SoxB1 transcription factors (Sox2/3) are well known for their role in early neural fate specification in the embryo, but little is known about functional roles for SoxB1 factors in non-neural ectodermal cell types, such as the neural plate border (NPB). Using Xenopus laevis, we set out to determine whether SoxB1 transcription factors have a regulatory function in NPB formation. Here, we show that SoxB1 factors are necessary for NPB formation, and that prolonged SoxB1 factor activity blocks the transition from a NPB to a neural crest state. Using ChIP-seq, we demonstrate that Sox3 is enriched upstream of NPB genes in early NPB cells and in blastula stem cells. Depletion of SoxB1 factors in blastula stem cells results in downregulation of NPB genes. Finally, we identify Pou5f3 factors as potential Sox3 partners in regulating the formation of the NPB and show that their combined activity is needed for normal NPB gene expression. Together, these data identify a role for SoxB1 factors in the establishment and maintenance of the NPB, in part through partnership with Pou5f3 factors.

UNASSIGNED: The remarkable diversity observed in the structure and development of the molluscan nervous system raises intriguing questions regarding the molecular mechanisms underlying neurogenesis in Mollusca. The expression of SoxB family transcription factors plays a pivotal role in neuronal development, thereby offering valuable insights into the strategies of neurogenesis.
UNASSIGNED: In this study, we conducted gene expression analysis focusing on SoxB-family transcription factors during early neurogenesis in the gastropod Lymnaea stagnalis. We employed a combination of hybridization chain reaction in situ hybridization (HCR-ISH), immunocytochemistry, confocal microscopy, and cell proliferation assays to investigate the spatial and temporal expression patterns of LsSoxB1 and LsSoxB2 from the gastrula stage to hatching, with particular attention to the formation of central ring ganglia.
UNASSIGNED: Our investigation reveals that LsSoxB1 demonstrates expanded ectodermal expression from the gastrula to the hatching stage, whereas expression of LsSoxB2 in the ectoderm ceases by the veliger stage. LsSoxB1 is expressed in the ectoderm of the head, foot, and visceral complex, as well as in forming ganglia and sensory cells. Conversely, LsSoxB2 is mostly restricted to the subepithelial layer and forming ganglia cells during metamorphosis. Proliferation assays indicate a uniform distribution of dividing cells in the ectoderm across all developmental stages, suggesting the absence of distinct neurogenic zones with increased proliferation in gastropods.
UNASSIGNED: Our findings reveal a spatially and temporally extended pattern of SoxB1 expression in a gastropod representative compared to other lophotrochozoan species. This prolonged and widespread expression of SoxB genes may be interpreted as a form of transcriptional neoteny, representing a preadaptation to prolonged neurogenesis. Consequently, it could contribute to the diversification of nervous systems in gastropods and lead to an increase in the complexity of the central nervous system in Mollusca.

Hemichordate enteropneust worms regenerate extensively in a manner that resembles the regeneration for which planaria and hydra are well known. Although hemichordates are often classified as an extant phylogenetic group that may hold ancestral deuterostome body plans at the base of the deuterostome evolutionary line leading to chordates, mammals, and humans, extensive regeneration is not known in any of these more advanced groups. Here we investigated whether hemichordates deploy functional homologs of canonical Yamanaka stem cell reprogramming factors, Oct4, Sox2, Nanog, and Klf4, as they regenerate. These reprogramming factors are not expressed during regeneration of limbs, fins, eyes or other structures that represent the best examples of regeneration in chordates. We first examined Ptychodera flava EST libraries and identified Pf-Pou3, Pf-SoxB1, Pf-Msxlx, and Pf-Klf1/2/4 as most closely related to the Yamanaka factors, respectively. In situ hybridization analyses revealed that all these homologs are expressed in a distinct manner during head regeneration. Furthermore, Pf-Pou3 partially rescued the loss of endogenous Oct4 in mouse embryonic stem cells in maintaining the pluripotency gene expression program. Based on these results, we propose that hemichordates may have co-opted these reprogramming factors for their extensive regeneration or that chordates may have lost the ability to mobilize these factors in response to damage. The robustness of these pluripotency gene circuits in the inner cell mass and in formation of induced pluripotent stem cells from mammalian somatic cells shows that these programs are intact in humans and other mammals and that these circuits may respond to as yet unknown gene regulatory signals, mobilizing full regeneration in hemichordates.

