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We describe an optimized protocol for application of expansion microscopy (ExM) on chick neural tube (NT) which enables different oriented nanoscale resolution imaging of the centrosomes/cilia. We explain embryo NT transversal sections and open-book preparations, immunohistochemistry for labeling, and sample preparation for 5-fold tissue expansion. Further, we detail sample orientation and Fast Airyscan confocal acquisition and show that NT-ExM retains fluorescence signals and overcomes biomolecules crowding in structural features that to date were only imaged with electron microscopy on tissues.

HOX transcription factors are members of an evolutionarily conserved family of proteins required for the establishment of the anteroposterior body axis during bilaterian development. Although they are often deregulated in cancers, the molecular mechanisms by which they act as oncogenes or tumor suppressor genes are only partially understood. Since the MAPK/ERK signaling pathway is deregulated in most cancers, we aimed at apprehending if and how the Hox proteins interact with ERK oncogenicity. Using an in vivo neoplasia model in the chicken embryo consisting in the overactivation of the ERK1/2 kinases in the trunk neural tube, we analyzed the consequences of the HOXB8 gain of function at the morphological and transcriptional levels. We found that HOXB8 acts as a tumor suppressor, counteracting ERK-induced neoplasia. The HOXB8 tumor suppressor function relies on a large reversion of the oncogenic transcriptome induced by ERK. In addition to showing that the HOXB8 protein controls the transcriptional responsiveness to ERK oncogenic signaling, our study identified new downstream targets of ERK oncogenic activation in an in vivo context that could provide clues for therapeutic strategies.

Most human cancers demonstrate activated MAPK/ERK pathway signaling as a key tumor initiation step, but the immediate steps of further oncogenic progression are poorly understood due to a lack of appropriate models. Spinal cord differentiation follows caudal elongation in vertebrate embryos; both processes are regulated by a FGF8 gradient highest in neuromesodermal progenitors (NMP), where kinase effectors ERK1/2 maintain an undifferentiated state. FGF8/ERK signal attenuation is necessary for NMPs to progress to differentiation. We show that sustained ERK1/2 activity, using a constitutively active form of the kinase MEK1 (MEK1ca) in the chicken embryo, reproducibly provokes neopasia in the developing spinal cord. Transcriptomic data show that neoplasia not only relies on the maintenance of NMP gene expression, and the inhibition of genes expressed in the differentiating spinal cord, but also on a profound change in the transcriptional signature of the spinal cord cells leading to a complete loss of cell-type identity. MEK1ca expression in the developing spinal cord of the chicken embryo is therefore a tractable in vivo model to identify the critical factors fostering malignancy in ERK-induced tumorigenesis.

Differential Hox gene expression is central for specification of axial neuronal diversity in the spinal cord. Here, we uncover an additional function of Hox proteins in the developing spinal cord, restricted to B cluster Hox genes. We found that members of the HoxB cluster are expressed in the trunk neural tube of chicken embryo earlier than Hox from the other clusters, with poor antero-posterior axial specificity and with overlapping expression in the intermediate zone (IZ). Gain-of-function experiments of HoxB4, HoxB8 and HoxB9, respectively, representative of anterior, central and posterior HoxB genes, resulted in ectopic progenitor cells in the mantle zone. The search for HoxB8 downstream targets in the early neural tube identified the leucine zipper tumor suppressor 1 gene (Lzts1), the expression of which is also activated by HoxB4 and HoxB9. Gain- and loss-of-function experiments showed that Lzts1, which is expressed endogenously in the IZ, controls neuronal delamination. These data collectively indicate that HoxB genes have a generic function in the developing spinal cord, controlling the expression of Lzts1 and neuronal delamination.

Differential Hox gene expression is central for specification of axial neuronal diversity in the spinal cord. Here, we uncover an additional function of Hox proteins in the developing spinal cord, restricted to B cluster Hox genes. We found that members of the HoxB cluster are expressed in the trunk neural tube of chicken embryo earlier than Hox from the other clusters, with poor antero-posterior axial specificity and with overlapping expression in the intermediate zone (IZ). Gain-of-function experiments of HoxB4, HoxB8 and HoxB9, respectively, representative of anterior, central and posterior HoxB genes, resulted in ectopic progenitor cells in the mantle zone. The search for HoxB8 downstream targets in the early neural tube identified the leucine zipper tumor suppressor 1 gene (