An amphioxus neurula stage cell atlas supports a complex scenario for the emergence of vertebrate head mesoderm.

The emergence of new structures can often be linked to the evolution of novel cell types that follows the rewiring of developmental gene regulatory subnetworks. Vertebrates are characterized by a complex body plan compared to the other chordate clades and the question remains of whether and how the emergence of vertebrate morphological innovations can be related to the appearance of new embryonic cell populations. We previously proposed, by studying mesoderm development in the cephalochordate amphioxus, a scenario for the evolution of the vertebrate head mesoderm. To further test this scenario at the cell population level, we used scRNA-seq to construct a cell atlas of the amphioxus neurula, stage at which the main mesodermal compartments are specified. Our data allowed us to validate the presence of a prechordal-plate like territory in amphioxus. Additionally, the transcriptomic profile of somite cell populations supports the homology between specific territories of amphioxus somites and vertebrate cranial/pharyngeal and lateral plate mesoderm. Finally, our work provides evidence that the appearance of the specific mesodermal structures of the vertebrate head was associated to both segregation of pre-existing cell populations, and co-option of new genes for the control of myogenesis.

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.

Exploring tissue morphodynamics using the photoconvertible Kaede protein in amphioxus embryos.

Photoconvertible proteins are powerful tools widely used in cellular biology to study cell dynamics and organelles. Over the past decade, photoconvertible proteins have also been used for developmental biology applications to analyze cell lineage and cell fate during embryonic development. One of these photoconvertible proteins called Kaede, from the stony coral Trachyphyllia geoffroyi, undergoes irreversible photoconversion from green to red fluorescence when illuminated with UV light. Undertaking a cell tracing approach using photoconvertible proteins can be challenging when using unconventional animal models. In this protocol, we describe the use of Kaede to track specific cells during embryogenesis of the cephalochordate Branchiostoma lanceolatum. This protocol can be adapted to other unconventional models, especially marine animals.

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

The 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.

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'.

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

The 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.