The precise control of neural crest stem cell delamination, migration and differentiation ensures proper craniofacial and head development. Sox2 shapes the ontogeny of the cranial neural crest to ensure precision of the cell flow in the developing head. Here, we review how Sox2 orchestrates signals that control these complex developmental processes.

Despite the unprecedented gene editing capability of CRISPR-Cas9-mediated targeted knock-in, the efficiency and precision of this technology still require further optimization, particularly for multicellular model organisms, such as the zebrafish (Danio rerio). Our study demonstrated that an ∼200 base-pair sequence encoding a composite tag can be efficiently \"knocked-in\" into the zebrafish genome using a combination of the CRISPR-Cas9 ribonucleoprotein complex and a long single-stranded DNA (lssDNA) as a donor template. Here, we targeted the sox3, sox11a, and pax6a genes to evaluate the knock-in efficiency of lssDNA donors with different structures in somatic cells of injected embryos and for their germline transmission. The structures and sequence characteristics of the lssDNA donor templates were found to be crucial to achieve a high rate of precise and heritable knock-ins. The following were our key findings: (1) lssDNA donor strand selection is important; however, strand preference and its dependency appear to vary among the target loci or their sequences. (2) The length of the 3\' homology arm of the lssDNA donor affects knock-in efficiency in a site-specific manner; particularly, a shorter 50-nt arm length leads to a higher knock-in efficiency than a longer 300-nt arm for the sox3 and pax6a knock-ins. (3) Some DNA sequence characteristics of the knock-in donors and the distance between the CRISPR-Cas9 cleavage site and the tag insertion site appear to adversely affect the repair process, resulting in imprecise editing. By implementing the proposed method, we successfully obtained precisely edited sox3, sox11a, and pax6a knock-in alleles that contained a composite tag composed of FLAGx3 (or PAx3), Bio tag, and HiBiT tag (or His tag) with moderate to high germline transmission rates as high as 21%. Furthermore, the knock-in allele-specific quantitative polymerase chain reaction (qPCR) for both the 5\' and 3\' junctions indicated that knock-in allele frequencies were higher at the 3\' side of the lssDNAs, suggesting that the lssDNA-templated knock-in was mediated by unidirectional single-strand template repair (SSTR) in zebrafish embryos.

Sox transcription factors play many diverse roles during development, including regulating stem cell states, directing differentiation, and influencing the local chromatin landscape. Of the twenty vertebrate Sox factors, several play critical roles in the development the neural crest, a key vertebrate innovation, and the subsequent formation of neural crest-derived structures, including the craniofacial complex. Herein, we review the specific roles for individual Sox factors during neural crest cell formation and discuss how some factors may have been essential for the evolution of the neural crest. Additionally, we describe how Sox factors direct neural crest cell differentiation into diverse lineages such as melanocytes, glia, and cartilage and detail their involvement in the development of specific craniofacial structures. Finally, we highlight several SOXopathies associated with craniofacial phenotypes.

Specification of the main axes of polarity of the embryo is an essential process during embryonic development. In many species, this process is achieved by the localization of maternal factors into discrete regions of the egg. However, in other animals, like in amniotes and in echinoderms, the considerable plasticity of the early blastomeres seems to preclude the existence of maternal determinants and the mechanisms by which the radial symmetry of the egg is broken remain largely enigmatic. In this chapter, we review recent progress on the identification of maternal components involved in symmetry breaking and dorsal-ventral (D/V) axis formation of the sea urchin embryo. We will first review some key experiments on D/V axis formation from classical embryologists that provided evidence for a weak maternal D/V prepattern. We will then detail more recent molecular analyses that established the critical role played by Nodal signaling in allocating cell fates along the secondary axis and led to the discovery that maternal transcription factors such as the Sry-related HMG box B1 (SoxB1), the Octamer binding factor1/2 (Oct1/2), the T-cell factor/Lymphoid enhancer-binding factor (TCF/LEF) and the Erythroblastosis virus E26 Oncogene Homolog (ETS) domain transcriptional repressor Translocation-Ets-Leukemia virus protein (Yan/Tel) as well as maternal signaling molecules like Univin are essential for the initiation of nodal expression. Finally, we will describe recent advances that uncovered a role in symmetry breaking and dorsal-ventral axis orientation for the transforming growth factor beta (TGF-beta)-like factor Panda, which appears to be both necessary and sufficient for D/V axis orientation. Therefore, even in the highly regulative sea urchin embryo, the activity of localized maternal factors provides the embryo with a blueprint of the D/V axis.

