Defining progressive stages in the commitment process leading to embryonic lens formation

Abstract

Summary The commitment of regions of the embryo to form particular tissues or organs is a central concept in development, but the mechanisms controlling this process remain elusive. The well-studied model of lens induction is ideal for dissecting key phases of the commitment process. We find in Xenopus tropicalis, at the time of specification of the lens, i.e. when presumptive lens ectoderm (PLE) can be isolated, cultured and will differentiate into a lens, that the PLE is not yet irreversibly committed, or determined, to form a lens. When transplanted into the posterior of a host embryo lens development is prevented at this stage, while approximately 3 hr later, using the same assay, determination is complete. Interestingly, we find that specified lens ectoderm, when cultured, acquires the ability to become determined without further tissue interactions. Further, we show that specified PLE has a different gene expression pattern than determined PLE, and that determined PLE can maintain expression of essential regulatory genes (e.g. foxe3, mafB) in an ectopic environment while specified PLE cannot. These observations set the stage for a detailed mechanistic study of the genes and signals controlling tissue commitment.

Special Considerations for Making Explants and Transplants with Xenopus tropicalis

“…The techniques described here, although focused on the PLE, apply to any preplacodal head ectoderm as well as to early neural plate/neural tube regions, and with slight modifications, to any embryonic stage. We have used these techniques to prepare PLE explants and transplants for studying aspects of PLE determination in wild-type and transgenic X. laevis (e.g., see Grainger et al [1988]) and in wildtype, mutant and transgenic X. tropicalis (e.g., see Jin et al [2012]). The key point is that PLE from both species responds similarly.…”

“…In the case of the PLE, the degree of difficulty of separating the ectoderm from the underlying tissues will increase with the increasing age of embryo and the degree of interaction between the PLE and ultimately the optic vesicle which comes to underlie it. By stage 21, coincidently the age of determination of the PLE (Jin et al 2012), it can be extremely difficult to separate the ectoderm from the underlying optic vesicle. 2× Steinberg's solution can be used in place of MBS during dissection to ease tissue separation.…”

“…For some studies it might be desirable to transplant tissues to another embryo, either hetero-or homotopically. In many cases, it is advisable to use host and donor marking to distinguish transplanted tissue from host tissue (e.g., see Jin et al [2012]). Here the PLE is dissected from a neurula-stage embryo and then transplanted to a neurula-stage host.…”

Six3acts independently ofPax6to provide an essential contribution to lens development

“…Owing to the limitations in performing quantitative assessments by histology, we examined representative markers expressed during lens development at st.37/38, by when, for example, a lens fiber marker, cryg1, is highly expressed in WT embryos (e.g., Jin et al, 2012).…”

“…As described here, both Pax6 and Six3 contribute to lens formation, a process in Xenopus that has been described as involving multiple stages, beginning at gastrulation, when presumptive lens ectoderm (PLE) is first competent to form a lens, followed by the “bias” stage when lens-inducing signals emanate from the neural plate/retina area, then determination at the neural tube stage and subsequently by differentiation, when crystallin synthesis occurs (Grainger, 1992; Grainger et al, 1997). Gene regulation studies during the lens induction process (Gunhaga, 2011; Jin et al, 2012; Ogino et al, 2012) reveal essential signaling and TF networks, e.g., in X. tropicalis showing that Delta/Notch signaling from the optic vesicle leads to activation in the PLE of foxe3 , a forkhead class TF essential for lens development (Ogino et al, 2008). Other studies on signaling from the developing retina show that BMP4 functions as an optic vesicle-derived signal required for lens formation (Furuta and Hogan, 1998; Huang et al, 2015).…”