SummaryRadial glial progenitors (RGPs) are responsible for producing nearly all neocortical neurons. To gain insight into the patterns of RGP division and neuron production, we quantitatively analyzed excitatory neuron genesis in the mouse neocortex using Mosaic Analysis with Double Markers, which provides single-cell resolution of progenitor division patterns and potential in vivo. We found that RGPs progress through a coherent program in which their proliferative potential diminishes in a predictable manner. Upon entry into the neurogenic phase, individual RGPs produce ∼8–9 neurons distributed in both deep and superficial layers, indicating a unitary output in neuronal production. Removal of OTX1, a transcription factor transiently expressed in RGPs, results in both deep- and superficial-layer neuron loss and a reduction in neuronal unit size. Moreover, ∼1/6 of neurogenic RGPs proceed to produce glia. These results suggest that progenitor behavior and histogenesis in the mammalian neocortex conform to a remarkably orderly and deterministic program.

No abstract

SummaryNeurons in the mammalian neocortex arise from asymmetric divisions of progenitors residing in the ventricular zone. While in most progenitor divisions, the mitotic spindle is parallel to the ventricular surface, some progenitors reorient the spindle and divide in oblique orientations. Here, we use conditional deletion and overexpression of mouse Inscuteable (mInsc) to analyze the relevance of spindle reorientation in cortical progenitors. Mutating mInsc almost abolishes oblique and vertical mitotic spindles, while mInsc overexpression has the opposite effect. Our data suggest that oblique divisions are essential for generating the correct numbers of neurons in all cortical layers. Using clonal analysis, we demonstrate that spindle orientation affects the rate of indirect neurogenesis, a process where progenitors give rise to basal progenitors, which in turn divide symmetrically into two differentiating neurons. Our results indicate that the orientation of progenitor cell divisions is important for correct lineage specification in the developing mammalian brain.

Asymmetric cell divisions allow stem cells to balance proliferation and differentiation. During embryogenesis, murine epidermis expands rapidly from a single layer of unspecified basal layer progenitors to a stratified, differentiated epithelium. Morphogenesis involves perpendicular (asymmetric) divisions and the spindle orientation protein LGN, but little is known about how the apical localization of LGN is regulated. Here, we combine conventional genetics and lentiviral-mediated in vivo RNAi to explore the functions of the LGN-interacting proteins Par3, mInsc and Gαi3. Whereas loss of each gene alone leads to randomized division angles, combined loss of Gnai3 and mInsc causes a phenotype of mostly planar divisions, akin to loss of LGN. These findings lend experimental support for the hitherto untested model that Par3–mInsc and Gαi3 act cooperatively to polarize LGN and promote perpendicular divisions. Finally, we uncover a developmental switch between delamination-driven early stratification and spindle-orientation-dependent differentiation that occurs around E15, revealing a two-step mechanism underlying epidermal maturation.

The thyroid-stimulating hormone͞thyrotropin (TSH) is the most relevant hormone in the control of thyroid gland physiology in adulthood. TSH effects on the thyroid gland are mediated by the interaction with a specific TSH receptor (TSHR). We studied the role of TSH͞TSHR signaling on gland morphogenesis and differentiation in the mouse embryo using mouse lines deprived either of TSH (pit dw ͞pit dw ) or of a functional TSHR (tshr hyt ͞tshr hyt and TSHRknockout lines). The results reported here show that in the absence of either TSH or a functional TSHR, the thyroid gland develops to a normal size, whereas the expression of thyroperoxidase and the sodium͞iodide symporter are reduced greatly. Conversely, no relevant changes are detected in the amounts of thyroglobulin and the thyroid-enriched transcription factors TTF-1, TTF-2, and Pax8. These data suggest that the major role of the TSH͞TSHR pathway is in controlling genes involved in iodide metabolism such as sodium͞iodide symporter and thyroperoxidase. Furthermore, our data indicate that in embryonic life TSH does not play an equivalent role in controlling gland growth as in the adult thyroid. T he mouse thyroid gland begins to develop at embryonic day (E)8.5 as an endodermal thickening in the floor of the primitive pharynx. After loosing all connections with the pharynx, the thyroid bud migrates caudally, reaching its final position in front of the trachea ϷE13 (1). Only after completion of migration do thyroid follicular cells begin their differentiative program and express thyroid-specific genes such as thyroglobulin (Tg), thyroid-stimulating hormone͞thyrotropin (TSH) receptor (TSHR), thyroperoxidase (TPO), and the sodium͞iodide symporter (NIS) (2). Finally, primitive follicles appear, and the gland displays its final morphological organization. Since E8.5, thyroid precursor cells express a combination of transcription factors such as TTF-1 (encoded by the titf1͞nkx2.1 gene) (3), TTF-2 (encoded by the titf2͞foxe1 gene) (4), and Pax8 (5). Gene-targeting experiments demonstrated that all these factors are required for the early stages of thyroid development (6-8). However, it still is unclear what the mechanisms are that lead to the initiation of functional differentiation that only occurs at E14.TSH is known as the main regulator of the adult thyroid gland. Indeed, after binding to its receptor, TSH stimulates the thyroid cells in almost every aspect of their metabolism including synthesis and secretion of thyroid hormones (9). Several groups have demonstrated clearly that TSH regulates mRNA levels of several thyroid-specific genes such as Tg (10-13), TPO (13-15), and NIS (16,17).TSH also stimulates the aggregation of porcine thyroid cells in follicles (18), and its presence is necessary to maintain the follicular architecture (19). In the rat, there is a temporal correlation between the increased expression of TSHR and the formation of follicles. Indeed, TSHR mRNA is expressed by E15 (3, 20), and its expression increases on E17-E18. At this stage, thyroid-speci...

