Laura Howard | Cardiff University (original) (raw)

Papers by Laura Howard

Neuroscience Letters, 2015

The extracellular calcium-sensing receptor (CaSR) is a G-protein coupled receptor that monitors t... more The extracellular calcium-sensing receptor (CaSR) is a G-protein coupled receptor that monitors the systemic extracellular free ionized calcium level ([Ca(2+)]o) in organs involved in systemic [Ca(2+)]o homeostasis. CaSR is widely expressed in the nervous system and its activation promotes axon and dendrite growth during development, but the mechanism by which it does this is not known. Here we show that enhanced axon growth and branching from cultured embryonic sympathetic neurons by activation of the endogenous CaSR depends on the presence of nerve growth factor (NGF). Our observation that activation of overexpressed CaSR promotes axon growth in NGF-free medium has enabled us to investigate CaSR downstream signaling contributing to axon growth in the absence of NGF signaling. We show that activation of overexpressed CaSR leads to activation of ERK1 and ERK2, and pharmacological inhibition of CaSR-dependent ERK1/ERK2 activation prevents CaSR-dependent axon growth. Analysis of axon growth from cultured neurons expressing deletion mutants of the CaSR cytoplasmic tail revealed that the region between alanine 877 and glycine 907 is required for promoting axon growth that is distinct from the high-affinity filamin-A binding site that has previously been implicated in ERK1/ERK2 activation.

Cell reports, Jan 3, 2015

The regulation of innervation by target-derived factors like nerve growth factor (NGF) is the cor... more The regulation of innervation by target-derived factors like nerve growth factor (NGF) is the cornerstone of neurotrophic theory. Whereas autocrine signaling in neurons affecting survival and axon growth has been described, it is difficult to reconcile autocrine signaling with the idea that targets control their innervation. Here, we report that an autocrine signaling loop in developing mouse sympathetic neurons involving CD40L (TNFSF5) and CD40 (TNFRSF5) selectively enhances NGF-promoted axon growth and branching, but not survival, via CD40L reverse signaling. Because NGF negatively regulates CD40L and CD40 expression, this signaling loop operates only in neurons exposed to low levels of NGF. Consequently, the sympathetic innervation density of tissues expressing low NGF is significantly reduced in CD40-deficient mice, whereas the innervation density of tissues expressing high levels of NGF is unaffected. Our findings reveal how differential regulation of autocrine signaling in neu...

EMBO reports, 2008

Expression of the basic helix-loop-helix transcription factor HAND2 begins early in sympathetic n... more Expression of the basic helix-loop-helix transcription factor HAND2 begins early in sympathetic neuron development and is essential for the differentiation of noradrenergic neurons. Here, we show that the expression of HAND2 and related HAND1 are maintained in sympathetic neurons throughout fetal and postnatal development when these neurons depend on target-derived nerve growth factor (NGF) for survival. Short interfering RNA knockdown of endogenous HAND2 and, to a lesser extent, HAND1 in neonatal sympathetic neurons cultured with NGF, reduced the expression of the NGF receptor tyrosine kinase TrkA (tropomyosin-related kinase A), as well as neuronal survival. Chromatin immunoprecipitation analysis showed that NGF promotes HAND2 binding to the TrkA minimal enhancer and that transfection of sympathetic neurons with a TrkA expression plasmid rescued the neurons from HAND knockdown. These findings show that HAND transcription factors have a crucial function in sustaining the survival of neonatal sympathetic neurons with NGF by a feed-forward loop that maintains the expression of TrkA.

Developmental Biology, 2007

The gene encoding the Sox F-group transcription factor Xsox17alpha(1) is specifically expressed t... more The gene encoding the Sox F-group transcription factor Xsox17alpha(1) is specifically expressed throughout the entire region of the Xenopus blastula fated to become endoderm, and is important in controlling endodermal development. Xsox17alpha(1) is a direct target of the maternal endodermal determinant VegT and of Sox17 itself. We have analysed the promoter of the Xenopus laevis Xsox17alpha(1) gene by transgenesis, and have identified two important control elements which reside about 9 kb upstream at the start of transcription. These elements individually drive transgenic endodermal expression in the blastula and gastrula. One contains functional, cooperating VegT and Sox-binding consensus sites. The Sox sites in this region are occupied in vivo. The other responds to TGF-beta signals like Activin or Nodals that act through Smad2/3. We propose that these two regions co-operate in regulating the early endodermal expression of the Xsox17alpha(1) gene.

