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Cerebrospinal fluid (CSF) provides vital support for the brain. Abnormal CSF accumulation, such as hydrocephalus, can negatively affect perinatal neurodevelopment. The mechanisms regulating CSF clearance during the postnatal critical period are unclear. Here, we show that CSF K+, accompanied by water, is cleared through the choroid plexus (ChP) during mouse early postnatal development. We report that, at this developmental stage, the ChP showed increased ATP production and increased expression of ATP-dependent K+ transporters, particularly the Na+, K+, Cl−, and water cotransporter NKCC1. Overexpression of NKCC1 in the ChP resulted in increased CSF K+ clearance, increased cerebral compliance, and reduced circulating CSF in the brain without changes in intracranial pressure in mice. Moreover, ChP-specific NKCC1 overexpression in an obstructive hydrocephalus mouse model resulted in reduced ventriculomegaly. Collectively, our results implicate NKCC1 in regulating CSF K+ clearance through the ChP in the critical period during postnatal neurodevelopment in mice., Abnormal CSF accumulation in the brain can lead to hydrocephalus. The mechanisms regulating CSF clearance during early development are unclear. Here, the authors show that NKCC1 regulates the clearance of both CSF K+ and fluid volume through the choroid plexus during postnatal development in mice.

During late embryonic development of the cerebral cortex, the major class of cortical output neurons termed subcerebral projection neurons (SCPN; including the predominant population of corticospinal neurons, CSN) and the class of interhemispheric callosal projection neurons (CPN) initially express overlapping molecular controls that later undergo subtype-specific refinements. Such molecular refinements are largely absent in heterogeneous, maturation-stalled, neocortical-like neurons (termed “cortical” here) spontaneously generated by established embryonic stem cell (ES) and induced pluripotent stem cell (iPSC) differentiation. Building on recently identified central molecular controls over SCPN development, we used a combination of synthetic modified mRNA (modRNA) for Fezf2, the central transcription factor controlling SCPN specification, and small molecule screening to investigate whether distinct chromatin modifiers might complement Fezf2 functions to promote SCPN-specific differentiation by mouse ES (mES)-derived cortical-like neurons. We find that the inhibition of a specific histone deacetylase, Sirtuin 1 (SIRT1), enhances refinement of SCPN subtype molecular identity by both mES-derived cortical-like neurons and primary dissociated E12.5 mouse cortical neurons. In vivo, we identify that SIRT1 is specifically expressed by CPN, but not SCPN, during late embryonic and postnatal differentiation. Together, these data indicate that SIRT1 has neuronal subtype-specific expression in the mouse cortex in vivo, and that its inhibition enhances subtype-specific differentiation of highly clinically relevant SCPN / CSN cortical neurons in vitro.

During late embryonic development of the cerebral cortex, the major class of cortical output neurons termed subcerebral projection neurons (SCPN; including the predominant population of corticospinal neurons, CSN) and the class of interhemispheric callosal projection neurons (CPN) initially express overlapping molecular controls that later undergo subtype-specific refinements. Such molecular refinements are largely absent in heterogeneous, maturation-stalled, neocortical-like neurons (termed “cortical” here) spontaneously generated by established embryonic stem cell (ES) and induced pluripotent stem cell (iPSC) differentiation. Building on recently identified central molecular controls over SCPN development, we used a combination of synthetic modified mRNA (modRNA) for Fezf2, the central transcription factor controlling SCPN specification, and small molecule screening to investigate whether distinct chromatin modifiers might complement Fezf2 functions to promote SCPN-specific differentiation by mouse ES (mES)-derived cortical-like neurons. We find that the inhibition of a specific histone deacetylase, Sirtuin 1 (SIRT1), enhances refinement of SCPN subtype molecular identity by both mES-derived cortical-like neurons and primary dissociated E12.5 mouse cortical neurons. In vivo, we identify that SIRT1 is specifically expressed by CPN, but not SCPN, during late embryonic and postnatal differentiation. Together, these data indicate that SIRT1 has neuronal subtype-specific expression in the mouse cortex in vivo, and its inhibition enhances subtype-specific differentiation of highly clinically relevant SCPN / CSN cortical neurons in vitro.

