Genetic regulation of amphioxus somitogenesis informs the evolution of the vertebrate head mesoderm

The evolution of vertebrates from an ancestral chordate was accompanied by the acquisition of a predatory lifestyle closely associated to the origin of a novel anterior structure, the highly specialized head. While the vertebrate head mesoderm is unsegmented, the paraxial mesoderm of the earliest divergent chordate clade, the cephalochordates (amphioxus), is fully segmented in somites. We have previously shown that fibroblast growth factor signalling controls the formation of the most anterior somites in amphioxus; therefore, unravelling the fibroblast growth factor signalling downstream effectors is of crucial importance to shed light on the evolutionary origin of vertebrate head muscles. By using a comparative RNA sequencing approach and genetic functional analyses, we show that several transcription factors, such as Six1/2, Pax3/7 and Zic, act in combination to ensure the formation of three different somite populations. Interestingly, these proteins are orthologous to key regulators of trunk, and not head, muscle formation in vertebrates. Contrary to prevailing thinking, our results suggest that the vertebrate head mesoderm is of visceral and not paraxial origin and support a multistep evolutionary scenario for the appearance of the unsegmented mesoderm of the vertebrates new 'head'

Evolution of somitogenesis in chordates and the appearence of the vertebrate head

Une question centrale dans l'histoire de l'évolution des vertébrés est de comprendre l'origine de leur tête complexe. L'apparition de nouvelles structures de la tête, telles que les cellules de la crête neurale, a déjà été longuement étudiée. Cependant, comment le mésoderme non segmenté de la tête chez les vertébrés a émergé à partir d’un mésoderme entièrement segmenté reste une question non résolue. En raison de leur position phylogénétique, de leurs caractéristiques morphologiques, développementales et génomiques, les céphalochordés (c'est-à-dire les amphioxus) représentent le meilleur proxy existant de l'ancêtre des chordés. De plus, l'amphioxus possède un mésoderme paraxial entièrement segmenté tout le long du corps, caractéristique proposée comme ancestrale. Comparer la somitogenèse entre amphioxus et vertébrés pourrait résoudre la question de savoir comment le mésoderme non segmenté de la tête des vertébrés a évolué. Les travaux réalisés dans notre laboratoire ont montré le rôle central du signal FGF, via la voie MAPK, dans la formation des somites les plus antérieurs chez l'amphioxus. L'inhibition de cette voie de signalisation au cours de la gastrulation induit une perte spécifique de ces structures. Afin de comprendre le destin du mésoderme paraxial antérieur présomptif après inhibition de la signalisation FGF, j’ai analysé l’expression de différents gènes marqueurs, j’ai réalisé un traçage des cellules de ce territoire et, enfin, j’ai mis en œuvre des analyses morphologiques. Nous avons conclu que ce territoire acquiert un destin axial dorsal antérieur au cours de la gastrulation lorsque le signal FGF est inhibé et que les cellules correspondantes intègrent ensuite la notochorde. L'étude morphologique de la notochorde chez ces embryons traités nous permet de proposer une hypothèse quant à l'apparition de la plaque préchordale. Pour la deuxième partie de mon doctorat, des études avaient préalablement montré que la formation des somites antérieurs et postérieurs repose sur la fonction de facteurs de transcription (Six1/2 et Pax3/7) orthologues d’acteurs majeurs de la formation des muscles du tronc chez les vertébrés. De plus, les gènes principalement impliqués dans la formation du mésoderme de la tête et du mésoderme latéral chez les vertébrés sont exprimés dans la partie ventrale des somites d'amphioxus. A partir de ces données, il a été proposé l'hypothèse que le mésoderme de la tête des vertébrés soit homologue à la partie ventrale des somites d’amphioxus. D’une part, j’ai analysé chez l'amphioxus l’expression de gènes connus comme jouant un rôle dans le développement des dérivés du mésoderme latéral chez les vertébrés. D’autre part, j’ai montré que les séquences cis-régulatrices de certains gènes d'amphioxus exprimés dans les somites ventraux dirigent l’expression dans les dérivés du mésoderme de la tête et latéral chez le poisson zèbre, un vertébré. En conclusion, ces résultats permettent