Cédric Francius

Renshaw cell interneuron specialization is controlled by a temporally restricted transcription factor program

The spinal cord contains a diverse array of physiologically distinct interneuron cell types that subserve specialized roles in somatosensory perception and motor control. The mechanisms that generate these specialized interneuronal cell types from multipotential spinal progenitors are not known. In this study, we describe a temporally regulated transcriptional program that controls the differentiation of Renshaw cells (RCs), an anatomically and functionally discrete spinal interneuron subtype. We show that the selective activation of the Onecut transcription factors Oc1 and Oc2 during the first wave of V1 interneuron neurogenesis is a key step in the RC differentiation program. The development of RCs is additionally dependent on the forkhead transcription factor Foxd3, which is more broadly expressed in postmitotic V1 interneurons. Our demonstration that RCs are born, and activate Oc1 and Oc2 expression, in a narrow temporal window leads us to posit that neuronal diversity in the developing spinal cord is established by the composite actions of early spatial and temporal determinants.

The GATA2 transcription factor negatively regulates the proliferation of neuronal progenitors.

Postmitotic neurons are produced from a pool of cycling progenitors in an orderly fashion that requires proper spatial and temporal coordination of proliferation, fate determination, differentiation and morphogenesis. This probably relies on complex interplay between mechanisms that control cell cycle, specification and differentiation. In this respect, we have studied the possible implication of GATA2, a transcription factor that is involved in several neuronal specification pathways, in the control of the proliferation of neural progenitors in the embryonic spinal cord. Using gain- and loss-of-function manipulations, we have shown that Gata2 can drive neural progenitors out of the cycle and, to some extent, into differentiation. This correlates with the control of cyclin D1 transcription and of the expression of the p27/Kip1 protein. Interestingly, this functional aspect is not only associated with silencing of the Notch pathway but also appears to be independent of proneural function. Consistently, GATA2 also controls the proliferation capacity of mouse embryonic neuroepithelial cells in culture. Indeed, Gata2 inactivation enhances the proliferation rate in these cells. By contrast, GATA2 overexpression is sufficient to force such cells and neuroblastoma cells to stop dividing but not to drive either type of cell into differentiation. Furthermore, a non-cell autonomous effect of Gata2 expression was observed in vivo as well as in vitro. Hence, our data have provided evidence for the ability of Gata2 to inhibit the proliferation of neural progenitors, and they further suggest that, in this regard, Gata2 can operate independently of neuronal differentiation.

Dynamic expression of the Onecut transcription factors HNF-6, OC-2 and OC-3 during spinal motor neuron development

The Onecut (OC) transcription factors, namely Hepatocyte nuclear factor 6 (HNF-6), OC-2 and OC-3, are transcriptional activators expressed in liver, pancreas and nervous system during development. Although their expression and roles in endoderm-derived tissues and in the trigeminal ganglia have been investigated, their expression in the CNS has not been described yet. In this study, we report a qualitative and quantitative expression profile of the OC factors in the developing spinal motor neurons (MN). We provide evidence that OC expression is initiated in newly-born MN. At later stages, they are differentially and dynamically expressed in subsets of differentiating motor neuron within the four motor columns. We also show that the expression profile of HNF-6 in spinal MN is conserved in chick embryos. Together, our data unveil a complex and dynamic expression profile of the OC proteins in spinal MN, which suggests that these factors may participate in regulatory networks that control different steps of motor neuron development.

Onecut transcription factors act upstream of Isl1 to regulate spinal motoneuron diversification

