The brain plays a central role in controlling energy, glucose, and lipid homeostasis, with specialized neurons within nuclei of the mediobasal hypothalamus, namely the arcuate (ARC) and ventromedial (VMH), tasked with proper signal integration. Exactly how the exquisite cytoarchitecture and underlying circuitry become established within these nuclei remains largely unknown, in part because hypothalamic developmental programs are just beginning to be elucidated. Here, we demonstrate that the Retinal and anterior neural fold homeobox (Rax) gene plays a key role in establishing ARC and VMH nuclei in mice. First, we show that Rax is expressed in ARC and VMH progenitors throughout development, and accordingly, genetic fate mapping studies reveal that Rax+ lineages give rise to VMH neurons. Second, the conditional ablation of Rax in a subset of VMH progenitors using a Shh∷Cre driver leads to a fate switch from a VMH neuronal phenotype to a hypothalamic but non-VMH identity, suggesting that Rax is a selector gene for VMH cellular fates. Finally, the broader elimination of Rax throughout ARC/VMH progenitors using Six3∷Cre leads to a severe loss of both VMH and ARC cellular phenotypes, demonstrating a role for Rax in both VMH and ARC fate specification. Combined, our study illustrates that Rax is required in ARC/VMH progenitors to specify neuronal phenotypes within this hypothalamic brain region. Rax thus provides a molecular entry point for further study of the ontology and establishment of hypothalamic feeding circuits.

A derepression mode of cell-fate specification involving the transcriptional repressors Tbr1, Fezf2, Satb2, and Ctip2 operates in neocortical projection neurons to specify six layer identities in sequence. Less well understood is how laminar fate transitions are regulated in cortical progenitors. The proneural genes Neurog2 and Ascl1 cooperate in progenitors to control the temporal switch from neurogenesis to gliogenesis. Here we asked whether these proneural genes also regulate laminar fate transitions. Several defects were observed in the derepression circuit in Neurog2−/− mutants: an inability to repress expression of Tbr1 (a deep layer VI marker) during upper-layer neurogenesis, a loss of Fezf2+ layer V neurons, and precocious differentiation of normally late-born, Satb2 + layer II-IV neurons. Conversely, in stable gain-of-function transgenics, Neurog2 promoted differentiative divisions and extended the period of Tbr1 deep-layer neurogenesis while reducing Satb2+ upper-layer neurogenesis. Similarly, acute misexpression of Neurog2 in early cortical progenitors promoted Tbr1 expression, whereas both Neurog2 and Ascl1 induced Ctip2. However, Neurog2 was unable to influence the derepression circuit when misexpressed in late cortical progenitors, and Ascl1 repressed only Satb2. Nevertheless, neurons derived from late misexpression of Neurog2 and, to a lesser extent, Ascl1, extended aberrant subcortical axon projections characteristic of early-born neurons. Finally, Neurog2 and Ascl1 altered the expression of Ikaros and Foxg1, known temporal regulators. Proneural genes thus act in a context-dependent fashion as early determinants, promoting deeplayer neurogenesis in early cortical progenitors via input into the derepression circuit while also influencing other temporal regulators.neocortex | laminar fate specification | derepression circuit | proneural genes | temporal identity N eocortical neurons project to nearby or distant targets, depending on their molecular identity and correlating with laminar position. Deep-layer corticofugal neurons project subcortically and include layer VI corticothalamic neurons, which target the thalamus, and layer V subcerebral neurons, which project to the spinal cord, basal ganglia, and other distant targets (1). Conversely, layer IV granular neurons are the major site of thalamic input, whereas layer II/III callosal neurons form corticocortical connections. A cross-repressive gene-regulatory network operates in postmitotic projection neurons to specify laminar fates and to repress competing laminar identities (Fig. 1A) (2). Tbr1, a T-box transcription factor expressed in layer VI, specifies a corticothalamic neuronal fate (3) and also represses the expression of Fezf2, a zinc finger transcription factor that specifies a layer V subcerebral identity (4, 5). Fezf2 represses Tbr1 expression and a corticothalamic fate in layer V neurons (6, 7) and also represses the expression of Satb2 (8), an AT-rich DNA-binding protein that specifies a layer II-IV callosal identity (9, 10). In laye...

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental pathways. Several powerful and well established transgenic technologies are available to manipulate gene expression levels in mouse, allowing for the generation of both loss- and gain-of-function models. However, the generation of mouse transgenics is both costly and time consuming. Alternative methods of gene manipulation have therefore been widely sought. In utero electroporation is a method of gene delivery into live mouse embryos(1,2) that we have successfully adapted(3,4). It is largely based on the success of in ovo electroporation technologies that are commonly used in chick(5). Briefly, DNA is injected into the open ventricles of the developing brain and the application of an electrical current causes the formation of transient pores in cell membranes, allowing for the uptake of DNA into the cell. In our hands, embryos can be efficiently electroporated as early as embryonic day (E) 11.5, while the targeting of younger embryos would require an ultrasound-guided microinjection protocol, as previously described(6). Conversely, E15.5 is the latest stage we can easily electroporate, due to the onset of parietal and frontal bone differentiation, which hampers microinjection into the brain. In contrast, the retina is accessible through the end of embryogenesis. Embryos can be collected at any time point throughout the embryonic or early postnatal period. Injection of a reporter construct facilitates the identification of transfected cells. To date, in utero electroporation has been most widely used for the analysis of neocortical development(1,2,3,4). More recent studies have targeted the embryonic retina(7,8,9) and thalamus(10,11,12). Here, we present a modified in utero electroporation protocol that can be easily adapted to target different domains of the embryonic CNS. We provide evidence that by using this technique, we can target the embryonic telencephalon, diencephalon and retina. Representative results are presented, first showing the use of this technique to introduce DNA expression constructs into the lateral ventricles, allowing us to monitor progenitor maturation, differentiation and migration in the embryonic telencephalon. We also show that this technique can be used to target DNA to the diencephalic territories surrounding the 3(rd) ventricle, allowing the migratory routes of differentiating neurons into diencephalic nuclei to be monitored. Finally, we show that the use of micromanipulators allows us to accurately introduce DNA constructs into small target areas, including the subretinal space, allowing us to analyse the effects of manipulating gene expression on retinal development.