Ventral Neural Tube Cells Differentiate into Craniofacial Skeletal Muscles G. S. Sohal, 1 A. A. Ali, and M. M. Ali Department of Cellular Biology and Anatomy, Medical College of Georgia, Augusta, Georgia 30912 Received October 19, 1998 Craniofacial skeletal muscle cells are believed to de- velop from mesoderm. A population of ventral neural tube cells has recently been shown to migrate out of the hindbrain and populate the craniofacial mesen- chyme in chick embryos. Since skeletal muscle cells develop from this mesenchyme, we sought to deter- mine if the emigrated neural tube cells contributed to their development. Ventral neural tube cells in the hindbrain of chick embryos were labeled on embry- onic day 2 with replication-deficient retroviral vectors containing the gene LacZ, which provides a perma- nent marker for the progeny. On day 7 embryos were processed for the detection of labeled cells. Labeled cells were seen in craniofacial skeletal muscles. By using muscle-specific markers, the labeled cells were confirmed to be skeletal muscle cells. Thus, some mus- cle cells are derived from the ventral neural tube cells of the hindbrain. © 1998 Academic Press Key Words: ventral neural tube cells; skeletal muscle; myocytes; development; chick embryo. It is generally believed that the craniofacial skeletal muscles develop from two cellular sources. Mesoderm is believed to provide the muscle cells and neural crest the connective tissue cells (1). This is based on the understanding that the neural tube provides a single population of cells, the neural crest, for the formation of structures developing outside the central nervous system (CNS). Neural crest cells originate from the dorsal portion of the developing neural tube (2, 3). After the emigration of neural crest, the neural tube cells are believed to give rise to only neurons and supporting cells of the CNS. In contrast to the aforementioned traditional view, several investigators have reported that some neural tube cells also emigrate and contribute to the forma- tion of structures developing outside the CNS. For example in the avian spinal cord, both the dorsal and the ventral neural tube cells emigrate, via their respec- tive nerve roots, and give rise to neural and non-neural cell types (4 –7). Emigration of neural tube cells also occurs from the cranial portion of the neural tube. Studies utilizing focal application of the vital dye DiI, homeobox gene islet-1 expression pattern, and retrovi- ral labeling have shown that some cells originating in the ventral portion of the hindbrain emigrate at the site of attachment of cranial nerves (8 –10). They con- tribute to the formation of structures developing out- side the CNS (8-10). The ventral neural tube cells emigrate considerably after the emigration of neural crest is finished and they do not express the neural crest cell antigen, HNK-1 (8 –10). We have recently followed the emigration of ventral neural tube cells from the rostral hindbrain. While some of the emigrated cells populate the trigeminal ganglion others migrate into the craniofacial mesen- chyme (10). Since craniofacial muscles develop from this mesenchyme and the possibility that neural tube cells may give rise to muscle cells has been suggested by numerous observations (described in the Discussion section), we sought to determine if the ventral neural tube cells contributed to the formation of muscles. MATERIALS AND METHODS Fertilized Arbor Acre chicken eggs were incubated at 37.5°C. Dur- ing the second day of incubation an opening in the eggshell was made to acquire access to the embryo. Ventral neural tube cells were labeled with replication-deficient retroviral vectors containing the LacZ gene, as described in detail previously (9, 10). Briefly, about 0.2 l of the vector LZ12 or LZ14 (provided by Deni S. Galileo, Depart- ment of Cellular Biology and Anatomy, Medical College of Georgia) was introduced into embryos at stage 14 of Hamburger and Hamil- ton stage series (11). The vector was microinjected into the lumen of the rostral hindbrain neural tube. In control embryos, the virus was dropped on top of the hindbrain neural tube. Embryos were sacrificed on embryonic day 7. They were fixed in 2% formaldehyde in phosphate-buffered saline (PBS). Subsequently, they were processed for histochemical detection of LacZ positive cells with the substrate Bluo-Gal as described for X-gal (12, 13). Speci- mens were postfixed in the same fixative and processed for standard paraffin histology. Twenty-one embryos were serially sectioned at 15 m thickness and stained with eosin. 1 To whom correspondence should be addressed. Fax: (706) 721- 6839. BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 252, 675– 678 (1998) ARTICLE NO. RC989715 675 0006-291X/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.