An Nkx2-5/Bmp2/Smad1 negative feedback loop controls heart progenitor specification and proliferation

During heart development the second heart field (SHF) provides progenitor cells for most cardiomyocytes and expresses the homeodomain factor Nkx2-5. We now show that feedback repression of Bmp2/Smad1 signaling by Nkx2-5 critically regulates SHF proliferation and outflow tract (OFT) morphology. In the cardiac fields of Nkx2-5 mutants, genes controlling cardiac specification (including Bmp2) and maintenance of the progenitor state were upregulated, leading initially to progenitor overspecification, but subsequently to failed SHF proliferation and OFT truncation. In Smad1 mutants, SHF proliferation and deployment to the OFT were increased, while Smad1 deletion in Nkx2-5 mutants rescued SHF proliferation and OFT development. In Nkx2-5 hypomorphic mice, which recapitulate human congenital heart disease (CHD), OFT anomalies were also rescued by Smad1 deletion. Our findings demonstrate that Nkx2-5 orchestrates the transition between periods of cardiac induction, progenitor proliferation, and OFT morphogenesis via a Smad1-dependent negative feedback loop, which may be a frequent molecular target in CHD.     

Cortical interneuron specification and diversification in the era of big data

Inhibition in the mammalian cerebral cortex is mediated by a small population of highly diverse GABAergic interneurons. These largely local neurons are interspersed among excitatory projection neurons and exert pivotal regulation on the formation and function of cortical circuits. We are beginning to understand the extent of GABAergic neuron diversity and how this is generated and shaped during brain development in mice and humans. In this review, we summarise recent findings and discuss how new technologies are being used to further advance our knowledge. Understanding how inhibitory neurons are generated in the embryo is an essential pre-requisite of stem cell therapy, an evolving area of research, aimed at correcting human disorders that result in inhibitory dysfunction.     

Nkx2-5- and Isl1-expressing cardiac progenitors contribute to proepicardium

Correct delineation of the hierarchy of cardiac progenitors is a key step to understanding heart development, and will pave the way for future use of cardiac progenitors in the treatment of heart disease. Multipotent Nkx2-5 and Isl1 cardiac progenitors contribute to cardiomyocyte, smooth muscle, and endothelial lineages, which constitute the major lineages of the heart. Recently, progenitors located within the proepicardium and epicardium were reported to differentiate into cardiomyocytes, as well as smooth muscle and endothelial cells. However, the relationship of these proepicardial progenitors to the previously described Nkx2-5 and Isl1 cardiac progenitors is incompletely understood. To address this question, we performed in vivo Cre-loxP-based lineage tracing. Both Nkx2-5- and Isl1-expressing progenitors contributed to the proepicardium and expressed Wt1 and Tbx18, markers of proepicardial progenitor cells. Interestingly, Nkx2-5 knockout resulted in abnormal proepicardial development and decreased expression of Wt1, suggesting a functional role for Nkx2-5 in proepicardium formation. Taken together, these results suggest that Nkx2-5 and/or Isl1 cardiac progenitors contribute to proepicardium during heart development.     

Selective cortical interneuron and GABA deficits in cyclin D2-null mice

In contrast to cyclin D1 nulls (cD1-/-), mice without cyclin D2 (cD2-/-) lack cerebellar stellate interneurons; the reason for this is unknown. In the present study in cortex, we found a disproportionate loss of parvalbumin (PV) interneurons in cD2-/- mice. This selective reduction in PV subtypes was associated with reduced frequency of GABA-mediated inhibitory postsynaptic currents in pyramidal neurons, as measured by voltage-clamp recordings, and increased cortical sharp activity in the EEGs of awake-behaving cD2-/- mice. Cell cycle regulation was examined in the medial ganglionic eminence (MGE), the major source of PV interneurons in mouse brain, and differences between cD2-/- and cD1-/- suggested that cD2 promotes subventricular zone (SVZ) divisions, exerting a stronger inhibitory influence on the p27 Cdk-inhibitor (Cdkn1b) to delay cell cycle exit of progenitors. We propose that cD2 promotes transit-amplifying divisions in the SVZ and that these ensure proper output of at least a subset of PV interneurons.     