In early vertebrate embryos, the dorsal ectoderm is induced by the axial mesendoderm to form the neural plate, which is given competence to form neural cells by soxB1 genes. Subsequently, neurogenesis proceeds in proneural clusters that are generated by a gene network involving proneural genes and Notch signaling. However, what occurs between early neural induction and the later initiation of neurogenesis has not been fully revealed. In the present study, we demonstrated that during gastrulation, the expression of the Oct4-related PouV gene pou5f3 (also called pou2), which is widely observed at earlier stages, was rapidly localized to an array of isolated spotted domains, each of which coincided with individual proneural clusters. Two-color in situ hybridization confirmed that each pou5f3-expressing domain included a proneural cluster. Further analysis demonstrated that anterior pou5f3 domains straddled the boundaries between rhombomere 1 (r1) and r2, whereas posterior domains were included in r4. The effects of forced expression of an inducible negative dominant-interfering pou5f3 gene suggested that pou5f3 activated early proneural genes, such as neurog1 and ebf2, and also soxB1, but repressed the late proneural genes atoh1a and ascl1b. Furthermore, pou5f3 was considered to repress her4.1, a Notch-dependent Hairy/E(spl) gene involved in lateral inhibition in proneural clusters. These results suggest that pou5f3 promotes early neurogenesis in proneural clusters, but negatively regulates later neurogenesis. Suppression of pou5f3 also altered the expression of other her genes, including her3, her5, and her9, further supporting a role for pou5f3 in neurogenesis. In vitro reporter assays in P19 cells showed that pou5f3 was repressed by neurog1, but activated by Notch signaling. These findings together demonstrate the importance of the pou5f3-mediated gene regulatory network in neural development in vertebrate embryos.

Many marine larvae begin feeding within a day of fertilization, thus requiring rapid development of a nervous system to coordinate feeding activities. Here, we examine the patterning and specification of early neurogenesis in sea urchin embryos. Lineage analysis indicates that neurons arise locally in three regions of the embryo. Perturbation analyses showed that when patterning is disrupted, neurogenesis in the three regions is differentially affected, indicating distinct patterning requirements for each neural domain. Six transcription factors that function during proneural specification were identified and studied in detail. Perturbations of these proneural transcription factors showed that specification occurs differently in each neural domain prior to the Delta-Notch restriction signal. Though gene regulatory network state changes beyond the proneural restriction are largely unresolved, the data here show that the three neural regions already differ from each other significantly early in specification. Future studies that define the larval nervous system in the sea urchin must therefore separately characterize the three populations of neurons that enable the larva to feed, to navigate, and to move food particles through the gut.

SoxB1 genes play fundamental roles in neurodevelopmental processes and maintaining stem cell multipotency, but little is known about their function in regeneration. We addressed this question by analyzing the activity of the SoxB1 homolog soxB1-2 in the planarian Schmidtea mediterranea. Expression and functional analysis revealed that soxB1-2 marks ectodermal-lineage progenitors, and its activity is required for differentiation of subsets of ciliated epidermal and neuronal cells. Moreover, we show that inhibiting soxB1-2 or its candidate target genes leads to abnormal sensory neuron regeneration that causes planarians to display seizure-like movements or phenotypes associated with the loss of sensory modalities. Our analyses highlight soxB1-2-regulated genes that are expressed in sensory neurons and are homologous to factors implicated in epileptic disorders in humans and animal models of epilepsy, indicating that planarians can serve as a complementary model to investigate genetic causes of epilepsy.