Cell Identity Switching Regulated by Retinoic Acid Signaling Maintains Homogeneous Segments in the Hindbrain

摘要

class="head no_bottom_margin" id="sec1title">IntroductionThe complex organization of the adult body arises during development by formation of distinct tissues at different locations, many of which are then further subdivided into domains with a specific regional identity. Such regionalization occurs along the anterior-posterior (A-P) axis of the vertebrate central nervous system to form subdivisions that are demarcated by sharp borders. A-P patterning of the neural epithelium is achieved through graded cell signaling, mediated by retinoic acid (RA) and members of the Fgf and Wnt families (), which regulates the spatial expression of transcription factors that specify regional identity (, ). However, at early stages the borders between different subdivisions are ragged and there can be overlapping expression of transcription factors that confer different identities. This imprecision is likely due in part to variability in the formation and interpretation of gradients of signals. In addition, the proliferation and intercalation of cells during tissue growth and morphogenesis can potentially scramble the pattern by causing intermingling of cells between adjacent regions. This raises the questions of how, despite these challenges, a sharp border forms at the interface of subdivisions, and each subdivision acquires a homogeneous regional identity. Insights into underlying mechanisms have come from studies of the vertebrate hindbrain.The hindbrain is subdivided into seven segments, termed rhombomeres (r1–r7), which underlie the organization and A-P specification of neurons and branchial neural crest cells (). Regionalization of the hindbrain is established by graded Fgf and RA signaling, which regulates the spatial expression of a network of transcription factors that underlie the formation and A-P identity of hindbrain segments, including Egr2 (Krox20), MafB, and Hox family members (). Initially, there is some overlap at borders between hoxb1 expression in r4, and egr2 expression in r3 and r5, which is resolved bdevcel_4183_gr4_4c.eps - y mutual repression such that cells express one or the other transcription factor (, , ). The borders of egr2 expression in r3 and r5 are ragged when first detected, and then progressively become sharp and straight (, , ). This sharpening is driven by signaling between segmentally expressed Eph receptors and ephrins that segregates cells and prevents intermingling across borders (, , , , href="#bib77" rid="bib77" class=" bibr popnode">Xu et al., 1999), potentially through regulation of cell adhesion, tension, and/or repulsion (href="#bib7" rid="bib7" class=" bibr popnode">Calzolari et al., 2014, href="#bib8" rid="bib8" class=" bibr popnode">Cayuso et al., 2015, href="#bib16" rid="bib16" class=" bibr popnode">Fagotto et al., 2014, href="#bib63" rid="bib63" class=" bibr popnode">Taylor et al., 2017). Computer simulations suggest that cell segregation and the resolution of cell identity have synergistic roles in border sharpening (href="#bib70" rid="bib70" class=" bibr popnode">Wang et al., 2017).A further mechanism required to establish segments with homogeneous identity was suggested by the results of clonal analyses in the chick hindbrain. Once rhombomeres are seen at the morphological level, intermingling of cells is restricted across segment borders, but the progeny of individual cells labeled at earlier stages can contribute to adjacent segments (href="#bib17" rid="bib17" class=" bibr popnode">Fraser et al., 1990). The finding that some intermingling occurs between hindbrain segments implies that cells that move into another segment acquire an identity in accordance with their new A-P location. Direct evidence for an ability of hindbrain cells to switch A-P identity has come from transplantation experiments in mouse and zebrafish embryos. It was found that when single cells are transplanted between hindbrain segments, they downregulate markers of their site of origin and switch to the identity of their new location (href="#bib25" rid="bib25" class=" bibr popnode">Kemp et al., 2009, href="#bib51" rid="bib51" class=" bibr popnode">Schilling et al., 2001, href="#bib66" rid="bib66" class=" bibr popnode">Trainor and Krumlauf, 2000). In zebrafish, cells can switch identity at early stages of segmentation (11.5 hr post fertilization [hpf]), but this plasticity progressively decreases at later stages (14–16.5 