Development, 2006

A conserved molecular pathway has emerged controlling endoderm formation in Xenopus zebrafish and... more A conserved molecular pathway has emerged controlling endoderm formation in Xenopus zebrafish and mice. Key genes in this pathway include Nodal ligands and transcription factors of the Mix-like paired homeodomain class, Gata4-6 zinc-finger factors and Sox17 HMG domain proteins. Although a linear epistatic pathway has been proposed, the precise hierarchical relationships between these factors and their downstream targets are largely unresolved. Here, we have used a combination of microarray analysis and loss-of-function experiments to examine the global regulatory network controlling Xenopus endoderm formation. We identified over 300 transcripts enriched in the gastrula endoderm, including most of the known endoderm regulators and over a hundred uncharacterized genes. Surprisingly only 10% of the endoderm transcriptome is regulated as predicted by the current linear model. We find that Nodal genes, Mixer and Sox17 have both shared and distinct sets of downstream targets, and that a number of unexpected autoregulatory loops exist between Sox17 and Gata4-6, between Sox17 and Bix1/Bix2/Bix4, and between Sox17 and Xnr4. Furthermore, we find that Mixer does not function primarily via Sox17 as previously proposed. These data provides new insight into the complexity of endoderm formation and will serve as valuable resource for establishing a complete endoderm gene regulatory network.

Differentiation, 2004

The Endodermin gene is expressed in the early endoderm and the Spemann organizer of Xenopus embry... more The Endodermin gene is expressed in the early endoderm and the Spemann organizer of Xenopus embryos. It has previously been shown to be a direct target of the early endodermal transcription factor Xsox17 (Clements et al., 2003, Mech Dev 120:337-348). Here we identify two adjacent control elements in the Endodermin promoter; these drive transcription of the gene in late-gastrula endoderm and contain consensus Soxbinding sites. We have analyzed one element in detail and show that it responds directly to Xsox17 and that the Sox sites are essential for endodermal expression in transgenic embryos. However, flanking regions on both sides are also essential, indicating that Xsox17 acts in concert with several DNA-binding partners.

Neuroscience Letters, 2015

The extracellular calcium-sensing receptor (CaSR) is a G-protein coupled receptor that monitors t... more The extracellular calcium-sensing receptor (CaSR) is a G-protein coupled receptor that monitors the systemic extracellular free ionized calcium level ([Ca(2+)]o) in organs involved in systemic [Ca(2+)]o homeostasis. CaSR is widely expressed in the nervous system and its activation promotes axon and dendrite growth during development, but the mechanism by which it does this is not known. Here we show that enhanced axon growth and branching from cultured embryonic sympathetic neurons by activation of the endogenous CaSR depends on the presence of nerve growth factor (NGF). Our observation that activation of overexpressed CaSR promotes axon growth in NGF-free medium has enabled us to investigate CaSR downstream signaling contributing to axon growth in the absence of NGF signaling. We show that activation of overexpressed CaSR leads to activation of ERK1 and ERK2, and pharmacological inhibition of CaSR-dependent ERK1/ERK2 activation prevents CaSR-dependent axon growth. Analysis of axon growth from cultured neurons expressing deletion mutants of the CaSR cytoplasmic tail revealed that the region between alanine 877 and glycine 907 is required for promoting axon growth that is distinct from the high-affinity filamin-A binding site that has previously been implicated in ERK1/ERK2 activation.

Cell reports, Jan 3, 2015

The regulation of innervation by target-derived factors like nerve growth factor (NGF) is the cor... more The regulation of innervation by target-derived factors like nerve growth factor (NGF) is the cornerstone of neurotrophic theory. Whereas autocrine signaling in neurons affecting survival and axon growth has been described, it is difficult to reconcile autocrine signaling with the idea that targets control their innervation. Here, we report that an autocrine signaling loop in developing mouse sympathetic neurons involving CD40L (TNFSF5) and CD40 (TNFRSF5) selectively enhances NGF-promoted axon growth and branching, but not survival, via CD40L reverse signaling. Because NGF negatively regulates CD40L and CD40 expression, this signaling loop operates only in neurons exposed to low levels of NGF. Consequently, the sympathetic innervation density of tissues expressing low NGF is significantly reduced in CD40-deficient mice, whereas the innervation density of tissues expressing high levels of NGF is unaffected. Our findings reveal how differential regulation of autocrine signaling in neu...