Cerebrospinal fluid (CSF) provides vital support for the brain. Abnormal CSF accumulation is deleterious for perinatal neurodevelopment, but how CSF leaves the brain during this critical period is unknown. We found in mice a postnatal neurodevelopmental transition phase featuring precipitous CSF K+ clearance, accompanied by water, through the choroid plexus (ChP). The period corresponds to a human fetal stage when canonical CSF clearance pathways have yet to form and congenital hydrocephalus begins to manifest. Unbiased ChP metabolic and ribosomal profiling highlighted this transition phase with increased ATP yield and activated energy-dependent K+ transporters, in particular the Na+-K+-Cl− and water cotransporter NKCC1. ChP-targeted NKCC1 overexpression enhanced K+-driven CSF clearance and enabled more permissive cerebral hydrodynamics. Moreover, ventriculomegaly in an obstructive hydrocephalus model was improved by ChP-targeted NKCC1 overexpression. Collectively, we identified K+-driven CSF clearance through ChP during a transient but critical neurodevelopmental phase, with translational value for pathologic conditions.

The choroid plexus (ChP) epithelium is a source of secreted signaling factors in cerebrospinal fluid (CSF) and a key barrier between blood and brain. Here, we develop imaging tools to interrogate these functions in adult lateral ventricle ChP in wholemount explants and in awake mice. By imaging epithelial cells in intact ChP explants, we observed calcium activity and secretory events that increased in frequency following delivery of serotonergic agonists. Using chronic two-photon imaging in awake mice, we observed spontaneous subcellular calcium events as well as strong agonist-evoked calcium activation and cytoplasmic secretion into CSF. Three-dimensional imaging of motility and mobility of multiple types of ChP immune cells at baseline and following immune challenge or focal injury revealed a range of surveillance and defensive behaviors. Together, these tools should help illuminate the diverse functions of this understudied body-brain interface.

The choroid plexus (ChP) epithelium is a source of secreted signaling factors in cerebrospinal fluid (CSF) and a key barrier between blood and brain. Here, we develop imaging tools to interrogate these functions in adult lateral ventricle ChP in whole-mount explants and in awake mice. By imaging epithelial cells in intact ChP explants, we observed calcium activity and secretory events that increased in frequency following delivery of serotonergic agonists. Using chronic two-photon imaging in awake mice, we observed spontaneous subcellular calcium events as well as strong agonist-evoked calcium activation and cytoplasmic secretion into CSF. Three-dimensional imaging of motility and mobility of multiple types of ChP immune cells at baseline and following immune challenge or focal injury revealed a range of surveillance and defensive behaviors. Together, these tools should help illuminate the diverse functions of this understudied body-brain interface.

Two existing and widely applied protocols of embryonic stem (ES) cell differentiation have been developed to enable in vitro generation of neurons resembling neocortical projection neurons in monolayer culture and from embryoid bodies. The monolayer approach offers advantages for detailed in vitro characterizations and potential mechanistic and therapeutic screening. We investigated whether mouse ES cells undergoing largely undirected neocortical differentiation in monolayer culture recapitulate progressive developmental programs of in vivo progenitor and postmitotic differentiation and whether they develop into specific neocortical subtypes. We find that ES-derived mitotic cells that have been dorsalized by the sonic hedgehog antagonist cyclopamine, and that express, as a total population, cardinal markers of telencephalic progenitors, are, in fact, molecularly heterogeneous. We next show that these progenitors subsequently generate small numbers of heterogeneous neocortical-like neurons that are "stalled" at an immature stage of differentiation, based on multiple developmental criteria. Although some aspects of neocortical development are recapitulated by existing protocols of ES cell differentiation, these data indicate that mouse ES-derived neocortical progenitors both are more heterogeneous than their in vivo counterparts and seemingly include many incorrectly specified progenitors. Furthermore, these ES-derived progenitors spontaneously differentiate into sparse, and incompletely and largely imprecisely differentiated, neocortical-like neurons that fail to adopt specific neuronal identities in vitro. These results provide both foundation and motivation for refining and enhancing directed differentiation of clinically important neocortical projection neuron subtypes.

Directing the differentiation of induced pluripotent stem cells into motor neurons has allowed investigators to develop new models of amyotrophic lateral sclerosis (ALS). However, techniques vary between laboratories and the cells do not appear to mature into fully functional adult motor neurons. Here we discuss common developmental principles of both lower and upper motor neuron development that have led to specific derivation techniques. We then suggest how these motor neurons may be matured further either through direct expression or administration of specific factors or coculture approaches with other tissues. Ultimately, through a greater understanding of motor neuron biology, it will be possible to establish more reliable models of ALS. These in turn will have a greater chance of validating new drugs that may be effective for the disease.

