The development and origins of vertebrate meninges

Meninges comprise three distinct layers, the dura mater, arachnoid, and pia mater that surround the brain, spinal cord and some parts of the nerves. Traditionally the meninges were believed to serve only as protection for tissues that they encase. However recent work shows they have other important functions related to development and regulation of the nervous system. Given the importance of the meninges, it is surprising that we know very little about their development. The embryological origin of the meninges has been debated for over a hundred years. Some studies imply that the meninges develop from the neural crest, while others suggest that they come from the somites. Here, we investigated the temporal development of meninges in birds and mice and found they form at comparable stages. We investigated the origin of avian spinal meninges using chick/quail cell tracing protocols and found they do not develop from the somites as previously thought. We propose that meningeal epithelial blood vessels may have been mistaken as meninges and led to an erroneous conclusion by previous investigators. We present data that show that avian spinal meninges originated from the neural crest supported by data demonstrating that they express the neural crest marker HNK1. Finally using the Wnt1-Cre mouse we show that trunk meninges of mammals also originate from neural crest

Characterisation of development and electrophysiological mechanisms underlying rhythmicity of the avian lymph heart

Despite significant advances in tissue engineering such as the use of scaffolds, bioreactors and pluripotent stem cells, effective cardiac tissue engineering for therapeutic purposes has remained a largely intractable challenge. For this area to capitalise on such advances, a novel approach may be to unravel the physiological mechanisms underlying the development of tissues that exhibit rhythmic contraction yet do not originate from the cardiac lineage. Considerable attention has been focused on the physiology of the avian lymph heart, a discrete organ with skeletal muscle origins yet which displays pacemaker properties normally only found in the heart. A functional lymph heart is essential for avian survival and growth in ovo. The histological nature of the lymph heart is similar to skeletal muscle although molecular and bioelectrical characterisation during development to assess mechanisms that contribute towards lymph heart contractile rhythmicity have not been undertaken. A better understanding of these processes may provide exploitable insights for therapeutic rhythmically contractile tissue engineering approaches in this area of significant unmet clinical need. Here, using molecular and electrophysiological approaches, we describe the molecular development of the lymph heart to understand how this skeletal muscle becomes fully functional during discrete in ovo stages of development. Our results show that the lymph heart does not follow the normal transitional programme of myogenesis as documented in most skeletal muscle, but instead develops through a concurrent programme of precursor expansion, commitment to myogenesis and functional differentiation which offers a mechanistic explanation for its rapid development. Extracellular electrophysiological field potential recordings revealed that the peak-to-peak amplitude of electrically evoked local field potentials elicited from isolated lymph heart were significantly reduced by treatment with carbachol; an effect that could be fully reversed by atropine. Moreover, nifedipine and cyclopiazonic acid both significantly reduced peak-to-peak local field potential amplitude. Optical recordings of lymph heart showed that the organ’s rhythmicity can be blocked by the HCN channel blocker, ZD7288; an effect also associated with a significant reduction in peak-to-peak local field potential amplitude. Additionally, we also show that isoforms of HCN channels are expressed in avian lymph heart. These results demonstrate that cholinergic signalling and L-type Ca2+ channels are important in excitation and contraction coupling, while HCN channels contribute to maintenance of lymph heart rhythmicity

Expression of HCN1 and HCN4 at HH36.

<p>Whole mount in-situ hybridisation showing expression of <i>HCN1</i> and <i>HCN4</i> in avian lymph heart at HH36 as indicated by red arrow. The expression of these channels was not detected in other stages.</p

Summary of the genes regulated during lymph heart development and involvement of ion channels in excitability and rhythmicity of the lymph heart.

<p>The lymph heart predominantly expresses skeletal muscle markers and their expression is evident as early as HH30 and decline<b>s</b> by HH38. By HH36, the lymph heart contracts rhythmically and the basis for their underlying rhythmicity is most likely mediated by HCN channels while their excitation and contraction coupling is facilitated by cholinergic and L-type Ca²⁺ channels.</p

Expression of Prox-1, and Cav1.1 during lymph heart development.

<p>Whole mount in-situ hybridisation showing developmental expression of <i>Prox-1</i> (A1-A4) and <i>Cav1</i>.<i>1</i> (B1-B4) in the lymph heart as indicated by red arrow. The expression of <i>Prox-1</i> and <i>Cav1</i>.<i>1</i> was detected as early as HH30 and declined by HH38.</p

Expression of cadherins during lymph heart development.

<p>Whole mount in-situ hybridisation showing developmental expression of <i>M-cadherin</i> (A1-A4), <i>N-cadherin</i> (B1-B4), <i>R-cadherin</i> (C1-C4) and <i>T-cadherin</i> (D1-D4) in the lymph heart as indicated by red arrow. The expression of <i>M</i>, <i>N</i>, and <i>R</i>, and <i>T-cadherin</i> was detected as early as HH30 and only <i>M-cadherin</i> was expressed at HH38.</p

Expression of skeletal muscle markers during development of lymph heart.

<p>Whole mount in-situ hybridisation showing developmental expression of <i>Pax-7</i> (A1-A4), <i>MyoD</i> (B1-B4), <i>Myogenin</i> (C1-C4) and Engrailed-1 (<i>En-1</i>) (D1-D4) in the lymph heart as indicated by red arrow. The expression of <i>Pax-7</i>, <i>MyoD</i>, <i>Myogenin and En-1</i> was detected as early as HH30 and declined by HH38.</p

Evoked local field potential recorded from lymph heart before and after addition of carbachol followed by atropine in the presence of carbachol.

<p>A) Representative trace of evoked local field potential (LFP) obtained from control, in the presence of carbachol (10μM) B) and after adding atropine (5μM) in the presence of carbachol C). D) Bar charts show average peak to peak of evoked LFP amplitude calculated from control, in the presence of carbachol and after adding atropine in the presence of carbachol (n = 9). The data for evoked LFP in the presence of carbachol and the data for evoked LFP of atropine in the presence of carbachol was normalised to data obtained from control. After adding carbachol the average peak to peak of evoked <b>LFP</b> amplitude was significantly reduced (*** p<0.001) compared to the control. On addition of atropine in the presence of carbachol the effects were significantly reversed (*** p<0.001). Error bars represent mean ± SEM (n = 9). E) Bar chart shows average peak to peak of evoked LFP amplitude calculated from control, in the presence of TTX (n = 3). The data for evoked LFP in the presence of TTX was normalised to data obtained from control. After adding TTX the average peak to peak of evoked LFP was reduced compared to control however it was not statistically significant (p>0.05). Error bars represent mean ± SEM.</p

Evoked local field potential recorded from lymph heart before and after addition of cyclopiazonic acid followed by calcium.

<p>A) Representative trace of evoked local field potential (LFP) obtained from control, in the presence of CPA (10μM) B) and after adding Ca²⁺ (2mM) C). D) Bar charts show average peak to peak of evoked LFP amplitude calculated from control, in the presence of CPA and after adding Ca²⁺ (n = 13). The data for the evoked LFP in the presence of CPA and the data for the evoked LFP of Ca²⁺ was normalised to data obtained from control. After adding CPA the average peak to peak of evoked LFP amplitude was significantly reduced (* p<0.05) compared to control. On addition of Ca²⁺ the average peak to peak of evoked LFP amplitude significantly increased compared to CPA (*** p<0.001) and control (* p<0.05). Error bars represent mean ± SEM (n = 13).</p