A disrupted compartment boundary underlies abnormal cardiac patterning and congenital heart defects.

Failure of septation of the interventricular septum (IVS) is the most common congenital heart defect (CHD), but mechanisms for patterning the IVS are largely unknown. We show that a Tbx5+/Mef2cAHF+ progenitor lineage forms a compartment boundary bisecting the IVS. This coordinated population originates at a first- and second heart field interface, subsequently forming a morphogenetic nexus. Ablation of Tbx5+/Mef2cAHF+ progenitors cause IVS disorganization, right ventricular hypoplasia and mixing of IVS lineages. Reduced dosage of the CHD transcription factor TBX5 disrupts boundary position and integrity, resulting in ventricular septation defects (VSDs) and patterning defects, including Slit2 and Ntn1 misexpression. Reducing NTN1 dosage partly rescues cardiac defects in Tbx5 mutant embryos. Loss of Slit2 or Ntn1 causes VSDs and perturbed septal lineage distributions. Thus, we identify essential cues that direct progenitors to pattern a compartment boundary for proper cardiac septation, revealing new mechanisms for cardiac birth defects.

A Mesp1-dependent developmental breakpoint in transcriptional and epigenomic specification of early cardiac precursors.

Transcriptional networks governing cardiac precursor cell (CPC) specification are incompletely understood owing, in part, to limitations in distinguishing CPCs from non-cardiac mesoderm in early gastrulation. We leveraged detection of early cardiac lineage transgenes within a granular single-cell transcriptomic time course of mouse embryos to identify emerging CPCs and describe their transcriptional profiles. Mesp1, a transiently expressed mesodermal transcription factor, is canonically described as an early regulator of cardiac specification. However, we observed perdurance of CPC transgene-expressing cells in Mesp1 mutants, albeit mislocalized, prompting us to investigate the scope of the role of Mesp1 in CPC emergence and differentiation. Mesp1 mutant CPCs failed to robustly activate markers of cardiomyocyte maturity and crucial cardiac transcription factors, yet they exhibited transcriptional profiles resembling cardiac mesoderm progressing towards cardiomyocyte fates. Single-cell chromatin accessibility analysis defined a Mesp1-dependent developmental breakpoint in cardiac lineage progression at a shift from mesendoderm transcriptional networks to those necessary for cardiac patterning and morphogenesis. These results reveal Mesp1-independent aspects of early CPC specification and underscore a Mesp1-dependent regulatory landscape required for progression through cardiogenesis.

Graded mesoderm assembly governs cell fate and morphogenesis of the early mammalian heart.

Using four-dimensional whole-embryo light sheet imaging with improved and accessible computational tools, we longitudinally reconstruct early murine cardiac development at single-cell resolution. Nascent mesoderm progenitors form opposing density and motility gradients, converting the temporal birth sequence of gastrulation into a spatial anterolateral-to-posteromedial arrangement. Migrating precardiac mesoderm does not strictly preserve cellular neighbor relationships, and spatial patterns only become solidified as the cardiac crescent emerges. Progenitors undergo a mesenchymal-to-epithelial transition, with a first heart field (FHF) ridge apposing a motile juxta-cardiac field (JCF). Anchored along the ridge, the FHF epithelium rotates the JCF forward to form the initial heart tube, along with push-pull morphodynamics of the second heart field. In Mesp1 mutants that fail to make a cardiac crescent, mesoderm remains highly motile but directionally incoherent, resulting in density gradient inversion. Our practicable live embryo imaging approach defines spatial origins and behaviors of cardiac progenitors and identifies their unanticipated morphological transitions.

Sinusoidal and lymphatic vessel growth is controlled by reciprocal VEGF-C-CDH5 inhibition.

Sinusoids are specialized, low pressure blood vessels in the liver, bone marrow, and spleen required for definitive hematopoiesis. Unlike other blood endothelial cells (ECs), sinusoidal ECs express high levels of VEGFR3. VEGFR3 and its ligand VEGF-C are known to support lymphatic growth, but their function in sinusoidal vessels is unknown. In this study, we define a reciprocal VEGF-C/VEGFR3-CDH5 (VE-cadherin) signaling axis that controls growth of both sinusoidal and lymphatic vessels. Loss of VEGF-C or VEGFR3 resulted in cutaneous edema, reduced fetal liver size, and bloodless bone marrow due to impaired lymphatic and sinusoidal vessel growth. Mice with membrane-retained VE-cadherin conferred identical lymphatic and sinusoidal defects, suggesting that VE-cadherin opposes VEGF-C/VEGFR3 signaling. In developing mice, loss of VE-cadherin rescued defects in sinusoidal and lymphatic growth caused by loss of VEGFR3 but not loss of VEGF-C, findings explained by potentiated VEGF-C/VEGFR2 signaling in VEGFR3-deficient lymphatic ECs. Mechanistically, VEGF-C/VEGFR3 signaling induces VE-cadherin endocytosis and loss of function via SRC-mediated phosphorylation, while VE-cadherin prevents VEGFR3 endocytosis required for optimal receptor signaling. These findings establish an essential role for VEGF-C/VEGFR3 signaling during sinusoidal vascular growth, identify VE-cadherin as a powerful negative regulator of VEGF-C signaling that acts through both VEGFR3 and VEGFR2 receptors, and suggest that negative regulation of VE-cadherin is required for effective VEGF-C/VEGFR3 signaling during growth of sinusoidal and lymphatic vessels. Manipulation of this reciprocal negative regulatory mechanism, e.g. by reducing VE-cadherin function, may be used to stimulate therapeutic sinusoidal or lymphatic vessel growth.