d'améliorer la robustesse de notre hypothèse proposant l'homologie entre le mésoderme latéral/de la tête des vertébrés et la partie ventrale des somites d’amphioxus.A central question in the evolutionary history of vertebrates is to understand the origin of their complex head. The emergence of new head structures, such as neural crest cells, has already been extensively studied. However, how the unsegmented mesoderm of the head in vertebrates emerged from a fully segmented mesoderm remains an unresolved question. Because of their phylogenetic position, morphological, developmental, and genomic characteristics, cephalochordates (i.e., amphioxus) represent the best existing proxy for the ancestor of chordates. Furthermore, amphioxus has a fully segmented paraxial mesoderm, a feature proposed as ancestral. Comparing somitogenesis between amphioxus and vertebrates could resolve the question of how the unsegmented mesoderm of the vertebrate head evolved. Work in our laboratory has shown the central role of FGF signaling, via the MAPK pathway, in the formation of the most anterior somites in amphioxus. Inhibition of this signaling pathway during gastrulation induces a specific loss of these structures. In order to understand the fate of the presumptive anterior paraxial mesoderm after inhibition of FGF signaling, I analyzed the expression of different marker genes, performed cell tracing of this territory, and finally implemented morphological analyses. We concluded that this territory acquires an anterior dorsal axial fate during gastrulation when the FGF signal is inhibited and that the corresponding cells subsequently integrate the notochord. The morphological study of the notochord in these treated embryos allows us to propose a hypothesis for the appearance of the prechordal plate. For the second part of my PhD, studies had previously shown that the formation of the anterior and posterior somites relies on the function of transcription factors (Six1/2 and Pax3/7) orthologous to major players in the formation of trunk muscles in vertebrates. In addition, genes primarily involved in the formation of head mesoderm and lateral mesoderm in vertebrates are expressed in the ventral part of amphioxus somites. Based on these data, it was proposed that the vertebrate head mesoderm is homologous to the ventral part of amphioxus somites. On the one hand, I analyzed in amphioxus the expression of genes known to play a role in the development of lateral mesoderm derivatives in vertebrates. On the other hand, I showed that cis-regulatory sequences of some amphioxus genes expressed in ventral somites direct the expression of a reporter gene in the head and in lateral mesoderm derivatives in the vertebrate zebrafish. In conclusion, these results improve the robustness of our hypothesis proposing homology between vertebrate lateral/head mesoderm and the ventral region of amphioxus somites

Functions of the FGF signalling pathway in cephalochordates provide insight into the evolution of the prechordal plate

International audienceThe fibroblast growth factor (FGF) signalling pathway plays various roles during vertebrate embryogenesis, from mesoderm formation to brain patterning. This diversity of functions relies on the fact that vertebrates possess the largest FGF gene complement among metazoans. In the cephalochordate amphioxus, which belongs to the chordate clade together with vertebrates and tunicates, we have previously shown that the main role of FGF during early development is the control of rostral somite formation. Inhibition of this signalling pathway induces the loss of these structures, resulting in an embryo without anterior segmented mesoderm, as in the vertebrate head. Here, by combining several approaches, we show that the anterior presumptive paraxial mesoderm cells acquire an anterior axial fate when FGF signal is inhibited and that they are later incorporated in the anterior notochord. Our analysis of notochord formation in wild type and in embryos in which FGF signalling is inhibited also reveals that amphioxus anterior notochord presents transient prechordal plate features. Altogether, our results give insight into how changes in FGF functions during chordate evolution might have participated to the emergence of the complex vertebrate head

Hindbrain rhombomere centers harbor a heterogenous population of dividing progenitors which rely on Notch signaling