Two highly related homeodomain proteins, Nkx5-1 and Nkx5-2, display different DNA binding specificities.

The mouse Nkx5-1 and Nkx5-2 genes are related to NK genes in Drosophila and encode proteins with very similar homeodomains. In higher vertebrates Nkx5 genes are specifically expressed in the inner ear. Inactivation of the mouse Nkx5-1 gene by homologous recombination revealed a critical role for the formation of vestibular inner ear structures. Here, we investigated biochemical properties of the proteins encoded by the Nkx5 genes. A similar consensus binding sequence was isolated for both Nkx5 proteins using binding site selection. This sequence is related to target sequences previously identified for other Nkx proteins and contains the conserved homeodomain binding core TAAT. An additional, novel and unrelated high affinity binding sequence could be identified for the Nkx5-2 protein.     

Developmental mechanisms underlying the generation of cortical interneuron diversity

GABAergic interneurons are critical components of cortical circuits. However, understanding their function has become extremely challenging because they constitute one of the most diverse groups of cells in the central nervous system. Indeed, cortical GABAergic interneurons are heterogeneous in so many different ways--morphology, molecular profiling, electrical properties--that even attempts to discern what parameters should be used to identify cortical interneuron subtypes have failed to generate broad consensus among experts in the field. The extent to which cortical interneuron diversity emerges during development is largely unknown, but it is likely that insights on how this process takes place may help us understand their role as integrative and synchronizing elements in cortical function. Here, we review recent data on how the large variety of distinct classes of cortical interneurons may arise during development.     

Expression of Nkx2-5-GFP bacterial artificial chromosome transgenic mice closely resembles endogenous Nkx2-5 gene activity

Mouse Nkx2-5 gene is essential for early heart development and it is regulated by a complex array of regulatory modules. In order to establish an efficient in vivo system for mapping the Nkx2-5 genomic locus for regulatory regions, we developed improved homologous recombination technology for use in Escherichia coli and then knocked an IRES-hrGFP reporter gene into Nkx2-5 gene in a 120 kb Nkx2-5 bacterial artificial chromosome (BAC) clone. We employed the recombination genes redalpha and redbeta under the pBAD promoter, which was specifically induced by the addition of L-arabinose. Recombination was selected for by our universal targeting cassette which conferred kanamycin resistance in bacterial cells and neomycin resistance in mammalian cells. Transgenic mouse lines generated from this modified BAC clone closely resembled the endogenous Nkx2-5 expression in the heart, pylorus sphincter, and spleen, but expression was not detected in the tongue. Nkx2-5 BAC-GFP expression was copy number-dependent and locus site-independent. BAC transgenics using the GFP reporter offers an efficient model system to study gene expression and regulation.     

Cortical interneuron specification: the juncture of genes,time and geometry

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Directional guidance of interneuron migration to the cerebral cortex relies on subcortical Slit1/2-independent repulsion and cortical attraction

Tangential migration from the basal telencephalon to the cortex is a highly directional process, yet the mechanisms involved are poorly understood. Here we show that the basal telencephalon contains a repulsive activity for tangentially migrating cells, whereas the cerebral cortex contains an attractive activity. In vitro experiments demonstrate that the repulsive activity found in the basal telencephalon is maintained in mice deficient in both Slit1 and Slit2, suggesting that factors other than these are responsible for this activity. Correspondingly, in vivo analysis demonstrates that interneurons migrate to the cortex in the absence of Slit1 and Slit2, or even in mice simultaneously lacking Slit1, Slit2 and netrin 1. Nevertheless, loss of Slit2 and, even more so, Slit1 and Slit2 results in defects in the position of other specific neuronal populations within the basal telencephalon, such as the cholinergic neurons of the basal magnocellular complex. These results demonstrate that whereas Slit1 and Slit2 are not necessary for tangential migration of interneurons to the cortex, these proteins regulate neuronal migration within the basal telencephalon by controlling cell positioning close to the midline.