EMBO reports, 2008

Expression of the basic helix-loop-helix transcription factor HAND2 begins early in sympathetic n... more Expression of the basic helix-loop-helix transcription factor HAND2 begins early in sympathetic neuron development and is essential for the differentiation of noradrenergic neurons. Here, we show that the expression of HAND2 and related HAND1 are maintained in sympathetic neurons throughout fetal and postnatal development when these neurons depend on target-derived nerve growth factor (NGF) for survival. Short interfering RNA knockdown of endogenous HAND2 and, to a lesser extent, HAND1 in neonatal sympathetic neurons cultured with NGF, reduced the expression of the NGF receptor tyrosine kinase TrkA (tropomyosin-related kinase A), as well as neuronal survival. Chromatin immunoprecipitation analysis showed that NGF promotes HAND2 binding to the TrkA minimal enhancer and that transfection of sympathetic neurons with a TrkA expression plasmid rescued the neurons from HAND knockdown. These findings show that HAND transcription factors have a crucial function in sustaining the survival of neonatal sympathetic neurons with NGF by a feed-forward loop that maintains the expression of TrkA.

Developmental Biology, 2007

The gene encoding the Sox F-group transcription factor Xsox17alpha(1) is specifically expressed t... more The gene encoding the Sox F-group transcription factor Xsox17alpha(1) is specifically expressed throughout the entire region of the Xenopus blastula fated to become endoderm, and is important in controlling endodermal development. Xsox17alpha(1) is a direct target of the maternal endodermal determinant VegT and of Sox17 itself. We have analysed the promoter of the Xenopus laevis Xsox17alpha(1) gene by transgenesis, and have identified two important control elements which reside about 9 kb upstream at the start of transcription. These elements individually drive transgenic endodermal expression in the blastula and gastrula. One contains functional, cooperating VegT and Sox-binding consensus sites. The Sox sites in this region are occupied in vivo. The other responds to TGF-beta signals like Activin or Nodals that act through Smad2/3. We propose that these two regions co-operate in regulating the early endodermal expression of the Xsox17alpha(1) gene.

Development, 2006

A conserved molecular pathway has emerged controlling endoderm formation in Xenopus zebrafish and... more A conserved molecular pathway has emerged controlling endoderm formation in Xenopus zebrafish and mice. Key genes in this pathway include Nodal ligands and transcription factors of the Mix-like paired homeodomain class, Gata4-6 zinc-finger factors and Sox17 HMG domain proteins. Although a linear epistatic pathway has been proposed, the precise hierarchical relationships between these factors and their downstream targets are largely unresolved. Here, we have used a combination of microarray analysis and loss-of-function experiments to examine the global regulatory network controlling Xenopus endoderm formation. We identified over 300 transcripts enriched in the gastrula endoderm, including most of the known endoderm regulators and over a hundred uncharacterized genes. Surprisingly only 10% of the endoderm transcriptome is regulated as predicted by the current linear model. We find that Nodal genes, Mixer and Sox17 have both shared and distinct sets of downstream targets, and that a number of unexpected autoregulatory loops exist between Sox17 and Gata4-6, between Sox17 and Bix1/Bix2/Bix4, and between Sox17 and Xnr4. Furthermore, we find that Mixer does not function primarily via Sox17 as previously proposed. These data provides new insight into the complexity of endoderm formation and will serve as valuable resource for establishing a complete endoderm gene regulatory network.

Differentiation, 2004

The Endodermin gene is expressed in the early endoderm and the Spemann organizer of Xenopus embry... more The Endodermin gene is expressed in the early endoderm and the Spemann organizer of Xenopus embryos. It has previously been shown to be a direct target of the early endodermal transcription factor Xsox17 (Clements et al., 2003, Mech Dev 120:337-348). Here we identify two adjacent control elements in the Endodermin promoter; these drive transcription of the gene in late-gastrula endoderm and contain consensus Soxbinding sites. We have analyzed one element in detail and show that it responds directly to Xsox17 and that the Sox sites are essential for endodermal expression in transgenic embryos. However, flanking regions on both sides are also essential, indicating that Xsox17 acts in concert with several DNA-binding partners.