Coordination of Neuron Production in Mouse and Human Cerebral Cortex by the Homolog of Drosophila Mastermind Protein.

The coordination of progenitor self-renewal, neuronal production, and migration is essential to the normal development and evolution of the cerebral cortex. Numerous studies have shown that the Notch, Wnt/beta-catenin, and Neurogenin pathways contribute separately to progenitor expansion, neurogenesis, and neuronal migration, but it is unknown how these signals are coordinated. In vitro studies suggested that the mastermind-like 1 (MAML1) gene, homologue of the Drosophila mastermind, plays a role in coordinating the aforementioned signaling pathways, yet its role during cortical development remains largely unknown. Here we show that ectopic expression of dominant-negative MAML (dnMAML) causes exuberant neuronal production in the mouse cortex without disrupting neuronal migration. Comparing the transcriptional consequences of dnMAML and Neurog2 ectopic expression revealed a complex genetic network controlling the balance of progenitor expansion versus neuronal production. Manipulation of MAML and Neurog2 in cultured human cerebral stem cells exposed interactions with the same set of signaling pathways. Thus, our data suggest that evolutionary changes that affect the timing, tempo, and density of successive neuronal layers of the small lissencephalic rodent and large convoluted primate cerebral cortex depend on similar molecular mechanisms that act from the earliest developmental stages.

Antibodies to cannabinoid type 1 receptor co-react with stomatin-like protein 2 in mouse brain mitochondria.

Anti-cannabinoid type 1 receptor (CB1 ) polyclonal antibodies are widely used to detect the presence of CB1 in a variety of brain cells and their organelles, including neuronal mitochondria. Surprisingly, we found that anti-CB1 sera, in parallel with CB1 , also recognize the mitochondrial protein stomatin-like protein 2. In addition, we show that the previously reported effect of synthetic cannabinoid WIN 55,212-2 on mitochondrial complex III respiration is not detectable in purified mitochondrial preparations. Thus, our study indicates that a direct relationship between endocannabinoid signaling and mitochondrial functions in the cerebral cortex seems unlikely, and that caution should be taken interpreting findings obtained using anti-CB1 antibodies.

POU-III transcription factors (Brn1, Brn2, and Oct6) influence neurogenesis, molecular identity, and migratory destination of upper-layer cells of the cerebral cortex.

The upper layers (II-IV) are the most prominent distinguishing feature of mammalian neocortex compared with avian or reptilian dorsal cortex, and are vastly expanded in primates. Although the time-dependent embryonic generation of upper-layer cells is genetically instructed within their parental progenitors, mechanisms governing cell-intrinsic fate transitions remain obscure. POU-homeodomain transcription factors Pou3f3 and Pou3f2 (Brn1 and Brn2) are known to label postmitotic upper-layer cells, and are redundantly required for their production. We find that the onset of Pou3f3/2 expression actually occurs in ventricular zone (VZ) progenitors, and that Pou3f3/2 subsequently label neural progeny switching from deep-layer Ctip2(+) identity to Satb2(+) upper-layer fate as they migrate to proper superficial positions. By using an Engrailed dominant-negative repressor, we show that sustained neurogenesis after the deep- to upper-layer transition requires the proneual action of Pou3fs in VZ progenitors. Conversely, single-gene overexpression of any Pou3f in early neural progenitors is sufficient to specify the precocious birth of Satb2(+) daughter neurons that extend axons to the contralateral hemisphere, as well as exhibit robust pia-directed migration that is characteristic of upper-layer cells. Finally, we demonstrate that Pou3fs influence multiple stages of neurogenesis by suppressing Notch effector Hes5, and promoting the expression of proneural transcription factors Tbr2 and Tbr1.

Transcriptional programs in transient embryonic zones of the cerebral cortex defined by high-resolution mRNA sequencing.

Characterizing the genetic programs that specify development and evolution of the cerebral cortex is a central challenge in neuroscience. Stem cells in the transient embryonic ventricular and subventricular zones generate neurons that migrate across the intermediate zone to the overlying cortical plate, where they differentiate and form the neocortex. It is clear that not one but a multitude of molecular pathways are necessary to progress through each cellular milestone, yet the underlying transcriptional programs remain unknown. Here, we apply differential transcriptome analysis on microscopically isolated cell populations, to define five transcriptional programs that represent each transient embryonic zone and the progression between these zones. The five transcriptional programs contain largely uncharacterized genes in addition to transcripts necessary for stem cell maintenance, neurogenesis, migration, and differentiation. Additionally, we found intergenic transcriptionally active regions that possibly encode unique zone-specific transcripts. Finally, we present a high-resolution transcriptome map of transient zones in the embryonic mouse forebrain.

Decision by division: making cortical maps.

In the past three decades, mounting evidence has revealed that specification of the basic cortical neuronal classes starts at the time of their final mitotic divisions in the embryonic proliferative zones. This early cell determination continues during the migration of the newborn neurons across the widening cerebral wall, and it is in the cortical plate that they attain their final positions and establish species-specific cytoarchitectonic areas. Here, the development and evolutionary expansion of the neocortex is viewed in the context of the radial unit and protomap hypotheses. A broad spectrum of findings gave insight into the pathogenesis of cortical malformations and the biological bases for the evolution of the modern human neocortex. We examine the history and evidence behind the concept of early specification of neurons and provide the latest compendium of genes and signaling molecules involved in neuronal fate determination and specification.