During development, spinal motoneurons (MNs) diversify into a variety of subtypes that are specifically dedicated to the motor control of particular sets of skeletal muscles or visceral organs. MN diversification depends on the coordinated action of several transcriptional regulators including the LIM-HD factor Isl1, which is crucial for MN survival and fate determination. However, how these regulators cooperate to establish each MN subtype remains poorly understood. Here, using phenotypic analyses of single or compound mutant mouse embryos combined with gain-of-function experiments in chick embryonic spinal cord, we demonstrate that the transcriptional activators of the Onecut family critically regulate MN subtype diversification during spinal cord development. We provide evidence that Onecut factors directly stimulate Isl1 expression in specific MN subtypes and are therefore required to maintain Isl1 production at the time of MN diversification. In the absence of Onecut factors, we observed major alterations in MN fate decision characterized by the conversion of somatic to visceral MNs at the thoracic levels of the spinal cord and of medial to lateral MNs in the motor columns that innervate the limbs. Furthermore, we identify Sip1 (Zeb2) as a novel developmental regulator of visceral MN differentiation. Taken together, these data elucidate a comprehensive model wherein Onecut factors control multiple aspects of MN subtype diversification. They also shed light on the late roles of Isl1 in MN fate decision.

Identification of Multiple Subsets of Ventral Interneurons and Differential Distribution along the Rostrocaudal Axis of the Developing Spinal Cord.

The spinal cord contains neuronal circuits termed Central Pattern Generators (CPGs) that coordinate rhythmic motor activities. CPG circuits consist of motor neurons and multiple interneuron cell types, many of which are derived from four distinct cardinal classes of ventral interneurons, called V0, V1, V2 and V3. While significant progress has been made on elucidating the molecular and genetic mechanisms that control ventral interneuron differentiation, little is known about their distribution along the antero-posterior axis of the spinal cord and their diversification. Here, we report that V0, V1 and V2 interneurons exhibit distinct organizational patterns at brachial, thoracic and lumbar levels of the developing spinal cord. In addition, we demonstrate that each cardinal class of ventral interneurons can be subdivided into several subsets according to the combinatorial expression of different sets of transcription factors, and that these subsets are differentially distributed along the rostrocaudal axis of the spinal cord. This comprehensive molecular profiling of ventral interneurons provides an important resource for investigating neuronal diversification in the developing spinal cord and for understanding the contribution of specific interneuron subsets on CPG circuits and motor control.

IFN-ε Is Constitutively Expressed by Cells of the Reproductive Tract and Is Inefficiently Secreted by Fibroblasts and Cell Lines

Type-I interferons (IFNs) form a large family of cytokines that primarily act to control the early development of viral infections. Typical type-I IFN genes, such as those encoding IFN-α or IFN-β are upregulated by viral infection in many cell types. In contrast, the gene encoding IFN-ε was reported to be constitutively expressed by cells of the female reproductive tract and to contribute to the protection against vaginal infections with herpes simplex virus 2 and Chlamydia muridarum. Our data confirm the lack of induction of IFN-ε expression after viral infection and the constitutive expression of IFN-ε by cells of the female but also of the male reproductive organs. Interestingly, when expressed from transfected expression plasmids in 293T, HeLa or Neuro2A cells, the mouse and human IFN-ε precursors were inefficiently processed and secretion of IFN-ε was minimal. Analysis of chimeric constructs produced between IFN-ε and limitin (IFN-ζ) showed that both the signal peptide and the mature moiety of IFN-ε contribute to poor processing of the precursor. Immunofluorescent detection of FLAG-tagged IFN-ε in transfected cells suggested that IFN-ε and chimeric proteins were defective for progression through the secretory pathway. IFN-ε did not, however, act intracellularly and impart an antiviral state to producing cells. Given the constitutive expression of IFN-ε in specialized cells and the poor processing of IFN-ε precursor in fibroblasts and cell lines, we hypothesize that IFN-ε secretion may require a co-factor specifically expressed in cells of the reproductive organs, that might secure the system against aberrant release of this IFN.

Generating spinal motor neuron diversity: a long quest for neuronal identity

Understanding how thousands of different neuronal types are generated in the CNS constitutes a major challenge for developmental neurobiologists and is a prerequisite before considering cell or gene therapies of nervous lesions or pathologies. During embryonic development, spinal motor neurons (MNs) segregate into distinct subpopulations that display specific characteristics and properties including molecular identity, migration pattern, allocation to specific motor columns, and innervation of defined target. Because of the facility to correlate these different characteristics, the diversification of spinal MNs has become the model of choice for studying the molecular and cellular mechanisms underlying the generation of multiple neuronal populations in the developing CNS. Therefore, how spinal motor neuron subpopulations are produced during development has been extensively studied during the last two decades. In this review article, we will provide a comprehensive overview of the genetic and molecular mechanisms that contribute to the diversification of spinal MNs.