Tissue growth and morphogenesis are interrelated processes, whose tight coordination is essential for the production of different cell fates and the timely precise allocation of stem cell capacities. The zebrafish embryonic brainstem, the hindbrain, exemplifies such coupling between spatiotemporal cell diversity acquisition and tissue growth as the neurogenic commitment is differentially distributed over time. Here, we combined cell lineage and in vivo imaging approaches to reveal the emergence of specific cell population properties within the rhombomeres. We studied the molecular identity of hindbrain rhombomere centers and showed that they harbor different progenitor capacities that change over time. By clonal analysis, we revealed that cells within the center of rhombomeres decrease the proliferative capacity to remain mainly in the G1 phase. Proliferating progenitors give rise to neurons by asymmetric and symmetric neurogenic divisions while maintaining the pool of progenitors. The proliferative capacity of these cells differs from their neighbors, and they are delayed in the onset of Notch activity. Through functional studies, we demonstrated that they rely on Notch3 signaling to be maintained as non-committed progenitors. In this study, we show that cells in rhombomere centers, despite the neurogenic asynchrony, might share steps of a similar program with the rhombomere counterparts, to ensure proper tissue growth.The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This work was funded by grants BFU2015-67400-P, PGC2018-095663-B-I00, and PID2021-123261NB-I00 from Ministerio de Ciencia e Innovación (MICIN), Agencia Estatal de Investigación (AEI, DOI: 10.13039/501100011033), and Fondo Europeo de Desarrollo Regional (FEDER) to CP. The Department of Medicine and Life Sciences (UPF) is a Unidad de Excelencia María de Maeztu (CEX2018-000792-M) funded by MICIN and AEI. CBM was the recipient of a predoctoral fellowship from MICIN (FPI, BES-2016-076664). CP is a recipient of the ICREA Academia award (Generalitat de Catalunya)

Developmental evolution and developmental plasticity of the olfactory epithelium and olfactory skills in Mexican cavefish

International audienceThe fish Astyanax mexicanus comes in two forms: the normal surface-dwelling (SF) and the blind depigmented cave-adapted (CF) morphs. Among many phenotypic differences, cavefish show enhanced olfactory sensitivity to detect amino-acid odors and they possess large olfactory sensory organs. Here, we questioned the relationship between the size of the olfactory organ and olfactory capacities. Comparing olfactory detection abilities of CF, SF and F1 hybrids with various olfactory epithelium (OE) sizes in behavioral tests, we concluded that OE size is not the only factor involved. Other possibilities were envisaged. First, olfactory behavior was tested in SF raised in the dark or after embryonic lens ablation, which leads to eye degeneration and mimics the CF condition. Both absence of visual function and absence of visual organs improved the SF olfactory detection capacities, without affecting the size of their OE. This suggested that developmental plasticity occurs between the visual and the olfactory modalities, and can be recruited in SF after visual deprivation. Second, the development of the olfactory epithelium was compared in SF and CF in their first month of life. Proliferation, cell death, neuronal lifespan, and olfactory progenitor cell cycling properties were identical in the two morphs. By contrast, the proportions of the three main olfactory sensory neurons subtypes (ciliated, microvillous and crypt) in their OE differed. OMP-positive ciliated neurons were more represented in SF, TRPC2-positive microvillous neurons were proportionately more abundant in CF, and S100-positive crypt cells were found in equal densities in the two morphs. Thus, general proliferative properties of olfactory progenitors are identical but neurogenic properties differ and lead to variations in the neuronal composition of the OE in SF and CF. Together, these experiments suggest that there are at least two components in the evolution of cavefish olfactory skills: (1) one part of eye-dependent developmental phenotypic plasticity, which does not depend on the size of the olfactory organ, and (2) one part of developmental evolution of the OE, which may stem from embryonic specification of olfactory neurons progenitor pools

Genetic regulation of amphioxus somitogenesis informs the evolution of the vertebrate head mesoderm

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