hpf) (href="#bib51" rid="bib51" class=" bibr popnode">Schilling et al., 2001). In contrast to single cells, groups of cells transplanted between segments maintain their original identity, suggestive of a community regulation of cell identity (href="#bib51" rid="bib51" class=" bibr popnode">Schilling et al., 2001, href="#bib66" rid="bib66" class=" bibr popnode">Trainor and Krumlauf, 2000). Such community effects have been found in other contexts to be mediated by positive feedback between transcription factors and intercellular signals that regulate cell identity (href="#bib3" rid="bib3" class=" bibr popnode">Bolouri and Davidson, 2010, href="#bib6" rid="bib6" class=" bibr popnode">Buckingham, 2003, href="#bib14" rid="bib14" class=" bibr popnode">Cossu et al., 1995, href="#bib21" rid="bib21" class=" bibr popnode">Gurdon, 1988, href="#bib61" rid="bib61" class=" bibr popnode">Standley et al., 2001). Through non-autonomous induction of transcription factor expression, this feedback promotes a homogeneous identity within a field of cells (href="#bib3" rid="bib3" class=" bibr popnode">Bolouri and Davidson, 2010). Interestingly, mosaic overexpression of egr2 in the chick hindbrain induces egr2 expression in neighboring cells (href="#bib19" rid="bib19" class=" bibr popnode">Giudicelli et al., 2001), but the molecular basis of this non-autonomous induction is not known.The findings from transplantation experiments have led to the idea that cell identity switching could act in parallel with cell segregation to establish sharp and homogeneous segments (href="#bib13" rid="bib13" class=" bibr popnode">Cooke and Moens, 2002, href="#bib46" rid="bib46" class=" bibr popnode">Pasini and Wilkinson, 2002). However, it is unclear to what extent intermingling of cells between segments occurs during normal development. egr2 has a key role in hindbrain segmentation through specification of r3 and r5 identity (href="#bib54" rid="bib54" class=" bibr popnode">Schneider-Maunoury et al., 1993, href="#bib69" rid="bib69" class=" bibr popnode">Voiculescu et al., 2001) and is a direct transcriptional regulator of ephA4 (href="#bib65" rid="bib65" class=" bibr popnode">Theil et al., 1998), which underlies cell segregation (href="#bib12" rid="bib12" class=" bibr popnode">Cooke et al., 2005, href="#bib75" rid="bib75" class=" bibr popnode">Xu et al., 1995, href="#bib77" rid="bib77" class=" bibr popnode">Xu et al., 1999). It is therefore likely that intermingling between segments is confined to the time period before there has been sufficient upregulation of EphA4 to drive cell segregation. Consistent with findings in chick (href="#bib17" rid="bib17" class=" bibr popnode">Fraser et al., 1990), some isolated cells expressing egr2 or egr2-cre reporter are detected in even-numbered segments in the mouse hindbrain (href="#bib24" rid="bib24" class=" bibr popnode">Irving et al., 1996, href="#bib69" rid="bib69" class=" bibr popnode">Voiculescu et al., 2001). However, recent work has suggested that there is no intermingling between hindbrain segments in zebrafish, and therefore cell identity switching does not occur (href="#bib7" rid="bib7" class=" bibr popnode">Calzolari et al., 2014). In this study, tracking of cells in time-lapse movies from 11 hpf did not detect intermingling between segments, and fluorescent reporter expression driven downstream of egr2 was not detected in any cells in adjacent segments (href="#bib7" rid="bib7" class=" bibr popnode">Calzolari et al., 2014). However, interpretation of these findings may be limited by timing of the analyses, as mechanisms that restrict cell intermingling may already be in place by 11 hpf and prior to detectable expression of the transgenic reporters.We set out to analyze the role and mechanisms of cell identity switching in establishment of homogeneous segmental identity. By using genome modification to create an early reporter of egr2 expression, we show that cell intermingling and identity switching occurs during hindbrain segmentation in zebrafish. egr2 expression is regulated by a combination of A-P location and non-autonomous mechanisms that depend upon the number of neighbors that express egr2. We uncover a crucial role of RA-degrading enzymes, cyp26b1 and cyp26c1, which we show are regulated by egr2 and are required for identity switching of r3 and r5 cells that intermingle into adjacent segments. These findings reveal that coupling between segment identity and retinoid signaling enables homogeneous segmental identity to be maintained despite intermingling of cells.