The thyroid gland originates from the ventral floor of the foregut as a thickening of the endodermal cell layer. The molecular mechanisms underlying the early steps of thyroid morphogenesis are not known. Gene targeting experiments have contributed to the identification of several transcription factors, in general playing a role in the proliferation, survival, and migration of the thyroid cell precursors. The experiments reported here analyze the expression of the transcription factors Titf1, Hhex, Pax8, and Foxe1 in the thyroid primordium of null mutants of each of them. We found that most of these transcription factors are linked in an integrated regulatory network, each of them controlling the presence of other members of the network. The expression of Foxe1 is regulated in an intriguing fashion as it is strongly dependent on the presence of Pax8 in thyroid precursor cells, while the expression of the same gene in the pharyngeal endoderm surrounding the primordium is dependent on Sonic hedgehog (Shh)-derived signaling. Moreover, by the generation of mouse mutants expressing Foxe1 exclusively in the thyroid primordium, we provide a better understanding of the role of Foxe1 in these cells in order to acquire the competence to migrate into the underlying mesenchyme. In conclusion, we provide the first evidence of gene expression programs, controlled by a hierarchy of transcription factors expressed in the thyroid presumptive gut domain and directing the progression of thyroid morphogenesis.

The concerted production of neurons and glia by neural stem cells (NSCs) is essential for neural circuit assembly. In the developing cerebral cortex, radial glia progenitors (RGPs) generate nearly all neocortical neurons and certain glia lineages. RGP proliferation behavior shows a high degree of non-stochasticity, thus a deterministic characteristic of neuron and glia production. However, the cellular and molecular mechanisms controlling RGP behavior and proliferation dynamics in neurogenesis and glia generation remain unknown. By using mosaic analysis with double markers (MADM)-based genetic paradigms enabling the sparse and global knockout with unprecedented single-cell resolution, we identified Lgl1 as a critical regulatory component. We uncover Lgl1-dependent tissue-wide community effects required for embryonic cortical neurogenesis and novel cell-autonomous Lgl1 functions controlling RGP-mediated glia genesis and postnatal NSC behavior. These results suggest that NSC-mediated neuron and glia production is tightly regulated through the concerted interplay of sequential Lgl1-dependent global and cell intrinsic mechanisms.

SummaryThe developmental mechanisms regulating the number of adult neural stem cells (aNSCs) are largely unknown. Here we show that the cleavage plane orientation in murine embryonic radial glia cells (RGCs) regulates the number of aNSCs in the lateral ganglionic eminence (LGE). Randomizing spindle orientation in RGCs by overexpression of Insc or a dominant-negative form of Lgn (dnLgn) reduces the frequency of self-renewing asymmetric divisions while favoring symmetric divisions generating two SNPs. Importantly, these changes during embryonic development result in reduced seeding of aNSCs. Interestingly, no effects on aNSC numbers were observed when Insc was overexpressed in postnatal RGCs or aNSCs. These data suggest a new mechanism for controlling aNSC numbers and show that the role of spindle orientation during brain development is highly time and region dependent.