Two existing and widely applied protocols of embryonic stem (ES) cell differentiation have been developed to enable in vitro generation of neurons resembling neocortical projection neurons in monolayer culture and from embryoid bodies. The monolayer approach offers advantages for detailed in vitro characterizations and potential mechanistic and therapeutic screening. We investigated whether mouse ES cells undergoing largely undirected neocortical differentiation in monolayer culture recapitulate progressive developmental programs of in vivo progenitor and postmitotic differentiation and whether they develop into specific neocortical subtypes. We find that ES-derived mitotic cells that have been dorsalized by the sonic hedgehog antagonist cyclopamine, and that express, as a total population, cardinal markers of telencephalic progenitors, are, in fact, molecularly heterogeneous. We next show that these progenitors subsequently generate small numbers of heterogeneous neocortical-like neurons that are “stalled” at an immature stage of differentiation, based on multiple developmental criteria. Although some aspects of neocortical development are recapitulated by existing protocols of ES cell differentiation, these data indicate that mouse ES-derived neocortical progenitors both are more heterogeneous than their in vivo counterparts and seemingly include many incorrectly specified progenitors. Furthermore, these ES-derived progenitors spontaneously differentiate into sparse, and incompletely and largely imprecisely differentiated, neocortical-like neurons that fail to adopt specific neuronal identities in vitro. These results provide both foundation and motivation for refining and enhancing directed differentiation of clinically important neocortical projection neuron subtypes.

Metalloproteinase cleavage of transmembrane proteins (ectodomain cleavage), including the epidermal growth factor (EGF) ligands heparin-binding EGF-like growth factor (HB-EGF), neuregulin (NRG), and transforming growth factor-alpha (TGF-α), is important in many cellular signaling pathways and is disregulated in many diseases. It is largely unknown how physiological stimuli of ectodomain cleavage—hypertonic stress, phorbol ester, or activation of G-protein-coupled receptors [e.g., by lysophosphatidic acid (LPA)]—are molecularly connected to metalloproteinase activation. To study this question, we developed a fluorescence-activated cell sorting (FACS) -based assay that measures cleavage of EGF ligands in single living cells. EGF ligands expressed in mouse lung epithelial cells are differentially and specifically cleaved depending on the stimulus. Inhibition of protein kinase C (PKC) isoenzymes or metalloproteinase inhibition by batimastat (BB94) showed that different regulatory signals are used by different stimuli and EGF substrates, suggesting differential effects that act on the substrate, the metalloproteinase, or both. For example, hypertonic stress led to strong cleavage of HB-EGF and NRG but only moderate cleavage of TGF-α. HB-EGF, NRG, and TGF-α cleavage was not dependent on PKC, and only HB-EGF and NRG cleavage were inhibited by BB94. In contrast, phorbol 12-myristate-13-acetate (TPA) -induced cleavage of HB-EGF, NRG, and TGF-α was dependent on PKC and sensitive to BB94 inhibition. LPA led to significant cleavage of only NRG and TGF-α and was inhibited by BB94; only LPA-induced NRG cleavage required PKC. Surprisingly, specific inhibition of atypical PKCs zeta and iota [not activated by diacylglycerol (DAG) and calcium] significantly enhanced TPA-induced NRG cleavage. Employed in a high-throughput cloning strategy, our cleavage assay should allow the identification of candidate proteins involved in signal transduction of different extracellular stimuli into ectodomain cleavage.—Herrlich, A., Klinman, E., Fu, J., Sadegh, C., Lodish, H. Ectodomain cleavage of the EGF ligands HB-EGF, neuregulin1-β, and TGF-α is specifically triggered by different stimuli and involves different PKC isoenzymes.

Erythropoietin (Epo) is the principal regulator of the erythropoietic response to hypoxic stress, through its receptor, EpoR. The EpoR signals mediating the stress response are largely unknown, and the spectrum of progenitors that are stress responsive is not fully defined. Here, we used flow cytometry to identify stress-responsive Ter119+CD71highfschigh early erythroblast subsets in vivo. In the mouse spleen, an erythropoietic reserve organ, early erythroblasts were present at lower frequencies and were undergoing higher rates of apoptosis than equivalent cells in bone marrow. A high proportion of splenic early erythroblasts coexpressed the death receptor Fas, and its ligand, FasL. Fas-positive early erythroblasts were significantly more likely to coexpress annexin V than equivalent, Fas-negative cells, suggesting that Fas mediates early erythroblast apoptosis in vivo. We examined several mouse models of erythropoietic stress, including erythrocytosis and β-thalassemia. We found a dramatic increase in the frequency of splenic early erythroblasts that correlated with down-regulation of Fas and FasL from their cell surface. Further, a single injection of Epo specifically suppressed early erythroblast Fas and FasL mRNA and cell-surface expression. Therefore, Fas and FasL are negative regulators of erythropoiesis. Epo-mediated suppression of erythroblast Fas and FasL is a novel stress response pathway that facilitates erythroblast expansion in vivo. (Blood. 2006;108:123-133)