaguanine (preQ(0)), an advanced precursor that is produced in a tRNA-independent portion of the biosynthesis, followed by its insertion into the tRNA by the enzyme tRNA-guanine transglycosylase (arcTGT), which replaces the target guanine base yielding preQ(0)-tRNA. The enzymes responsible for the biosynthesis of preQ(0) were recently identified, but the enzyme(s) catalyzing the conversion of preQ(0)-tRNA to G(+)-tRNA have remained elusive. Using a comparative genomics approach, we identified a protein family implicated in the late stages of archaeosine biosynthesis. Notably, this family is a paralog of arcTGT and is generally annotated as TgtA2. Structure-based alignments comparing arcTGT and TgtA2 reveal that TgtA2 lacks key arcTGT catalytic residues and contains an additional module. We constructed a Haloferax volcanii DeltatgtA2 derivative and demonstrated that tRNA from this strain lacks G(+) and instead accumulates preQ(0). We also cloned the corresponding gene from Methanocaldococcus jannaschii (mj1022) and characterized the purified recombinant enzyme. Recombinant MjTgtA2 was shown to convert preQ(0)-tRNA to G(+)-tRNA using several nitrogen sources and to do so in an ATP-independent process. This is the only example of the conversion of a nitrile to a formamidine known in biology and represents a new class of amidinotransferase chemistry.