Genetic dissection of Gata2 selective functions during specification of V2 interneurons in the developing spinal cord

Motor activities are controlled by neural networks in the ventral spinal cord and consist in motor neurons and a set of distinct cardinal classes of spinal interneurons. These interneurons arise from distinct progenitor domains (p0–p3) delineated according to a transcriptional code. Neural progenitors of each domain express a unique combination of transcription factors (TFs) that largely contribute to determine the fate of four classes of interneurons (V0–V3) and motor neurons. In p2 domain, at least four subtypes of interneurons namely V2a, V2b, V2c, and Pax6+ V2 are generated. Although genetic and molecular mechanisms that specify V2a and V2b are dependent on complex interplay between several TFs including Nkx6.1, Irx3, Gata2, Foxn4, and Ascl1, and signaling pathways such as Notch and TGF‐β, the sequence order of the activation of these regulators and their respective contribution are not completely elucidated yet. Here, we provide evidence by loss‐ or gain‐of‐function experiments that Gata2 is necessary for the normal development of both V2a and V2b neurons. We demonstrate that Nkx6.1 and Dll4 positively regulate the activation of Gata2 and Foxn4 in p2 progenitors. Gata2 also participates in the maintenance of p2 domain by repressing motor neuron differentiation and exerting a feedback control on patterning genes. Finally, Gata2 promotes the selective activation of V2b program at the expense of V2a fate. Thus our results provide new insights on the hierarchy and complex interactions between regulators of V2 genetic program.

Pax6 is expressed in subsets of V0 and V2 interneurons in the ventral spinal cord in mice.

The embryonic spinal cord in mice is organized into eleven progenitor domains. Cells in each domain first produce neurons and then switch to specifying glia. Five of these domains known as p3, pMN, p2, p1 and p0 are located in the ventral spinal cord and each expresses a unique code of transcription factors (TFs) that define the molecular profile of progenitor cells. This code is largely responsible for determining the subtype specification of neurons generated from each domain. Pax6 codes for a homedomain-containing TF that plays a central role in defining the molecular boundaries between the two ventral-most domains, p3 and pMN. Using fate mapping and gene expression studies we show that PAX6, in addition to each patterning function, is expressed in a group of late born interneurons that derive from the p2 and p0 domains. The p2-derived neurons represent a subset of late born V2b interneurons and their specification depends on Notch signaling. The V0 neurons represent V0v ventral neurons expressing Pax2. Our data demonstrate that interneuron diversity in the ventral spinal cord is more complex than originally appreciated and point to the existence of additional mechanisms that determine interneuron diversity, particularly in the p2 domain.

Onecut factors and Pou2f2 regulate diversification and migration of V2 interneurons in the mouse developing spinal cord

Acquisition of proper neuronal identity and position is critical for the formation of neural circuits. In the embryonic spinal cord, cardinal populations of interneurons diversify into specialized subsets and migrate to defined locations within the spinal parenchyma. However, the factors that control interneuron diversification and migration remain poorly characterized. Here, we show that the Onecut transcription factors are necessary for proper diversification and distribution of the V2 interneurons in the developing spinal cord. Furthermore, we uncover that these proteins restrict and moderate the expression of spinal isoforms of Pou2f2, a transcription factor known to regulate B-cell differentiation. By gain- or loss-of-function experiments, we show that Pou2f2 contribute to regulate the position of V2 populations in the developing spinal cord. Thus, we uncovered a genetic pathway that regulates the diversification and the distribution of V2 interneurons during embryonic development. Significance statement In this study, we identify the Onecut and Pou2f2 transcription factors as regulators of spinal interneuron diversification and migration, two events that are critical for proper CNS development.