Hindbrain morphogenesis

Abstract: A time course experiment is a widely used design in the study of cellular processes such as differentiation or response to stimuli. In this paper, we propose time course reg ulatory analysis (TimeReg) as a method for the analysis of gene regulatory networks based on paired gene expression and chromatin accessibility data from a time course. TimeReg can be used to prioritize regulatory elements, to extract core regulatory modules at each time point, to identify key regulators driving changes of the cellular state, and to causally connect the modules across different time points. We applied the method to analyze paired chromatin accessibility and gene expression data from a retinoic acid (RA)-induced mouse embryonic stem cells (mESCs) differentiation experiment. The analysis identified 57,048 novel regulatory elements regulating cerebellar development, synapse assembly, and hindbrain morphogenesis, which substantially extended our knowledge of cis-regulatory elements during differentiation. Using single-cell RNA-seq data, we showed that the core regulatory modules can reflect the properties of different subpopulations of cells. Finally, the driver regulators are shown to be important in clarifying the relations between modules across adjacent time points. As a second example, our method on Ascl1-induced direct reprogramming from fibroblast to neuron time course data identified Id1/2 as driver regulators of early stage of reprogramming.

Abstract: SUMMARY The early development of the exencephalic malformation in trisomy 12 (Tsl2) and trisomy 14 (Tsl4) mouse embryos was examined by means of scanning electron microscopy and compared with cranial neural tube formation in euploid litter-mates. Embryos from normal laboratory mice were used as additional controls. The euploid control embryos of the trisomy-inducing breeding system showed a slight delay and some variation in the timing of cranial neurulation. The pre-exencephalic trisomic embryos showed hypoplasia, and lower somite number when compared with euploid littermates; there was also a retardation of development of the whole neural tube, when related to the somite stage. External differences from the control embryos were observed at the late pre-somite stage, when the anterior part of the neural plate showed a crumpled appearance. At 6 somites the lateral edges of the forebrain were everted instead of elevated in Tsl2 and Tsl4 embryos. At later stages, however, the forebrain showed a tendency towards the normal morphogenetic pattern, so that the optic vesicles were eventually formed and the most anterior part fused. The caudal forebrain and the midbrain were more permanently affected by the disturbance of trisomic conditions and grew laterally, failing to appose or fuse in the midline in both Tsl2 and Tsl4 embryos. Hindbrain morphogenesis was different in Tsl2 and Tsl4 excencephaly: in Tsl2 embryos it did not close rostral to the otic pits, whereas in Tsl4 embryos it showed a normal closure up to the hindbrain/midbrain junction. These observations support the hypothesis that in mammalian embryos the mechanism of neural tube formation of the future brain region is more complex than that of the spinal neural tube and therefore may be more likely to react to a general delay of neurulation with a gross malformation. Tsl2 and Tsl4 exencephaly are due to a primary non-closure of the neural tube.

Abstract: The Lower Rhombic Lip (LRL) is a transient neuroepithelial structure of the dorsal hindbrain, which expands from r2 to r7, and gives rise to deep nuclei of the brainstem, such as the vestibular and auditory nuclei and most posteriorly the precerebellar nuclei. Although there is information about the contribution of specific proneural-progenitor populations to specific deep nuclei, and the distinct rhombomeric contribution, little is known about how progenitor cells from the LRL behave during neurogenesis and how their transition into differentiation is regulated. In this work, we investigated the atoh1 gene regulatory network operating in the specification of LRL cells, and the kinetics of cell proliferation and behavior of atoh1a-derivatives by using complementary strategies in the zebrafish embryo. We unveiled that atoh1a is necessary and sufficient for specification of LRL cells by activating atoh1b, which worked as a differentiation gene to transition progenitor cells towards neuron differentiation in a Notch-dependent manner. This cell state transition involved the release of atoh1a-derivatives from the LRL: atoh1a progenitors contributed first to atoh1b cells, which are committed non-proliferative precursors, and to the lhx2b-neuronal lineage as demonstrated by cell fate studies and functional analyses. Using in vivo cell lineage approaches we revealed that the proliferative cell capacity, as well as the mode of division, relied on the position of the atoh1a progenitors within the dorsoventral axis. We showed that atoh1a may behave as the cell fate selector gene, whereas atoh1b functions as a neuronal differentiation gene, contributing to the lhx2b neuronal population. atoh1a-progenitor cell dynamics (cell proliferation, cell differentiation, and neuronal migration) relies on their position, demonstrating the challenges that progenitor cells face in computing positional information from a dynamic two-dimensional grid in order to generate the stereotyped neuronal structures in the embryonic hindbrain.

Abstract: hlx1 is a related homeobox gene expressed in a dynamic spatiotemporal expression pattern during development of the zebrafish brain. The homologues of hlx1, mouse dbx1 and Xenopus Xdbx, are known to play a role in the specification of neurons in the spinal cord. However, the role of these molecules in the brain is less well known. We have used two different approaches to elucidate a putative function for hlx1 in the developing zebrafish brain. Blastomeres were injected with either synthetic hlx1 mRNA in gain-of-function experiments or with antisense morpholino oligonucleotides directed against hlx1 in loss-of-function experiments. Mis-expression of hlx1 produced severe defects in brain morphogenesis as a result of abnormal ventricle formation, a phenotype we referred to as "fused-brain". These animals also showed a reduction in the size of forebrain neuronal clusters as well as abnormal axon pathfinding. hlx1 antisense morpholinos specifically perturbed hindbrain morphogenesis leading to defects in the integrity of the neuroepithelium. While hindbrain patterning was in the most part unaffected there were select disruptions to the expression pattern of the neurogenic gene Zash1B in specific rhombomeres. Our results indicate multiple roles for hlx1 during zebrafish brain morphogenesis.