TheXenopusLIM-homeodomain protein Xlim5 regulates the differential adhesion properties of early ectoderm cells

Abstract

One of the earliest lineage restriction events in embryogenesis is the specification of the primary germ layers: ectoderm, mesoderm and endoderm. In Xenopus, germ layer specification occurs prior to gastrulation and requires the transcription factor VegT both for the cellautonomous specification of endoderm and the generation of mesoderm-inducing signals. In the absence of VegT, ectoderm is formed in all regions of the embryo. In this work, we show that VegT-depleted vegetal cells (prospective endoderm) behave like animal cells in sorting assays and ectopically express early markers of ectoderm. To gain insight into how ectoderm is specified, we looked for candidate ectoderm-specific genes that are ectopically expressed in VegT-depleted embryos, and examined the role of one of these, the LIM homeobox gene Xlim5, in ectoderm development. We show that overexpression of Xlim5 in prospective endoderm cells is sufficient to impair sorting of animal cells from vegetal cells but is not sufficient (at similar doses) to induce ectoderm-specific genes. In whole embryos, Xlim5 causes vegetal cells to segregate inappropriately to other germ layers and express late differentiation markers of that germ layer. Inhibition of Xlim5 function using an Engrailed repressor construct or a morpholino oligonucleotide causes loss of animal cell adhesion or delay in neural fold morphogenesis,respectively, without significantly affecting early ectoderm gene expression. Taken together, our results provide evidence that a primary role for Xlim5 is to specifically regulate differential cell adhesion behaviour of the ectoderm.

Lhx5 promotes forebrain development and activates transcription of secreted Wnt antagonists

“…In frogs or fish, lhx5 is broadly expressed by early gastrulation stages. Inhibition of Lhx5 function in frogs causes the dissociation of rostral ectodermal cells during gastrulation (Houston and Wylie, 2003), and our studies in zebrafish suggest that Lhx5 regulates Wnt antagonism in the gastrula. In mice, however, no gastrulation defects have been reported in Lhx5 knockout animals (Zhao et al, 1999).…”

“…Lhx5 belongs to a family of LIM-homeodomain transcription factors, and contains two LIM protein interaction domains and a homeodomain (Hobert and Westphal, 2000;Retaux and Bachy, 2002;Toyama et al, 1995). Previous studies suggested that Lhx5 regulates the differential adhesion of early ectodermal cells in Xenopus (Houston and Wylie, 2003), and morphogenesis and cell proliferation in the mouse hippocampus (Zhao et al, 1999). We show that Lhx5 gain of function in zebrafish inhibits Wnt signaling, whereas inhibition of Lhx5 results in ectopic activation of Wnt signaling and forebrain defects.…”

FoxI1e activates ectoderm formation and controls cell position in theXenopusblastula

“…Each experiment was repeated a minimum of three times in independent experiments to verify reproducibility of results. Sequences for markerspecific primers are listed in Houston and Wylie (Houston and Wylie, 2003). New primers were generated for NRP-1 (U: 5Ј-TGATCCGCTCTG-CACTCTTT-3Ј, D: 5Ј-TGCTTTGAATTTGCAGTACATTG-3Ј), AP-2 (U: 5Ј-GCAGCCACCAACTCTTCTCT-3Ј, D: 5Ј-CGTAGCTCCATTGCCT-GTTC-3Ј) and Sox-2 (U: 5Ј-TCTGCACATGAAGGAGCATC-3Ј, D: 5Ј-CGTTCATGTGGGCATAAGTG-3Ј).…”

“…Cell mixing across this boundary is prevented by expression of surface proteins that control cell position by differential adhesion. Many elements of this model require further testing, including the functional relationship between FoxI1e and other factors known to be important in ectoderm differentiation such as ectodermin (Dupont et al, 2005) Xoom (Hasegawa et al, 2001) and Xlim5 (Houston and Wylie, 2003), as well as the epidermal proteins AP-2 (Luo et al, 2002) and grainyhead-like1 (Tao et al, 2005), and the many early CNS factors including Sox2 and Sox3. It is also likely that there will be signaling pathways in addition to nodals that control the spatial and temporal expression of FoxI1e, and we know little detail concerning its downstream targets.…”

“…The molecular mechanisms of this differential adhesion are not yet known. However, it has been shown that the ability of animal and vegetal cells to sort from one another is lost when VegT is depleted (Houston and Wylie, 2003). Further, singlecell transplantation experiments have shown that animal cells, which are able to contribute to all three germ layers during the early and mid-blastula stages, become gradually restricted to ectodermal fates from the late blastula to gastrula stages (Snape et al, 1987).…”