Responses of Epibranchial Placodes to Disruptions of the FGF and BMP Signaling Pathways in Embryonic Mice

Abstract

Placodes are ectodermal thickenings of the embryonic vertebrate head. Their descendants contribute to sensory organ development, but also give rise to sensory neurons of the cranial nerves. In mammals, the signaling pathways which regulate the morphogenesis and neurogenesis of epibranchial placodes, localized dorsocaudally to the pharyngeal clefts, are poorly understood. Therefore, we performed mouse whole embryo culture experiments to assess the impact of pan-fibroblast growth factor receptor (FGFR) inhibitors, anti-FGFR3 neutralizing antibodies or the pan-bone morphogenetic protein receptor (BMPR) inhibitor LDN193189 on epibranchial development. We demonstrate that each of the three paired epibranchial placodes is regulated by a unique combination of FGF and/or bone morphogenetic protein (BMP) signaling. Thus, neurogenesis depends on fibroblast growth factor (FGF) signals, albeit to different degrees, in all epibranchial placodes (EP), whereas only EP1 and EP3 significantly rely on neurogenic BMP signals. Furthermore, individual epibranchial placodes vary in the extent to which FGF and/or BMP signals (1) have access to certain receptor subtypes, (2) affect the production of Neurogenin (Ngn)2+ and/or Ngn1+ neuroblasts, and (3) regulate either neurogenesis alone or together with structural maintenance. In EP2 and EP3, all FGF-dependent production of Ngn2+ neuroblasts is mediated via FGFR3 whereas, in EP1, it depends on FGFR1 and FGFR3. Differently, production of FGF-dependent Ngn1+ neuroblasts almost completely depends on FGFR3 in EP1 and EP2, but not in EP3. Finally, FGF signals turned out to be responsible for the maintenance of both placodal thickening and neurogenesis in all epibranchial placodes, whereas administration of the pan-BMPR inhibitor, apart from its negative neurogenic effects in EP1 and EP3, causes only decreases in the thickness of EP3. Experimentally applied inhibitors most probably not only blocked receptors in the epibranchial placodes, but also endodermal receptors in the pharyngeal pouches, which act as epibranchial signaling centers. While high doses of pan-FGFR inhibitors impaired the development of all pharyngeal pouches, high doses of the pan-BMPR inhibitor negatively affected only the pharyngeal pouches 3 and 4. In combination with partly concordant, partly divergent findings in other vertebrate classes our observations open up new approaches for research into the complex regulation of neurogenic placode development.

Patterns of senescence and apoptosis during development of branchial arches, epibranchial placodes, and pharyngeal pouches

“…24 When present, it is usually restricted to small dorsal apoptotic zones. 24,28 Our current investigations further show that senescent cells are virtually absent among densely clustered Ngn2 + premigratory neuroblasts, which fill central parts of the placode far more extensively than the currently detectable delamination sites (Figure 10B-G). Therefore, Ngn2 + senescent cells are likely to occur at most in the marginal zones of the placode and in very small numbers.…”

“…In the developmental period considered here as an example (E10.5 to E11, Figures 9 and 10), e2 and e3 are still neurogenetically active, while e1 is about to disappear. 24,28 In e2 and e3, a central cluster composed of strongly and weakly Pax8 + progenitors can be distinguished from the placodal periphery dominated by weakly Pax8 + cells (Figure 9B,C). Senescent cells are mainly found at the transition between these two placodal regions.…”

“…57 C-F, Postulated (C and D: mouse) and partially demonstrated (E and F: chicken, zebrafish) epibranchial signaling centers in the pharyngeal pouches express members of the bone morphogenetic protein (BMP) and fibroblast growth factor (FGF) gene families (see Table 1 for references). Five levels of evidence are presented: (1) Known rostral (ro) or caudal (ca) expression sites (genes expressed in the respective pharyngeal walls) with proven effects on epibranchial neurogenesis (green curved arrows), (2) Pharyngeal expression confirmed in principle (genes displayed in the pharyngeal lumen) with proven effects on epibranchial neurogenesis (green curved arrow), (3) Pharyngeal expression confirmed in principle (genes displayed in pharyngeal lumen), but either proven to have no effect on epibranchial neurogenesis or not yet tested in this context, (4) Known rostral and/or caudal expression sites (genes displayed in the respective pharyngeal walls), but not yet tested for their effects on epibranchial neurogenesis, (5) Proven negative effects on epibranchial neurogenesis 28,37 produced by externally applied pharmacological inhibitors of BMP receptors (BMPR) and/or FGFR (red inhibition symbols). C and D, From the synopsis of known properties of proven and postulated epibranchial signaling centers (C-F) and the developmental profiles of mouse epibranchial placodes (Figure 21), we hypothesize that pharyngeal signaling centers also promote epibranchial neurogenesis in mice (C) until they are "deactivated" (D) by caudal apoptosis (red) and/or rostral cellular senescence (blue).…”

“…[3][4][5][6][7][8][9][10][11][13][14][15][16][17][18][19][20][21][22] Second, through extensive preliminary work on the patterns and functions of apoptosis relevant to branchial and epibranchial development in mouse, chicken, and the primate-related tree shrew (Tupaia belangeri, Scandentia), we have established a satisfactory basis for follow-up studies of cellular senescence in a well-defined biological context. [23][24][25][26][27][28] Third, and finally, the incompletely understood functions of apoptosis during branchial and epibranchial development may be better elucidated by analyzing concurrent senescence events.…”

“…[40][41][42][43][44][45][46][47][48][49][50][51][52] In addition, individual responses of the three epibranchial placodes to perturbations of the FGF and BMP signaling pathways have recently been reported. 28 To adequately discuss this part of our findings, we have schematically summarized all available information on the location and molecular composition of epibranchial signaling centers in vertebrate pharyngeal pouches.…”